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May 6, 2013 - binding domain .... Structure Screen 1 cacodylate-free 50 x 10 mL MD1-01-CF ... cacodylate-free. Structure Screen 1 Single Reagent. 100 mL.
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MOLECULAR DIMENSIONS Providing you with the latest innovations in protein crystallography

Inside

For Protein Crystallography

Molecular Dimensions Ltd. Unit 6, Goodwin Business Park, Willie Snaith Road, Newmarket, Suffolk, CB8 7SQ, UK Telephone: +44 (0)1638561051 Fax: +44 (0)1638660674

Molecular Dimensions Inc. 849 Sunshine Lane, Altamonte Springs, Florida, 32714 USA Telephone: +1 877 479 4339 or +1 407 886 6901 Fax: +1 321 972-8896

Latest screens New additives Cryo cocktails In situ plates Sub µL DLS Crystal lead finder Academic articles Crystallization exercises

Intelligent solutions for

protein crystal growth

MD Journal Cover with Spine_MD Journal Build Cover with Spine 6/5/13 10:29 AM Page 2

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03

The Meaning of Quality

Welcome

ARTICLES

How to order You can place your order online, by fax, telephone or via e-mail. Our site includes a full e-commerce facility for selecting and ordering your product. Credit card orders can be placed securely on-line.

Returns Returns will not be accepted without prior authorization. Damaged items must be notified within 7 days.

Prices

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12

Towards High Efficiency Screening

Exploring More Crystallization Space

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26

The MemGold Family: the science behind the screens

Seeding Lab Exercises

Making Use of Counter Diffusion

64

52

67 Choosing the right Crystallization Plate

Advanced Methods

77

To place an order in Europe or the rest of the world, excluding the Americas please place your order with our UK office.

To place an order in the USA, Canada, Mexico or South America please place your order with our USA office.

Molecular Dimensions Ltd. Unit 6, Goodwin Business Park, Willie Snaith Road, Newmarket, Suffolk, CB8 7SQ, UK Telephone: +44 (0)1638561051 Fax: +44 (0)1638660674

Molecular Dimensions Inc. 849 Sunshine Lane, Altamonte Springs, Florida, 32714 USA Telephone: +1 877 479 4339 or +1 407 886 6901 Fax: +1 321 972-8896

To order by e-mail use [email protected]

To order by e-mail use [email protected]

To order on account please contact us to set up an account.

To order on account please contact us to set up an account.

Shipping

Shipping

Products are shipped by DHL.

Products are shipped by Fedex.

32 Identifying hits with Trace Fluorescent Labelling

Media Composition influences Recombinant Protein accumulation in E. Coli

Prices are subject to change without notice. There is no minimum order charge.

99 Understanding Radiation Damage

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

108 Systematic Approaches to Crystallization and Data Collection

visit: www.moleculardimensions.com

Distributors CHINA Bio-Direct Co. Ltd. Room 301, Building No. 2. 88 Darwin Road, Pudong, 201203 Shanghai, P.R. China Tel: +86 21 5197 3610

KOREA MDxK Molecular Diagnostics Korea Inc. T: +82 2 578 8848 F: +82 2 578 8838 4F Song B/D, 333-16, Yungjae-Dong, Seocho-Gu, Seoul 137-897, Korea (Republic of)

Beijing Seaskybio Technology Co.Ltd Room 904A Building, Maples International Centre, No. 32 Xizhimen North Street, Haidian District, Beijing, China, 100082 Tel: +8610-62220605 Fax: +8610-62168941

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INDIA RFCL Limited, a part of Avantor Performance Materials 12th Floor, Pinnacle Claridges Business Tower, Shooting Range Road, Surajkund, Faridabad-121009, India Tel: +91-129-4267059 Fax : +91-129-4267199

RUSSIAN FEDERATION Vladimir Chernyavsky Nagatinskaya Naberezhnaya 54, Suite 577 Moscow, Russia 115407 Tel. +79167575787 Tel. +79857773773

SINGAPORE Practical Mediscience Pte Ltd +65 6254 2633 551-C Baletier Road, Singapore 329868, Singapore Bio-Etc Pte Ltd (Email: [email protected]) 41 Science Park Road #04-06A The Gemini Singapore Science Park II Singapore 117610 Mobile: +65 8617 7360 Office: +65 6779 4149 Fax: +65 6779 4095 TAIWAN Yeong Jyi Chemical +886 (2) 2982 8796 Apparatus Co. Limited PO Box 117, Sanchung, Taipei, Taiwan

JAPAN Waken B Tech, Co., Ltd. Sales Promotion Dept. 1-12-8 Senbahigashi, Minoh-city, Osaka 562-0035, Japan Phone: +81-72-749-5300 Fax: +81-72-749-5600 http://www.wakenbtech.co.jp North & South America orders: +1 877 479 4339 email: [email protected]

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 1

CONTENTS 4

Crystal growth screens 5

14

16 17

25

Soluble proteins 5 Structure screen 1 6 Structure screen 2 6 Stura footprint screens 7 Mini screen 7 MultiXal™ screen 8 The solubility tool kit™ 8 Clear strategy screens™ I & II 9 PACT premier™ 9 JCSG plus™ 13 Morpheus® Protein complexes 14 Macrosol™ 15 Proplex™ 15 MIDAS™ Nuclear receptors 16 Nuclear receptor ligand binding domain Membrane proteins 17 MemStart™ 18 MemSys™ 18 MemPlus™ 19 MemGold™ 19 MemGold 2™ 22 Lipidic-sponge phase™ screen 22 PGA™ screen 23 MemMagic® bicelle screen kit 23 The Calixar™ Additive Kit 24 MemAdvantage™ Nucleic acids 25 Helix™

26 Optimization 30 31 31 35 36 36 37 38 39 39 40 40 41 43 43 44 45 45 46

Custom screens Naomi’s nucleant MicroSeed Beads™ Trace Fluorescent labelling kits Additive screen Single reagents PEG precipitants Alternative precipitiants Jeffamines® Volatiles – organics Non-volatiles – organics Polyamines Salts Other reagents Cryoprotectants Buffer stocks Special buffers Morpheus® mixes Morpheus® individual stock reagents 48 MIDAS™ reagents 49 MemAdvantage reagents 51 The Really Useful Buffer kit 51 Gelled Surface Kit 51 Microbatch Oils

60 Plates 61 XRL 24–well plates 61 24-well SBS plates 61 Grease 61 Sitting drop bridges 62 Coverslips 63 Counter diffusion 66 High-throughput plates 68 MRC Crystallization plates 68 Triple drop plates 68 MRC Maxi Optimization plates 69 Microbatch plates 69 In situ diffraction plates 72 Hanging drop plates & seals 72 Screw top hanging drop plate 73 Laminex for LCP 73 Sealing sheets and deep well blocks 76 Protein production 76 Protein production 80 Protein expression media 80 Animal product free media 81 Media optimization kit 81 LB Broth 82 Turbo Broth 82 Superior Broth 83 Power Broth 83 Hyper Broth 84 Glucose M9Y, & nutrient mix 84 Augmedium 85 LB Booster 85 Atholate 85 Perk expression rescue kit 86 Expressmax 86 Labelling media 87 SelenoMethionine media 87 SILAC media 88 Cell culture products 88 BRFF-BMZERO 89 BRFF-EPM2 89 BRFF-HPC1 90 BRFF-P4-8F 90 PET Cell dissociation formula 91 Freezing media pair 91 FNC Coating mix 92 HEPES buffered saline 92 DMEM / F12 serum free medium 93 MDM serum free medium 93 Bovine pituitary extract 94 SFM screening kit 94 Protein refolding reagents 95 Quickfold refolding kit 95 Detergent screening kit 96 Cyclodextrin kit 96 Dialysis & Accessories 97 Gebaflex dialysis tubes 97 Capillaries 97 Paper wicks 97 LCP mixing adaptor

North & South America orders: +1 877 479 4339 email: [email protected]

98 Cryocrystallography 102 102 102 103 104 105 105 109 110 110

LithoLoops™ Elliptical LithoLoops Mesh LithoLoops CryoMount Sets Magnetic CryoVials & CryoCaps DataMouse Pro Cryoprotectants CryoProtX™ Dewars Cryogenic shippers, & storage dewars 112 EMBL/ESRF sample changer kits 114 CryoTools & accessories

115 Books 116 Tools 116 Microtools 117 Greasing 118 Giftware 119 Crystal structure glass blocks 120Crystallography instrumentation 120 Incubators 120 Bench top incubators 122 Floor standing vibration free incubators 124 Liquid handling 124 Optimizer 126 Imaging 126 Crystal X2 automated lead finder 128 X-taLight™ 200 automated UV imaging system 129 X-taLight™ 100 UV fluorescence light source 130 Dynamic light scattering 130 SpectroSize™ 300 cuvette dls 131 SpectroLight™ 600 sub µL screening dls 132 Pre crystallization screening 133 In crystallization analysis 134 Product Index 145 How to order

visit: www.moleculardimensions.com

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 2

THE MEANING OF QUALITY QUALITY IS A WORD THAT IS OFTEN USED BUT RARELY CARRIES ANY REAL MEANING ISO 9001 Registered

Quality Management

Editorial We read about “quality products” but find little qualification of what is actually being described. Quality systems, and international quality assurance standards are, however, very different as they convey real and unequivocal meaning. To achieve accreditation to an internationally recognised standard requires the operation of a quality assurance system of processes and procedures that are regularly audited internally and externally assessed in order to maintain certification.

S

uch internationally agreed standards are regularly updated and an accredited company must adapt its systems in order to continue to meet the published criteria. Molecular Dimensions Limited has achieved accreditation to ISO 9001: 2008 from the international assessment organisation NQA. This is part of the latest version of the ISO 9000 standard and perhaps the biggest change from previous versions has been the emphasis on continual improvement. It is no longer good enough to continue to meet measurable performance levels alone; we now have to demonstrate that we have systems that help us to improve. Our measured trends also must show that we are constantly improving the way we run our business. Our Quality System is not just a system of internal procedures but is designed to deliver real benefits to our customers. Our quality policy states that we will win your business by having the best reputation for reliable products, advice and service information, including delivery. The Molecular Dimensions Quality System is a set of processes that not only establish conformance but, strive to continually improve our effectiveness as a business. Our system has three main components: system processes and monitoring processes which ensure that the system functions in a measurable way; but of most importance to you are the customer processes. We have standards for how we handle the complete product supply chain from how we answer customer inquiries to delivering an order. Customer related processes Enquiries Order processing Production Final product monitoring Goods received Stock control Packing & delivery Non-conforming products Non-conforming service Inside these processes there are procedures that are customer focused. For example our product standards are all set based on their expected performance in our customers’ laboratories. It is often said at conferences that there is a need for rationalisation of the number of conditions used for screening for crystal growth based on what have been successful. All of the Molecular Dimensions’ screens have been developed by our customers in just that way; as a result of selecting sets of conditions that have worked particularly well in their experience. As a result of this approach our product development programme is ‘truly customer focussed’.

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Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

visit: www.moleculardimensions.com

MD Journal Build_Layout 1 6/5/13 10:10 AM Page 3

Welcome

to the new Molecular Dimensions catalogue. Looking back to fifteen years ago when we started Molecular Dimensions it was our vision to offer crystallographers a choice of the very latest tools for their research and also bring new methods to a broader audience all over the world. As with everything we started small but rapidly became well recognised in Europe and within two years had opened a subsidiary company in the USA offering next day delivery from east to west coast. As the science of crystallography progressed we continued to foster and maintain strong alliances with leading crystallographers to develop and commercialise unique, proven ideas. Some of these have kindly contributed interesting and educational articles which can be found throughout this catalogue. In addition to this, Molecular Dimensions formed partnerships with entrepreneurial companies with novel technologies and ideas applicable to further bridging the gap between industrial and academic thinking. These collaborations have led to the introduction of crystallization methods for high efficiency screening, extended range crystallization space, regularly updated tools for membrane proteins, and stimulated high throughput methods for crystallization and data collection, to name but a few. Most importantly continually delivering proven results has given people the belief to try something new for the first time and also the ability to trust in our products for their day-to-day research.

Tony Savill President, Molecular Dimensions

We know that major progress in scientific research is driven by the continuous development of new technology and methods, structural biology is no exception. Over time we have built a team of trained customer service staff and experienced scientists to understand both the work you do and also provide reliable technical and customer service information. Today we are proud to be supported by equally well qualified personnel throughout the global community of crystallography who are able to provide local service and accurate advice in markets such as India, the Far East and South America. In 2013 Molecular Dimensions continues its commitment to bring forward new and exciting ideas with the introduction of a modern nucleic acid screen, a counter diffusion screen, the first membrane protein additive screen, calix(4)arene additives, fluorescent trace labelling, cryo-protection cocktails, high throughput plates for lipidic cubic phase, in situ screening plates, and micro DLS directed crystal growth. We hope both these and the future developments we are committed to delivering will enable you to get the positive results you need. We are sure you will find the catalogue both interesting and informative. Good luck with your research and we look forward to seeing you all again soon,

Tony Savill President, Molecular Dimensions North & South America orders: +1 877 479 4339 email: [email protected]

visit: www.moleculardimensions.com

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 4

CRYSTAL GROWTH SCREENS MOLECULAR DIMENSIONS SUPPLIES A WIDE RANGE OF CRYSTAL GROWTH SCREENS FROM CLASSIC SPARSE MATRICES TO MODERN HIGH EFFICIENCY PRODUCTS EXTENSIVELY COVERING CRYSTALLIZATION SPACE FOR GLOBULAR PROTEINS, MEMBRANE PROTEINS AND NUCLEIC ACIDS.

5 Soluble proteins 5 Structure screen 1 6 Structure screen 2 6 Stura footprint screens 7 Mini screen 7 MultiXal™ screen 8 The solubility tool kit™ 8 Clear strategy screens™ I & II 9 PACT premier™ 9 JCSG plus™ 13 Morpheus®

14 Protein complexes 14 Macrosol™ 15 Proplex™ 15 MIDAS™ 16 Nuclear receptors 16 Nuclear receptor ligand binding domain 17 Membrane proteins 17 MemStart™ 18 MemSys™ 18 MemPlus™

19 MemGold™ 19 MemGold 2™ 22 Lipidic-sponge phase™ screen 22 PGA™ screen 23 MemMagic® bicelle screen kit 23 The Calixar™ Additive Kit 24 MemAdvantage™ 25 Nucleic acids 25 Helix™

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Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

visit: www.moleculardimensions.com

MD Journal Build_Layout 1 6/5/13 10:10 AM Page 5

SOLUBLE PROTEINS

STRUCTURE SCREEN 1 STRUCTURE SCREEN 1 IS FORMULATED FOR THE CRYSTALLIZATION OF PROTEINS, PEPTIDES, NUCLEIC ACIDS, & WATER SOLUBLE SMALL MOLECULES.

Crystal Growth Screens

SOLUBLE PROTEINS

This classic screen was originally published by Jancarik & Kim from conditions found to be successful in the crystallization of biological macromolecules.

Features of Structure Screen 1: n The original sparse matrix screen1. n Enhanced buffer selection2. n Sparse matrix formula efficiently samples salts, polymers, organics & pH. n Proven effective with more than 1,000 biological macromolecules. n A complete kit designed to provide an effective and rapid screening method for the crystallization of biological macromolecules. n A simple and practical way to find initial crystallization conditions.

ORDER INFORMATION Description

Pack

Code

Structure Screen 1

50 x 10 mL

MD1-01

Structure Screen 1 cacodylate-free

50 x 10 mL

MD1-01-CF

The Structure Screen Combination

100 x 10 mL

MD1-03

The Structure Screen Combination cacodylate-free

100 x 10 mL

MD1-03-CF

Structure Screen I + II HT-96

96 x 1 mL

MD1-30

Structure Screen I + II HT-96 cacodylate-free

96 x 1 mL

MD1-30-CF

Structure Screen 1 Single Reagent

100 mL

MDSR-01

Structure Screen 1 Single Reagent

250 mL

MDSR-01-250

Structure Screen 1 + 2 HT-96 Single Reagent

100 mL

MDSR-30

References 1. Jancarik, J. & Kim, S.H. J. Appl. Cryst. (1991), 24, 409-411. 2. Wooh et al Acta Cryst. (2003) D59, 769 – 772.

North & South America orders: +1 877 479 4339 email: [email protected]

visit: www.moleculardimensions.com

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 6

Crystal Growth Screens

SOLUBLE PROTEINS

SOLUBLE PROTEINS

STRUCTURE SCREEN 2

STURA FOOTPRINT SCREENS

STRUCTURE SCREEN 2 IS FORMULATED FOR THE CRYSTALLIZATION OF PROTEINS, PEPTIDES, NUCLEIC ACIDS, & WATER SOLUBLE SMALL MOLECULES.

THE FOOTPRINT SCREENS ARE BASED ON THE CONCEPT OF SCREENING THE PROTEIN PRECIPITANT SOLUBILITY CURVE: A PROTEIN SOLUBILITY SCREEN.

A classic extension to the screen originally published by Jancarik & Kim1 from conditions found to be successful in the crystallization of biological macromolecules.

The screens are presented as two sets of 24 conditions at acidic, neutral and basic pH's for polyethylene glycols and salts.

Features of Structure Screen 2:

Features of Footprint Screening:

n An extension to the original sparse matrix screen with novel precipitants and combinations.

n Screen the protein precipitant solubility curve rather than a crystallization trial1.

n Structure Screen 2 contains 50 different and unique reagent combinations.

n Test the relative protein solubility with precipitants that have been used successfully in the crystallization of many proteins2.

n Sparse matrix formula efficiently samples salts, polymers, organics & pH.

n Once initial crystals or crystalline aggregates are obtained from the initial screening, streak seeding is recommended to determine the ranges of conditions under which crystal growth can proceed3.

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n Proven effective with more than 1,000 biological macromolecules. n A complete kit designed to provide an effective and rapid screening method for the crystallization of biological macromolecules. n A simple and practical way to find initial crystallization conditions.

ORDER INFORMATION

ORDER INFORMATION Description

Pack

Code

Description

Pack

Code

Structure Screen 2

50 x 10 mL

MD1-02

Stura Footprint Screens

48 x 10 mL

MD1-20

Structure Screen I + II HT-96

96 x 1 mL

MD1-30

Stura Footprint Screen cacodylate-free 48 x 10 mL

MD1-20-CF

Structure Screen I + II HT-96 cacodylate-free

96 x 1 mL

MD1-30-CF

The Stura Footprint Combination

96 x 10 mL

MD1-05 MD1-05-CF

100 x 10 mL

MD1-03

The Stura Footprint Combination cacodylate-free

48 x 10 mL

The Structure Screen Combination The Structure Screen Combination cacodylate-free

100 x 10 mL

MD1-03-CF

Stura Footprint Combination HT-96

96 x 1 mL

MD1-43 MD1-43-CF

100 mL

MDSR-02

Stura Footprint Combination HT-96 cacodylate-free

96 x 1 mL

Structure Screen 2 Single Reagent Structure Screen 1 + 2 HT-96 Single Reagent

100 mL

MDSR-30

Stura Footprint Screen Single Reagent 100 mL

MDSR-20-

Stura MacroSol HT-96 Single Reagent 100 mL

MDSR-43-

References 1. Jancarik, J & Kim, S.H., J. Appl. Cryst. (1991) 24, 409-411. 2. Cudney, R., Patel, S., Weisgraber, K., Newhouse, Y., and McPherson, A., Acta Cryst. (1994) D50, 414-423.

*Combination includes Stura Footprint Screens and Macrosol (p14). References 1. Stura E.A., Nemerow G.R., Wilson I.A, (1992),. Journal of Crystal Growth 122, 273-285. 2. Stura E.A. (1999) Strategy 3: Reverse Screening. In "Crystallization of Proteins: Techniques, Strategies and Tips. A laboratory manual" (Bergfors T. ed.) International University Line pp113-124. 3. Stura E.A, Satterthwait A.C., Calvo J.C., Kaslow D.C., Wilson I.A. (1994) Reverse Screening. Acta Cryst. D50: 448-455.

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Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

visit: www.moleculardimensions.com

MD Journal Build_Layout 1 6/5/13 10:10 AM Page 7

SOLUBLE PROTEINS

MINI SCREEN A MINIMISED SPARSE MATRIX SCREEN BASED ON THE CLASSIC JANCARIK AND KIM SCREEN1 BUT WITH MINIMAL REDUNDANCY.

MULTIXTAL™ CRYSTALLIZATION SCREEN THE FLEXIBLE CRYSTALLIZATION SCREEN

The kit contains a 24 condition, sparse matrix screen.

Crystal Growth Screens

SOLUBLE PROTEINS

Features of Mini Screen: n 24 conditions optimised for maximal probability of successfully obtaining a crystal with minimal redundancy2. n Particularly suitable as a cost effective start to crystallization experiments or where material is limited. n In a structural proteomics context, the kit works as an efficient, optimized screen that will give maximum samples for structure solution while minimising the amount of time and material wasted on unnecessary experiments.

Code

Use it as a high PEG screen for vapour diffusion as well as in counter-diffusion experiments and seeding experiments. Contains higher PEG concentrations than found in traditional standard screens; offering greater flexibility in its usage.

ORDER INFORMATION Description

Pack

A great addition to any crystallization lab – a 48 condition sparse matrix crystallization screen offering multiple uses for crystallization. For soluble and membrane proteins.

Mini Screen

24 x 10 mL

MD1-09

Features to come:

Mini Screen cacodylate-free

24 x 10 mL

MD1-09-CF

n High PEG concentration screen.

Mini Screen Single Reagent

100 mL

MDSR-09-

n Suitable for soluble and membrane proteins. n Ideal for counter-diffusion experiments – especially compatible with CrystalHarp™.

References 1. Jancarik J, Kim SH., (1991) , J. Appl. Cryst. 24, 409-411. 2. Kimber M.S. et al., (2003), Proteins: Structure, Function, and Genetics 51, 562-568.

n Use in seeding experiments.

3D STRUCTURE SCREEN

n Available as 10 mL, HT or prefilled microplate (FX) format.

3D STRUCTURE SCREEN IS FORMULATED FOR GROWING PROTEIN CRYSTALS IN THE METASTABLE ZONE USING THE VAPOUR DIFFUSION TECHNIQUE.

This method is presented in the 3D Structure Screen as an optimized sparse matrix crystal growth screen which allows the 24 “nucleation” conditions of the Mini Screen and 24 “backed-off” conditions for crystal growth to be tried. Formatted for HT blocks as the Heavy plus Light Twin Pack HT-96.

Features of 3D Structure Screen: n Separate nucleation from crystal growth. n A simple method for growing crystals in the metastable zone. n Obtain much larger and better diffracting crystals compared to conventional methods. ORDER INFORMATION

ORDER INFORMATION Description

Pack

Code

Description

Pack

Code

3D Structure Screen

48 x 10 mL

MD1-13

MultiXtal™

48 x 10 mL

MD1-65

3D Structure Screen cacodylate-free

48 x 10 mL

MD1-13-CF

MultiXtal™ HT-96

(48 x 1 mL) x 2

MD1-66

Heavy + Light Twin pack HT-96

2 x 48 x 1 mL

MD1-35

(48 x 0.1 mL) x 2 MD1-67

Heavy + Light Twin pack HT-96 cacodylate-free

2 x 48 x 1 mL

MD1-35-CF

MultiXtal™ FX (pre-filled conical bottom microplate) MultiXtal™ Single Reagent

100 mL

MDSR-65

3D Structure Screen Single Reagent

100 mL

MDSR-13-

MultiXtal™ HT-96 Single Reagent

100 mL

MDSR-66

MDSR-35-

MultiXtal™ FX Single Reagent

100 mL

MDSR-67

Heavy & Light HT-96 Single Reagent

100 mL

This product is manufactured under an exclusive licence from Imperial College of Science, Technology and Medicine, London U.K.

See also Crystal Harp (p65).

See page 57 for hints on screening using this screen.

North & South America orders: +1 877 479 4339 email: [email protected]

visit: www.moleculardimensions.com

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 8

Crystal Growth Screens

SOLUBLE PROTEINS

SOLUBLE PROTEINS

THE SOLUBILITY TOOL KIT™ A SYSTEMATIC ALTERNATIVE TO SPARSE MATRICES TO DETERMINE THE SOLUBILITY AND CRYSTALLIZATION POTENTIAL OF PROTEIN SALTS1 - 4.

CLEAR STRATEGY SCREEN™ I / CLEAR STRATEGY SCREEN™ II TWO COMPLIMENTARY 6 X 4 MATRIX SCREENS DEVELOPED BY DR. A. M. BRZOZOWSKI AND J. WALTON1 FROM THE LABORATORY OF STRUCTURAL BIOLOGY AT YORK UNIVERSITY.

Each kit contains 10 mL of 24 reagents buffered at pH 4.5, 24 reagents buffered at pH 9.0(box 1), 18 unbuffered polyethylene glycols, 6 unbuffered MPD’s, 21 salt concentrates and buffer concentrates from pH 5.5 – 9.5.(box 2)

Originally these screens were tested on a number of proteins, which had not been crystallized previously and yielded diffraction quality crystals. Now these are amongst our most popular products.

Features of The Solubility Tool Kit:

Features of Clear Strategy Screens:

n Provides the best starting conditions for the right buffer, pH and salt.

n Limit number of trials.

n Rapidly identifies likely crystallization reagents and their suitable range of concentration.

n Aid rational design of subsequent trials.

n Locate the nucleation zone of a protein and prepare phase diagrams.

n Use protein information.

n Can be performed with batch, vapour diffusion or dialysis technique. n Provides detailed information on the solubility profile of a protein.

Each screen contains 24 stock solutions and the following (1M) buffers: Sodium Acetate, pH 4.5; Sodium Acetate, pH 5.5; Sodium Cacodylate, pH 6.5; Tris, pH 7.5; and Tris, pH 8.5.

n User defined pH. n Maintain ‘folding homogeneity’ of protein. n Interchangeable components. n Potential anomalous scattering centres2. ORDER INFORMATION

ORDER INFORMATION

Description

Pack

Code

Clear Strategy Screen I

24 x 10 mL*

MD1-14

Clear Strategy Screen I cacodylate-free

24 x 10 mL*

MD1-14-CF

Clear Strategy Screen II

24 x 10 mL*

MD1-15

Clear Strategy Screen II cacodylate-free 24 x 10 mL*

MD1-15-CF

The Clear Strategy Screen Combination

48 x 1 mL*

MD1-16

The Clear Strategy Screen Combination- cacodylate-free

48 x 10 mL*

MD1-16-CF

Clear Strategy Screen Combination pH Premixed

240 x 10 mL

MD1-16LMB

Description

Pack

Code

Clear Strategy Screen I HT-96

96 x 1 mL

MD1-31

The Solubility Tool Kit (Box 1 + 2)

98 x 10 mL

MD1-17

Clear Strategy Screen I HT-96 cacodylate-free

96 x 1 mL

MD1-31-CF

Solubility Screen Box 1

48 x 10 mL

MD1-18

Clear Strategy Screen II HT-96

96 x 1 mL

MD1-32

Precipitant Screen Box 2

50 x 10 mL

MD1-19

MD1-32-CF

100 mL

MDSR-17-

Clear Strategy Screen II HT-96 cacodylate-free

96 x 1 mL

The Solubility Tool Kit Single Reagent

Clear Strategy Screen 1 Single Reagent

100 mL

MDSR-14-

Clear Strategy Screen 11 Single Reagent

100 mL

MDSR-15-

References 1. Riès-Kautt M, Ducruix A,. Methods Enzymol 1997, 276:23-59. 2. Riès-Kautt M, Ducruix A, In "Crystallization of Nucleic acids and proteins: A practical approach". (Ducruix A and Giegé R. ed.) IRL/Oxford Press. 3. Riès-Kautt M Strategy 2, In "Protein Crystallization Techniques, Strategies, and Tips" (Bergfors T. ed) IUL Biotechnology Series International University Line, La Jolla, California. 4. Vaney M. C. et al, Acta Cryst D (2001), D57, 929-940.

Clear Strategy Screen 1 HT-96 Single Reagent 100 mL

MDSR-31-

Clear Strategy Screen 2 HT-96 Single Reagent 100 mL

MDSR-32-

* Plus buffers. These products are manufactured under an exclusive license from the University of York, UK. References 1. Brzozowski A. M. and Walton J., (2001) J. Appl. Cryst. 34, 97-101. 2. Dauter Z., Dauter M. & Rajashankar K. R. (2000), Acta Cryst. D56, 232-237. 3. Selmer et al, (2006), Science 313, 1935-1442.

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Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

visit: www.moleculardimensions.com

MD Journal Build_Layout 1 6/5/13 10:10 AM Page 9

SOLUBLE PROTEINS

PACT PREMIER™ PACT PREMIER IS A pH, ANION, CATION CRYSTALLIZATION TRIAL DEVISED TO TEST PH WITHIN A PEG/ION SCREEN ENVIRONMENT. THIS IS ONE OF THE MOST EFFECTIVE SYSTEMATIC SCREENS AVAILABLE TO DATE.

JCSG-PLUS™ SCREEN AN OPTIMIZED SPARSE MATRIX SCREEN OF CLASSIC AND MODERN CONDITIONS DEVISED AT THE JOINT CENTRE FOR STRUCTURAL GENOMICS1 AND DEVELOPED FURTHER BY NEWMAN AND PERRAKIS2 AT THE NETHERLANDS CANCER INSTITUTE.

Building on the success of PEG/Ion screening, this PEG/ION/pH screen has been developed to systematically test the effect of pH, anions and cations, using PEG as the precipitant. This screen was originally implemented very successfully at the Netherlands Cancer Institute (NKI), and at the Oxford Protein Production Facility (OXPPF). The kit contains 96 reagents arranged as a cation/PEG screen, an anion/PEG screen and a pH/PEG screen.

The kit contains a 96 condition, sparse matrix screen.

Features of PACT Premier:

Features of JCSG-Plus:

n A modern, comprehensive PEG/ion screen.

n Optimized sparse matrix screen.

n This 96-well screen is really 3 screens: 1. 24-well pH/PEG screen. 2. 24-well cation/PEG screen. 3. 48-well anion/PEG screen.

n Reduced redundancy.

Crystal Growth Screens

SOLUBLE PROTEINS

n Screens classic PEG and salt conditions. n Access more areas of crystallization space. n Neutralised organic acids: Formate, acetate, citrate, succinate, malate, malonate3. n More organic and polyalcohol conditions. n Precipitant synergy. n Wide pH range 4.0 – 10.0.

ORDER INFORMATION Description

Pack

Code

PACT premier (10 mL kit)

96 x 10 mL

MD1-29

PACT premier cacodylate-free

96 x 10 mL

MD1-29-CF

PACT premier HT-96

96 x 1 mL

MD1-36

PACT premier HT-96 cacodylate-free

96 x 1 mL

MD1-36-CF

Description

Pack

Code

PACT premier HT-96 Green Screen

96 x 1 mL

MD1-52

JCSG plus

96 x 10 mL

MD1-37

PACT Green Screen (10 mL)

96 x 10 mL

MD1-55

JCSG plus cacodylate-free

96 x 10 mL

MD1-37-CF

PACT premier Screen Single Reagent

100 mL

MDSR-29-

JCSG plus HT-96

96 x 1 mL

MD1-40

PACT premier Screen HT-96 Single Reagent 100 mL

100 mL

MDSR-36-

JCSG plus HT-96 cacodylate-free

96 x 1 mL

MD1-40-CF

JCSG plus HT-96 Green Screen

96 x 1 mL

MD1-53

96 x 10 mL

MD1-56

ORDER INFORMATION

PACT premier HT-96 Green Screen Single Reagent

100 mL

MDSR-52-

JCSG Plus Green Screen (10 mL)

PACT premier Green Screen (10mL) Single Reagent

MDSR-55-

Super2 Combo Value Pack (JCSG+ & PACT)

2 x 96 x 10 mL MD1-75

100 mL

Super2 Combo Value Pack (JCSG+ & PACT premier)

JCSG plus Screen Single Reagent

100 mL

MDSR-37-

2 x 96 x 10 mL MD1-75

JCSG plus HT-96 Single Reagent

100 mL

MDSR-40-

JCSG plus HT-96 Green Screen Single Reagent

100 mL

MDSR-53-

JCSG Green Screen (10 mL) Single Reagent

100 x 10 mL

MDSR-56-

The screen was developed by Janet Newman, and was tested in the laboratory of Anastassis Perrakis at the NKI as part of the SPINE programme, and is manufactured under license by Molecular Dimensions. References 1. Newman et al, (2005), Acta Cryst. D61, 1426

References 1. Page et al (2003), Acta Cryst. D59, 1028-1037. 2. Newman et al (2005), Acta Cryst. D61, 1426-1431. 3. McPherson et al (2001), Protein Science 10, 418422.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 10

TOWARDS HIGH EFFICIENCY SCREENING “…conditions most essential for promoting crystal formation…” Page et al (2003) Acta Cryst. D59, 1028

Editorial SPARSE MATRICES The earliest sparse matrix screens were based on previously successful crystallization conditions. Since most researchers used these screens, the same sub-sets of conditions were used repeatedly. As a result of the published results of data-mining exercises from a number of structural genomics initiatives more efficient screens have been devised.

T

he Joint Centre for Structural Genomics (JCSG) analysed the crystallization of over 500 different proteins against available sparse matrices. Using a novel algorithm, members of the JCSG identified a core screen (JCSG) developed when data mining revealed massive redundancy between clusters of conditions particularly where high molecular weight PEGs were used as precipitants (1). The second issue to come to light was that even extensive suites of sparse matrix screens represent incomplete coverage of crystallisation space – 480 conditions failed to crystallise 15% of the target proteins. The JCSG-plus screen (p9) developed at the Netherlands Cancer Institute by Janet Newman during the SPINE project supplements the JCSG core conditions to provide a more complete coverage of crystallisation space (2), by in-filling the pH profile, introducing neutralised organic acids as the precipitant (3) and expanding the range of organic and polyalcohol conditions to create precipitant synergy.

Rational approaches There are a few screens that try to test crystallization space in a more rational manner – for example, the Clear Strategy Screen (4) (p8), and the Solubility Tool Kit (5) (p8). The PEG/Ion screen (6) has been reported to be very successful in producing crystal hits, with the caveat that many of the hits are redundant. With this in mind, the PEG/ION/pH screen (PACT-premier) (p9) was developed (2) to systematically and additionally test the effect of pH, as well as anions and cations, using PEG as the precipitant. This screen has been implemented very successfully at the Netherlands Cancer Institute (NKI), and at the Oxford Protein Production Facility (OXPPF). The JCSG-plus sparse matrix screen is highly effective when used alongside a systematic screen such as PACT-premier. The two screens provide a thorough exploration of crystallization conditions and the unique design of PACT-premier facilitates rational interpretation of results from both itself and JCSG-plus assisting the design of subsequent experiments.

References 1. Page et al (2003). Shotgun crystallization strategy for structural genomics: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome. Acta Cryst. D59, 1028-1037 2. Newman et al (2005). Towards rationalization of crystallization screening for smallto medium-sized academic laboratories: the PACT/JCSG+ strategy. Acta Cryst. D61, 1426-1431 3. McPherson et al (2001). A comparison of salts for the crystallisation of macromolecules, Protein Science 10, 418422 4. Brozowski & Walton (2001) J. Appl. Cryst. 34, 97-101 5. Ries-Kaut in Protein Crystallization techniques,. Strategies and Tips (Bergfors T Ed.) IUL Series. 6. A comparison of salts for the crystallization of macromolecules. Alexander McPherson. Protein Science (2001), 10:418-422.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 11

Dr Rebecca Page

Dr Janet Newman

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 12

EXPLORING MORE CRYSTALLIZATION SPACE Editorial

Dr Clemens Grimm (Würzburg).

O

ut of 8289 entries scanned in the PDB, almost half of the crystallization conditions contained a PEG component and most commercial screens contain PEGS. However, the success rate of PEGS might be biased due to their widespread dominance in crystallization screens. There are many alternatives to PEGs and a variety have recently been described as being useful for macromolecular crystallogenesis, but had only sporadically been introduced into standard crystallization screens. To close this gap, Clemens Grimm et al (1) at Würzburg University in Germany, have devised a protein crystallization screen (MIDAS) that systematically searches for crystallization conditions with alternative polymeric precipitants. MIDAS covers a relatively narrow range of pH and salt concentrations centred on physiological values to increase its suitability for sensitive macromolecular complexes, while every condition contains at least one alternative polymeric precipitant. MIDAS has been designed to complement PEG- and salt-based screens, and is ideal for protein, protein/protein complexes, proteinnucleic acid complexes and sensitive macromolecular complexes.

Dr AM Brzozowski (York)

FOR DECADES PEGS OR THEIR MONOMETHYL ETHERS (PEG MMES), HAVE DOMINATED CRYSTALLIZATION SCREENS 12

An alternative new systematic screen from the York Structural Biology Laboratory (YSBL) based on the poly-γ-glutamic acid (PGA) polymer has also been reported (2). This PGA Screen (p22) is suitable for both globular and membrane protein crystallization. This novel precipitant allows totally new crystallization space to be explored. PGAs present at least two new aspects in protein crystallization; they extend the range of existing PEG-based polymers into: new-chemical polymers that exploit poly-amino acids and broaden the range of molecular weight of polymer precipitants into regions over 1MDa.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 13

MORPHEUS® The high nucleation-precipitation potential of PGAs enables their use at very low concentrations and in combination with classical precipitants, thus scaling down the amount of precipitant necessary for crystal appearance and growth. This feature of PGAs makes them especially useful in applications for labile, easily precipitating proteins. Although they can be employed for all type/classes of proteins, current experience resulting from work in the YSBL suggests that PGA should be especially effective for crystallization of membrane proteins. Therefore, the PGA-based screens are recommended as targeted screens with membrane proteins as the main/primary subjects of their applicability.

A 96 CONDITION PROTEIN CRYSTALLIZATION SCREEN WITH ORIGINAL CHEMISTRY INCORPORATING A RANGE OF LOW MOLECULAR WEIGHT LIGANDS FOUND ORDERED IN MORE THAN 33000 PDB STRUCTURES.

Developed and utilized in the world renowned Laboratory of Molecular Biology, (Cambridge, UK) - Morpheus® carries on the tradition of being original and dynamic. This unique screen aims to unlock novel chemical space previously inaccessible using conventional screens.

Morpheus (right) aims to unlock novel chemical space previously inaccessible using conventional screens by incorporating a range of low molecular weight ligands found ordered in more than 33,000 deposited structures. This 96 condition 3D crystallization screen was developed and utilized in the world renowned Laboratory of Molecular Biology, Cambridge, UK.

The ligands most frequently reported in PDB structures are grouped into eight mixes of additives based on chemical class (e.g. alcohols, carboxylic acids, etc). Several of these top PDB ligands are “biological buffers” like HEPES and have been used to build three buffer systems. Nine precipitants are grouped into four mixes that contain at least a PEG (Polyethylene glycol) and a different type of precipitant that is also a cryo-protectant (e.g. Glycerol).

Morpheus is based on extensive data mining of the PDB. The aim is to explore different chemical space than that achieved with conventional screening. Morpheus incorporates 49 low molecular weight components. They are PDB ligands sharing four main characteristics; they are small (the largest being HEPES MW 238.30 g/mol and the smallest a lithium ion MW 6.94 g/mol), stable, inexpensive and are associated with at least five unrelated PDB structures. For instance, the two enantiomers of tartaric acid (PDB ID: TAR and TLA) are found ordered in 113 structures.

The kit is presented as a 96 condition screen.

References 1. Grimm, C., Chari, A., Reuter, K. & Fischer, U. (2010). Acta Cryst. D66, 685-697. 2. TC Hu, J Korczynska, DK Smith, AM Brzozowski - Acta Crystallographica Section D: Biological Crystallography, 2008. D64, 957-963 3. Gorrec F. (2009) J. Appl. Cryst. 42, 1035-1042

Crystal Growth Screens

SOLUBLE PROTEINS

Features of Morpheus®: n Simple and effective 3D grid design covering a range of pH, PEGs and salt additives. n 49 low molecular weight ligands promote both initial crystal formation and lattice stability. n Reduced crystal "stress" - all conditions are cryo-protected. n Derived from extensive data mining of over 33,000 PDB entries. n Available in both 10 mL tube and 96 condition deep well block formats. ORDER INFORMATION Description

Pack

Code

Morpheus

96 x 10 mL

MD1-46

Morpheus HT-96

96 x 1 mL

MD1-47

Morpheus HT-96 Green Screen

96 x 1 mL

MD1-47-Green

Morpheus Additive OptiMax Kit (10 mL) 43 x 10 mL Power Combo Value Pack (Morpheus & MIDAS)

MD1-58

2 x 96 x 10 mL MD1-76

Morpheus Screen Single Reagent

100 mL

MDSR-46-

Morpheus HT-96 Single Reagent

100 mL

MDSR-47-

This product has been designed and developed by Fabrice Gorrec, in collaboration with the scientists at the Medical Research Council Laboratory of Molecular Biology (LMB) at Cambridge and is manufactured exclusively under license by Molecular Dimensions Limited.

Fabrice Gorrec receives the MRC CEO's Award from John Savill for innovations in the field of macromolecular crystallization (including the Morpheus screen).

References Gorrec F. (2009). J. Appl. Cryst. 42, 1035-1042

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 14

PROTEIN COMPLEXES

Crystal Growth Screens

PROTEIN COMPLEXES

MACROSOL™ THIS FOOTPRINT SCREEN FOR THE CRYSTALLIZATION OF PROTEIN COMPLEXES AND OTHER SAMPLES WITH HETEROGENEOUS SOLUBILITY, SCREENS THE PROTEIN PRECIPITANT SOLUBILITY CURVE RATHER THAN SETTING UP A RANDOMIZED CRYSTALLIZATION TRIAL.1-4

The screens are presented as two sets of 24 conditions at acidic, neutral and basic pH's for polyethylene glycols and salts.

Features of Macrosol: n Screens the protein precipitant solubility curve rather than a crystallization trial. n Satisfies dual requirements: – Maintain protein-protein interactions. – Reduce solubility of complex. n Test the relative protein solubility with precipitants that have been used successfully in the crystallization of many proteins. n Once initial crystals or crystalline aggregates are obtained from the initial screening, streak seeding is recommended to determine the ranges of conditions under which crystal growth can proceed.

ORDER INFORMATION Description

Pack

Code

MacroSol

48 x 10 mL

MD1-22

MacroSol cacodylate-free

48 x 10 mL

MD1-22-CF

MacroSol Screen Single Reagent

100 mL

MDSR-22-

The Stura Footprint Combination

96 x 10 mL

MD1-05

The Stura Footprint Combination cacodylate-free

48 x 10 mL

MD1-05-CF

Stura Footprint Combination HT-96

96 x 1 mL

MD1-43

Stura Footprint Combination HT-96 cacodylate-free

96 x 1 mL

MD1-43-CF

References 1. Stura E.A., Nemerow G.R., Wilson I.A (1992), Journal of Crystal Growth 122, 273-285 2. Stura E.A. (1999) Strategy 3: Reverse Screening. In "Crystallization of Proteins: Techniques, Strategies and Tips. A laboratory manual" (Bergfors T. ed.) International University Line pp113-124. 3. Stura E.A, Satterthwait, A.C, Calvo, J.C, Kaslow, D.C, Wilson, I.A. (1994), Acta Cryst. D50, 448-455. 4. Stura E.A, "EMBO Workshop on the crystallization of Macromolecular Complexes", Grenoble 8-13 April 2001

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 15

PROTEIN COMPLEXES

PROPLEX™

MIDAS™

PROPLEX IS FORMULATED FOR THE CRYSTALLIZATION OF PROTEIN COMPLEXES.

MIDAS IS A MODERN INTELLIGENT DYNAMIC ALTERNATIVE SCREEN WITH 96 REVOLUTIONARY CRYSTALLIZATION SCREENING CONDITIONS BASED ON ALTERNATIVE POLYMERIC PRECIPITANTS DEVELOPED AND TESTED IN THE LABORATORY OF DR. CLEMENS GRIMM AT UNIVERSITY OF WÜRZBURG, GERMANY.

Developed and tested by Sergei Radaev et al1,2, at the NIH National Institute of Allergy and Infectious Diseases. The kit contains a 96 condition, targeted sparse matrix screen.

MIDAS is a revolutionary crystallization screen that has moved away from the reliance on polyethylene glycols (PEGs) as the main precipitant (only 3 conditions in MIDAS contain a PEG).

Features of ProPlex:

Features of MIDAS:

n Targeted sparse matrix screen.

n Ideal for both protein, protein/protein complexes, protein / nucleic acid complexes, and sensitive macromolecular complexes.

n Based on results of Protein Complex Crystallization Database. n Satisfies dual requirements: – Maintain protein-protein interactions.

Crystal Growth Screens

PROTEIN COMPLEXES

n Narrow range of pH and salt concentrations centered on physiological values. n Every condition contains at least one alternative polymeric precipitant.

– Reduce solubility of complex. n Medium and High MW PEGs.

n Compatible with liquid handling robots.

n Lower PEG concentrations. n Fewer organic precipitants. n Neutralised organic acids. n pH range from 4.0 – 8.5.

ORDER INFORMATION

ORDER INFORMATION Description

Pack

Code

Description

ProPlex

96 x 10 mL

MD1-38

ProPlex cacodylate-free

96 x 10 mL

MD1-38-CF

ProPlex HT-96

96 x 1 mL

MD1-42

ProPlex HT-96 cacodylate-free

96 x 1 mL

MD1-42-CF

ProPlex Screen Single Reagent

100 mL

MDSR-38-

ProPlex HT-96 Single Reagent

100 mL

MDSR-42-

References 1. Radaev S., Li S. and Sun P. D. (2006), Acta Cryst. D62, pp 605-612. 2. Radaev S., Li S. and Sun P. D. (2002), J. Appl. Cryst. 35, 674-676 3. Dafforn (2007) So how do you know you have a macromolecular complex? Acta Cryst. D63, 17-25. 4. Crystallisation of Nucleic Acids and Proteins, Edited by A. Ducruix and R. Giegé, The Practical Approach Series, Oxford Univ. Press, 1992. 5. Protein Crystallization Techniques Strategies & Tips, (Bergfors T. ed.) IUL 1999.

Pack

Code

MIDAS

96 x 10 mL

MD1-59

Power Combo Value Pack (Morpheus & MIDAS)

2 x 96 x 10 mL MD1-76

MIDAS HT-96

96 x 1 mL

MD1-60

MIDAS OptiMax Kit

27 x 10 mL

MD1-62

MIDAS Single Reagent

100 mL

MDSR-59

MIDAS HT-96 Single Reagent

100 mL

MDSR-60

MIDAS™ is manufactured and distributed under an exclusive license with Dr. C. Grimm and Prof. Dr. U. Fischer. References 1. Grimm, C., Chari, A., Reuter, K. & Fischer, U. (2010). Acta Cryst. D66, 685-697

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 16

NUCLEAR RECEPTORS

Crystal Growth Screens

NUCLEAR RECEPTORS

NUCLEAR RECEPTOR LIGAND BINDING DOMAIN SCREENS A RATIONAL APPROACH TO SCREENING INITIAL CRYSTALLIZATION CONDITIONS OF NUCLEAR RECEPTOR LIGAND BINDING DOMAINS.1

The NR-LBD Screen contains 48 conditions divided into two parts: 24 conditions using various PEGs as the precipitant and 24 conditions using various carboxylic acids as the precipitant. The extension contains a set of 48 unique reagents which not only allows the user to reproduce previously obtained results but also to crystallize new ligand binding domains.

Features of the Nuclear Receptor Ligand Binding Domain Screens: n Nuclear receptors usually crystallize in the presence of two reagent families: polyethylene glycol and carboxylic acids. n The pH range for crystallization is 6.0 to 9.0. n Includes PEGs 4000, 6000 and 8000. n Includes sodium formate, sodium citrate, sodium acetate carboxylic acids. n Ionic strength controlled with sodium chloride and ammonium acetate. n pH range is between 6.5 and 8.5 using Bis Tris (pH 6.5), PIPES (pH 7.0), Na HEPES (pH 7.5) and Tris (pH 8.0 and 8.5).

ORDER INFORMATION Description

Pack

Code

NR-LBD Screen

48 x 10 mL

MD1-24

NR-LBD Extension Screen

48 x 10 mL

MD1-26

Nuclear Receptor Combination

96 x 10 mL

MD1-27

NR-LBD + NR-LBD Extension HT-96

96 x 1 mL

MD1-34

NR-LBD Screen Single Reagent

100 mL

MDSR-24-

NR-LBD Extension Screen Single Reagent

100 mL

MDSR-26-

NR-LBD + NR-LBD Extension HT-96 Single Reagent

100 mL

MDSR-34-

The NR-LBD screens were developed by Denis Zeyer, Sylvie Duclaud, Dino Moras and Jean-Paul Renaud at The Institute of Genetics and Molecular and Cell Biology, CNRS, UMR 7104, Illkirch, France and is manufactured under an exclusive license from CNRS, France. References 1. Zeyer D., Duclaud S., Moras D. Renaud J-P., Billas I. M. et al. (2001) J. Biol. Chem. 276, 7465-7474.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 17

MEMBRANE PROTEINS

MEMSTART™ A STARTING POINT FOR SCREENING AND OPTIMIZING CRYSTALLIZATION CONDITIONS FOR ALPHA TYPE TRANSMEMBRANE PROTEINS USING VAPOUR DIFFUSION METHODS.1

Crystal Growth Screens

MEMBRANE PROTEINS

A sparse matrix of 48 conditions allowing the pH range, precipitants and salts used in membrane protein crystallization to be screened with detergent- containing protein drops. Molecular Dimensions acknowledges the work of Prof. S. Iwata, Dr. M. Iwata and Dr. J. Abramson in designing this product.

Features of MemStart: n Based on the reagents typically used in the laboratory of Prof. S. Iwata. n Optimized to span 33 reported successful crystallization conditions of membrane proteins for which atomic resolution structures have been determined. n Includes the pH, precipitant concentration and type, and salts found to be successful.

ORDER INFORMATION Description

Pack

Code

MemStart Kit

48 x 10 mL

MD1-21

MemStart Screen Single Reagent

100 mL

MDSR-21-

The Membrane Protein Combination

96 x 10 mL

MD1-04

MemStart + MemSys HT-96

96 x 1 mL

MD1-33

MemStart + MemSys HT-96 Single Reagent

100 mL

MDSR-33-

This product is manufactured under an exclusive licence from Imperial College of Science, Technology & Medicine, London, UK. References 1. Methods and Results in the crystallization of Membrane proteins (2003) Ed. Iwata S. (International University Line)

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 18

Crystal Growth Screens

MEMBRANE PROTEINS

MEMBRANE PROTEINS

MEMSYS™

MEMPLUS™

A SYSTEMATIC SCREEN SPANNING THE KEY VALUES OF pH, PRECIPITANT TYPE/ CONCENTRATION, AND SALTS FOR ALPHA TYPE TRANSMEMBRANE PROTEINS USING VAPOUR DIFFUSION METHODS.1

A FURTHER 48 CONDITIONS TARGETED AT MEMBRANE PROTEINS.

48 conditions allowing the pH range, precipitants and salts used in membrane protein crystallization to be screened with detergent containing protein drops. The reagents can be easily arranged in systematic array to facilitate the interpretation of results and the design of further optimization experiments.

A useful addition to MemGold. This screen further targets β-barrel type membane proteins – including porins and outer membrane active transporters. Crystallization conditions were data mined from the PDB.

Features of MemSys:

Features of MemPlus:

n A systematic approach to screening for initial crystallization conditions for membrane proteins using vapour diffusion methods.

n Based on successful crystallization conditions data mined from the Protein Data Bank.

n Membrane protein solubility is pushed to the limit to provide more information than previous sparse matrix type screens.

n Contains 48 conditions covering a range of pH, PEGs and salt additives.

n Includes the pH, precipitant concentration and type, and salts found to be successful. n Primarily designed for alpha type transmembrane proteins, but has also been successfully applied to beta type outer membrane proteins. Developed by members of the So Iwata Lab, Imperial College, London.

ORDER INFORMATION Description

ORDER INFORMATION Pack

Code

Description

Pack

Code

MemSys

48 x 10 mL

MD1-25

MemPlus

48 x 10 mL

MD1-44

The Membrane Protein Combination

96 x 10 mL

MD1-04

MemPlus cacodylate-free

48 x 10 mL

MD1-44-CF

MemSys Single Reagent

100 mL

MDSR-25-

MemPlus HT-96

96 x 1 mL

MD1-45

MemStart + MemSys HT-96

96 x 1 mL

MD1-33

MemPlus HT-96 cacodylate-free

96 x 1 mL

MD1-45-CF

MemStart + MemSys HT-96 Single Reagent

100 mL

MDSR-33-

MemPlus Single Reagent

100 mL

MDSR-44-

MemPlus HT-96 Single Reagent

100 mL

MDSR-45-

This product is manufactured under an exclusive licence from Imperial College of Science, Technology & Medicine, London, UK. References 1. Methods and Results in the crystallization of Membrane proteins (2003) Ed. Iwata S. (International University Line)

18

This product is manufactured under an exclusive licence from Imperial College of Science, Technology & Medicine, London, UK. References 1. Newstead, Simon; Hobbs, Jeanette; Jordan, Davina; Carpenter, Elisabeth; Iwata, So, "Insights into outer membrane protein crystallisation." Molecular Membrane Biology (2008). Dec, 25(8): 631-638.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 19

MEMGOLD™

MEMGOLD2™

THE MOST ADVANCED MEMBRANE PROTEIN CRYSTALLIZATION SCREEN TO DATE.

THE LATEST INNOVATION FOR CRYSTALLIZATION OF MEMBRANE PROTEINS. THIS SCREEN TARGETS ALL ALPHA HELICAL TYPES OF PROKARYOTIC AND EUKARYOTIC MEMBRANE PROTEINS.

In 2008 Molecular Dimensions released MemGold; a rationalized sparse matrix type membrane protein crystallization screen. MemGold was based on the crystallization conditions for 121 alpha helical Membrane Proteins deposited in the PDB.

Since MemGold, the number of structures has more than doubled. In response to this, MemGold2 has been developed. Memgold2 includes a further 96 crystallization conditions from unique alpha helical Membrane Protein structures including channel and transporter structures, GPCRs and ATPases. It is suitable for both Prokaryotic and Eukaryotic alpha helical membrane proteins.

Features of MemGold:

Features of the PGA Screen:

n Data mined from 300 crystallization conditions for a-helical transmembrane proteins.

n A brand new set of 96 of the most recent alpha helical membrane protein crystallization conditions.

n Covers conditions for approximately 130 different membrane protein structures in the PDB.

n Particularly suited for Prokaryotic and Eukaryotic alpha helical membrane proteins.

n Addresses the diversity of membrane proteins studied.

n Works well in conjunction with MemGold, MemStart & MemSys & MemPlus.

Developed by members of the So Iwata Lab, Imperial College, London.

Crystal Growth Screens

MEMBRANE PROTEINS

MEMBRANE PROTEINS

n Screening over a wider range of pH’s (4 - 10). n Addition of small MW PEGs. n Can be used in conjunction with Lipidic Sponge Phase and/or Lipidic Cubic Phases. ORDER INFORMATION

ORDER INFORMATION Description

Pack

Code

Description

Pack

Code

Memgold

96 x 10 mL

MD1-39

MemGold2 (10 mL kit)

96 x 10 mL

MD1-63

Memgold cacodylate-free

96 x 10 mL

MD1-39-CF

MemGold Combo Value Pack

2 x 96 x 10 mL MD1-74

MemGold HT-96

96 x 1 mL

MD1-41

MemGold Combo Value Pack HT-96

2 x 96 x 1 mL

MD1-74-HT

MemGold HT-96 cacodylate-free

96 x 1 mL

MD1-41-CF

MemGold2 Cacodylate-free

96 x 10 mL

MD1-63-CF

MemGold HT-96 Green Screen

96 x 1 mL

MD1-54

MemGold2 HT-96

96 x 1 mL

MD1-64

MemGold Green Screen (10 mL)

96 x 10 mL

MD1-57

MemGold2 HT-96 Cacodylate-free

96 x 1 mL

MD1-64-CF

MemGold Combo Value Pack

2 x 96 x 10 mL MD1-74

MemGold Combo Value Pack HT-96

2 x 96 x 1 mL

MemGold Combo Value Pack cacodylate-free

2 x 96 x 10 mL MD1-74-CF

MemGold Combo Value Pack cacodylate-free

MemGold2 Single Reagent

100 mL

MDSR-63-

2 x 96 x 10 mL MD1-74-CF

MemGold2 HT-96 Single Reagent

100 mL

MDSR-64-

MemGold Screen Single Reagent

100 mL

MDSR-39-

MemGold HT-96 Single Reagent

100 mL

MDSR-41-

This product was developed by Dr. Simon Newstead, University of Oxford, UK and is manufactured under an exclusive licence from Isis Innovation Ltd.

MemGold HT-96 Green Screen Single Reagent

100 mL

MDSR-54-

MemGold Green Screen Single Reagent 100 mL

MD1-74-HT

MDSR-57-

References 1. Newstead, S., Ferrandon, S., and Iwata, S. ‘Rationalizing alpha-helical membrane protein crystallization‘ Volume 17, Issue 3, pages 466–472, March 2008 - Protein Science, 2008 - Wiley Online Library. 2. Parker, J. and Newstead, S. ‘Current trends in alpha helical membrane protein crystallization: an up-date’, Protein Science, 2012, 21 (9): 1358-1365.

This product is manufactured under an exclusive licence from Imperial College of Science, Technology & Medicine, London, UK. References 1. Simon Newstead, Sébastien Ferrandon, and So Iwata Rationalizing α-helical membrane protein crystallization Protein Sci 2008 17: 466-472.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 20

Crystal Growth Screens

The MemGold family of alpha helical membrane protein crystallization reagents: …the science behind the screens.

Dr Simon Newstead In recent years significant progress has been made in determining the atomic structure of alpha helical membrane proteins using X-ray crystallography. However the identification of conditions that give crystals that are suitable for structure determination remains one of the major bottlenecks.

T

he most successful method for growing crystals of membrane proteins remains vapour diffusion from detergent solubilised proteins. We reasoned that important advances in membrane protein crystal screen design could be achieved by systematically mining the information present in the Protein Data Bank and associated research literature. In 2008 we undertook an analysis of the crystallization conditions for 121 alpha helical membrane proteins to design a rationalized sparse matrix crystallization screen, MemGold (Newstead et al, 2008). Our analysis has revealed a striking success for small MW PEGs in the crystallization of channels and transporters, with larger MW PEGs being more successful for respiratory complexes and other membrane proteins that typically contain larger hydrophilic domains (Figure 1). 35

No. of successful crystallisations

30

Organic Molecules Salts Large MW PEGs (3000 - 10, 000 Da) Medium MW PEGs (1000 - 2000 Da) Small MW PEGs (200 - 600 Da)

25

20

15

10

5

Others

Respiratory Complexes

ATPases

Photosynthetic & Light Harvesting Complexes

Transporters

Channels

GPCRs

Bacterial Rhodopsins

0

Figure 1. Success of small MW PEGs for crystallising alpha helical membrane proteins. (Reproduced with permission from Newstead et al., Protein Science 2008).

Dr Simon Newstead

20

Interestingly, when compared with similar analyses of successful crystallization data for bacterial non-membrane protein targets (Page & Stevens, 2004) from the Joint Centre for Structural Genomics in San Diego, CA and the University of Toronto, Canada, it was clear that a major difference is the success of small MW PEGs in promoting the crystallization of membrane proteins. By comparison, organic molecules such as MPD are much less successful.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 21

Core 67 JCSG (84 %) MemGold

45

Core 24 Toronto (94 %) 40 35 30 25

MemAdvantage: Additive screening for membrane proteins

20 15 10 5 0 Small MW PEGs (200 - 600 Da)

Medium MW PEGs (1000 - 2000 Da)

Large MW PEGs (3000 - 10, 000 Da)

Salts

Organic Molecules

Figure 2. Successful crystallization precipitants reported in our 2008 study for alpha helical membrane proteins compared with similar results reported for bacterial non-membrane proteins from the Joint Centre for Structural Genomics and University of Toronto. The reported conditions in the JCSG-67 and Toronto Core-24 were found to be the smallest subset of conditions that would have crystallised the maximum number of bacterial nonmembrane protein targets in these structural genomics programs in 2004.

The rate of new membrane protein structures being determined is steadily increasing, in line with previous predictions (White, 2004), providing an ever expanding source of new crystallization information. We are continuing to mine this information to address the conundrum faced by many structural biologists in this area, which is “how many conditions are enough to adequately cover ‘crystallization space’ for a given membrane protein sample”? This question is especially pertinent for eukaryotic MPs, when amounts are likely to be far scarcer compared to their prokaryotic counterparts. In 2012 our group undertook another detailed study of the crystallization conditions (Parker & Newstead, 2012). Many of the membrane proteins analyzed in this study crystallized in similar precipitant conditions to our 2008 study, with approximately two thirds being crystallized in a low MW PEG. However, the pH, salt and buffer components

We know from experience with non-membrane proteins that an initial crystal condition will often require optimization through the addition of small molecules, salts and specific ligands. To facilitate this task and again using information gathered from the present literature on membrane protein structures, we developed a compact, 96-reagent additive screen, MemAdvantage. Figure 3 shows the range of different small molecule and salt additives that have been reported to improve initial crystallization conditions for alpha helical membrane proteins. Multivalent salts and polyalcohols appear prominently, accounting for 10 and 15 % of all reported structures respectively. A substantial increase in the number of secondary detergents and non-volatile organic molecules are also now being recorded since we first started analyzing the data in 2008, accounting for 19 and 12 % respectively.

Crystal Growth Screens

50

of the crystallization conditions differed substantially, suggesting successful crystallization screens should be more varied in these parameters rather than precipitant type. Based on these data a new sparse matrix style screen, MemGold 2 was developed. Our analysis of the available crystallization data suggests MemGold and MemGold 2 provide a comprehensive set of initial screening conditions for alpha helical membrane protein crystallization.

The rationale behind the MemGold family of membrane protein crystallization products is to maximize the current information in the public databases to improve our success in crystallization and structure determination. Our goal in developing these screens is to provide useful products to assist the structural biology community in tackling the challenge of membrane protein crystallization, optimization and ultimately structure determination. References Newstead S, Ferrandon S & Iwata S (2008) Rationalizing alpha-helical membrane protein crystallization. Protein Sci 17: 466–472 Page R & Stevens R (2004) Crystallization data mining in structural genomics: using positive and negative results to optimize protein crystallization screens. Methods 34: 373 389 Parker JL & Newstead S (2012) Current trends in α-helical membrane protein crystallization: An update. Protein Sci 21: 1358–1365 White S (2004) The progress of membrane protein structure determination. Protein Sci 13: 1948–1949

Figure 3. Successful additives used for alpha helical membrane protein crystallization. The range of additives used for successful crystallizations are shown for each membrane protein family. The additives have been grouped into more general classes for clarity and colored according to the pie chart shown inset, illustrating the composition of the current database. (Reproduced with permission from Paker and Newstead., Protein Science 2012).

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 22

Crystal Growth Screens

MEMBRANE PROTEINS

MEMBRANE PROTEINS

PGA™ SCREEN

LIPIDIC-SPONGE PHASE™ SCREEN THIS PIONEERING SCREEN FOR MEMBRANE PROTEIN CRYSTALLIZATION CONTAINS 48 CONDITIONS BASED ON PROVEN CRYSTALLIZATIONS OF TARGET MEMBRANE PROTEINS IN AN ACTIVE RESEARCH LABORATORY.1

A REVOLUTIONARY NEW SYSTEMATIC SCREEN BASED ON THE POLY-γ-GLUTAMIC ACID (PGA) POLYMER. PGA IS THE FIRST OF THE NEW BREED OF CHEMICAL POLYMERS THAT EXPLOIT POLY-AMINO ACIDS TO UNLOCK NEW AREAS OF CRYSTALLIZATION SPACE. THE PGA SCREEN IS SUITABLE FOR BOTH GLOBULAR AND MEMBRANE PROTEIN CRYSTALLIZATION.

The sponge phase is an expanded mesophase where the aqueous channels are larger than in the cubic phase, thus it is able to accommodate membrane proteins with large extra- and/or intracellular domains. It is obtained by mixing Monoolein (MO) with a buffer solution, and a crystallization agent, such as PEG or Jeffamine M600. Salt is also added to the buffer and the pH varied, allowing a broad variety of distinct sponge phases to be formed.

The high nucleation-precipitation potential of PGAs enables their use at very low concentrations and in combination with classical precipitants, thus scaling down the amount of precipitant necessary for crystal appearance and growth. This feature of PGAs makes them especially useful in applications for labile, easily precipitating proteins. PGA Screen is suitable for both globular and membrane protein crystallization!

Features of the Lipidic-Sponge Phase Screen:

Features of the PGA Screen:

n Guaranteed lipidic-sponge phases, premixed and ready-to-use!

n Novel precipitant.

n Closely resembles the native lipidic environment of membrane proteins.

n Totally New Crystallization Space.

n Suitable for use in hanging or sitting-drop vapour diffusion experiments, and in lamina (p.73).

n Applicable to both globular and membrane protein crystallization.

n Easy to handle liquids.

n Easy mixing properties with other PEGs. n Especially useful for labile, easily precipitating proteins. n Non-toxic and non-denaturing.

n Easy retrieval of protein crystals.

n Compatible with liquid – handling robots.

n No lipase or cryoprotectants needed. n Can be used with additive and crystallization screens. n Easy to spot colourless crystals. n Readily compatible with high-throughput crystallization approaches.

ORDER INFORMATION

ORDER INFORMATION Description

Pack

Code

Description

Pack

Code

The Lipidic-Sponge Phase Screen

48 x 40 µL

MD1-48

PGA Screen premixed with buffers

96 x 10 mL

MD1-50

The Lipidic-Sponge Phase Reservoir Solution

100 mL

MD2-100-85

PGA Screen premixed with buffers cacodylate-free

96 x 10 mL

MD1-50-CF

The Lipidic-Sponge Phase Reservoir Solution

PGA Screen HT-96

96 x 1 mL

MD1-51

250 mL

MD2-250-85

PGA Screen HT-96 cacodylate-free

96 x 1 mL

MD1-51-CF

Monoolein

500 mg

MD2-67

Monoolein

1G

MD2-68

Lipidic-Cubic Phase Mixing Adapter

40 µL

The Lipidic-Sponge Phase Single Reagent

40 µL

PGA screen PREMIXED Single Reagent 100 mL

MDSR-50-

MD6-17

PGA screen PREMIXED HT-96 Single Reagent

MDSR-51-

MDSR-48-

This product is manufactured under an exclusive license from York Structural Biology Laboratory, University of York, UK.

References 1. Annemarie B. Wöhri, Linda C. Johansson, Pia Wadsten-Hindrichsen, Weixiao Y. Wahlgren, Gerhard Fischer, Rob Horsefield, Gergely Katona, Maria Nyblom, Fredrik Öberg, Gillian Young, Richard J. Cogdell, Niall J. Fraser, Sven Engström, Richard Neutze. “A lipidic-sponge phase screen for membrane protein crystallisation” Structure, Vol 16, 1003-1009 (2008).

22

100 mL

References 1. TC Hu, J Korczynska, DK Smith, AM Brzozowski - Acta Crystallographica Section D: Biological Crystallography, 2008. D64, 957-963.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 23

MEMBRANE PROTEINS

MEMMAGIC® BICELLE SCREEN KIT

THE CALIXAR™ ADDITIVE KIT

THE MEMMAGIC™ BICELLE SCREEN KIT IS BASED ON THE USE OF BICELLES AS AN ALTERNATIVE METHOD FOR THE CRYSTALLIZATION OF MEMBRANE PROTEINS IN A LIPIDIC ENVIRONMENT.

THIS KIT IS DESIGNED TO TEST THE EFFECT OF 24 NEW COMPOUNDS ON PROTEIN CRYSTAL GROWTH.

Membrane proteins can be readily reconstituted into bicelles and are maintained in a native-like bilayer environment, which can be manipulated with almost the same ease as for detergent solubilized membrane proteins, making it compatible with standard highthroughput screening methods.

New reagents have been designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These reagents behave as surfactants forming micelles of 5–24 nm, with the critical micellar concentration (CMC), (being as expected sensitive to pH), ranging from 0.05 to 1.5 mM. Such molecules were successfully used to promote crystallization of membrane proteins. The figure shows an example of 12 transmembrane protein (ABC transporter) that was concentrated to 10mg/mL, crystallized using the sitting drop vapour diffusion method in 0.2M KSCN, 20% PEG 3340 in the presence of these new compounds. This example illustrates that these additives can promote membrane protein crystallization and help to obtain diffracting protein crystals.

Features of the MemMagic® Bicelle Screen Kit: n Handles like detergent n Behaves like lipid n Robotically compatible

Applications: n Crystallography

Features of The Calixar Additive Kit:

n NMR

n Promotes crystallization of functional membrane proteins

n Assay Development MemMagic® Bicelle Screen kit provides four bicelle solutions of 40%, 35%, 30%, 25% DMPC:CHAPSO (2.8:1).

n Generates a saltbridge network around the protein and hydrophobic contacts This product is manufactured and distributed under an exclusive license using CALIXAR patented technology and is supplied for research use only. This product may not be used for the provision of commercial services for which a separate license must be obtained.

Bicelles are disk-like micelles formed by the mixture of a phosphatidylcholine lipid such as Dimyristoylphosphatidylcholine (DMPC) and a detergent such as 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy1-propanesulfonate (CHAPSO). The bicelle discs can be described as patches of lipid bilayers with detergent molecules lining the apolar edges of each bilayer.

Figure: CALIXAR compounds promote crystallization of a 12 transmembrane target protein (ABC Transporter) only in presence of these compounds at different concentration.

ORDER INFORMATION

ORDER INFORMATION

Description ®

Crystal Growth Screens

MEMBRANE PROTEINS

Pack

Code

Description

Pack

Code

24 x 50 µL

MD1-80

50 µL

MDSR-80-

MemMagic Bicelle Screen kit

4 x 100 µL

MD1-81

The Calixar Additive Kit

MemMagic® Bicelle Screen kit

4 x 250 µL

MD1-82

CALIXAR Single Reagent

MemMagic® and MemX® are registered trademarks of MemxBiosciences LLC. References Faham, S., Ujwal, R., Abramson, J. and Bowie, J. U. (2009) “Practical Aspects of Membrane Proteins Crystallization in Bicelles” Current Topics in Membranes, Volume 63, Chapter 5, 111-127. Faham, S., Boulting, G. L., Massey, E. A., Yohannan, S., Yang, D., & Bowie, J. U. (2005). “Crystallization of bacteriorhodopsin from bicelle formulations at room temperature”. Protein Science, 14, 836–840. Faham, S., & Bowie, J. U. (2002). “Bicelle crystallization: A new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure”. Journal of Molecular Biology, 316, 1–6.

CALIXAR single reagents are manufactured by Calixar SAS and exclusively licensed to Molecular Dimensions for the manufacture and distribution of this additive kit for final end users only. The use of this kit for commercial service purposes requires a separate licence from Calixar SAS. References Matar Merheb M et al., PLoSONE 2011

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 24

Crystal Growth Screens

MEMBRANE PROTEINS

MEMADVANTAGE™ AN ADDITIVE SCREEN DEVELOPED EXCLUSIVELY FOR MEMBRANE PROTEINS. THIS SCREEN TARGETS ALL ALPHA HELICAL TYPES OF PROKARYOTIC AND EUKARYOTIC MEMBRANE PROTEINS. MEMADVANTAGE WAS DEVELOPED FROM THE IDENTIFICATION OF SUCCESSFUL ADDITIVES (USING DATA MINING) CURRENTLY USED IN THE CRYSTALLIZATION OF MEMBRANE PROTEINS. IT CONTAINS A NOVEL SET OF CHEMICALS PRESENTED AS A 96-FORMAT SCREEN FOR IMPLEMENTATION IN ROBOTIC SCREENING PIPELINES.

The kit is designed to help test the effect of 96 different compounds on membrane protein crystal growth. Detergent selection is a critical parameter for growing well-ordered, well diffracting crystals and with so many choices of detergents/ligands to choose it can be both time consuming and expensive to investigate all possibilities. MemAdvantage takes the most successful ligands, detergents, multivalent salts, polyalcohols, non-volatile organics, organics, amphiliphiles and puts them all together in one easy-to-use additive screen. Additives may affect hydration and intermolecular interactions between protein molecules or between protein molecule and solvent and even ligands. This kit is a screen and results may need to be interpreted with a view to designing further additive experiments using different compounds of the same type as the kit reagent that gave a promising result.

Features of MemAdvantage: n A rational and intelligently designed additive screen targeted specifically for membrane proteins. n Allows easy screening of 96 different additives (12 different classes of the following: polyalcohols, detergents, multivalent salts, non-volatile organics etc.) found to be the most successful in membrane protein crystallization. n Particularly suited for Prokaryotic and Eukaryotic alpha helical membrane proteins. n For initial screening or optimization screening. n Ready-to-use deep-well block. References Parker, J. and Newstead, S. ‘Current trends in alpha helical membrane protein crystallization: an update’, Protein Science, 2012, 21 (9):1358-1365.

ORDER INFORMATION Description

Pack

MemAdvantage HT

96 x 0.25 mL MD1-70

Code

This product is manufactured under an exclusive licence from Isis Innovation Ltd, and was developed by Dr. Simon Newstead, University of Oxford, UK.

For MemAdvantage reagents see page 49.

Additives found in MemAdvantage and their successfulness in crystallization of membrane proteins.

24

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 25

NUCLEIC ACID

HELIX™ – THE LATEST IN NUCLEIC ACID SCREENING HELIX - PROVIDES A VARIETY OF APPROACHES TO ENHANCE SUCCESSFUL CRYSTALLISATIONS FOR A DIVERSE RANGE OF NUCLEIC ACID TOPOLOGIES AND MOLECULAR WEIGHTS.

Crystal Growth Screens

NUCLEIC ACID

HELIX is a 96 condition nucleic acid focused kit designed to maximize the diversity of conditions, and is modular in its design to allow for focused experiments. Conditions include: a 24-well subset covering cryo-cooling; a 24-well subset covering four-stranded DNA/RNA quadruplexes; a 12-well subset focused on low pH for i-motifs, and cytosine protonation; a 12-well subset focused on large molecular weight nucleic acids; a 24-well subset covering divalent metal ions and additives designed to be combined in various ways to rapidly generate customised orthogonal screens.

Features of HELIX: n Nucleic acid fragments, of all molecular weights n Double stranded DNA and RNA, pseudo knots, G-quadruplexes, i-motifs, triplex, ribozymes n Optimized for MAD, SAD, SIRAS data collections n Cryo-cooling optimization n Screening of additives n Adaptable for High Throughput Screening

ORDER INFORMATION Description

Pack

Code

HELIX

96 x 10 mL

MD1-68

HELIX HT-96

96 x 1 mL

MD1-69

HELIX single reagent

100 mL

MDSR-68-

HELIX HT-96 single reagent

100 mL

MDSR-69-

Developed by Dr. Gary Parkinson from School of Pharmacy, University College, London, UK and manufactured under an exclusive license from the University of London.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 26

OPTIMIZATION TO OPTIMIZE INITIAL HITS FROM SCREENING, MOLECULAR DIMENSIONS PROVIDES A COMPLETE RANGE OF BUFFERS, CRYSTALLIZATION STOCK SOLUTIONS AND SINGLE SCREEN REAGENTS.

30 31 31 35 36 36 37 38 39 39 40

Custom screens Naomi’s nucleant MicroSeed Beads™ Fluorescent labelling kits Additive screen Single reagents PEG precipitants Alternative precipitiants Jeffamines® Volatiles – organics Non-volatiles – organics

40 41 43 43 44 45 45 46

Polyamines Salts Other reagents Cryoprotectants Buffer stocks Special buffers Morpheus® mixes Morpheus® individual stock reagents 48 Midas™ reagents 49 MemAdvantage reagents

51 51 51

26

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The Really Useful Buffer kit Gelled Surface Kit Microbatch Oils

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 27

SEEDING LAB EXERCISES Dr Terese Bergfors

1. What is seeding? • The use of an existing nucleus (usually a small crystal or crystal fragment), introduced into a new drop, where it acts as a growth site.

2. Why seed? • To separate nucleation from growth and bypass the need for spontaneous nucleation. It is easier to add onto an already existing nucleus than create one de novo. See Fig. 1. Seeding and the phase diagram. • Improve the size of the crystals or control the number of crystals • Get more consistent results when crystals don’t always appear in known conditions • Speed up results if spontaneous nucleation is slow • To obtain a wider range of crystal forms (polymorphs) by seeding into totally different precipitants

3. What are the types of seeding? • Macroseeding is the transfer of a single, pre-grown, washed crystal.

In this article we will present two laboratory exercises: 1. Instantaneous streak seeding varying the protein concentration. 2. Comparison of jab and streak seeding with overnight equilibration.

• Microseeding is the transfer of microscopic crystals, crushed up into fragments. • Streak seeding is a form of microseeding that transfers the microseeds by a stroking motion with a whisker or hair of some sort. See Fig. 2. Streak seeding. Can be used on its own or in combination with microseeds in a dilution series. • Jab seeding is another variationon microseeding where the new drop is “innoculated” with a single jab of the seed transfer tool.

Reference: For a review article on seeding, see: Bergfors, T. “Seeds to Crystals” J. Structural Biol. 2003, vol. 142, 66-76

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 28

Probe

[Macromolecule]

Wax

[Crystallizing agent] Undersaturation Saturation

No seed crystal added

Metastable zone Labile zone

Seed crystal added

Precipitation zone

Figure 1: Seeding and the phase diagram. From Luft and DeTitta, Acta Cryst. (1999) D55, 988-993.

EXPERIMENT 1: Instant streak seeding

Reference: This exercise has been adapted from Enrico Stura’s chapter "Seeding" in Protein Crystallization: Strategies, Techniques, and Tips edited by T. Bergfors 1999 International University Line, La Jolla Ca.

Figure 2: Streak seeding from Enrico Stura, Published in “Crystallization of Nucleic Acids and Proteins” Edited by Ducruix and Giege, 1992, Oxford University Press

3. Once the parent crystals have formed in the drop with 100 mg/mL, you can set up the 10 µl drops of lysozyme at 80, 60, 40, and 20 mg/mL on the inside of a Petri dish lid.

Purpose of this experiment: 1. To learn how to generate new seeds by the easy, fast, and simple method of streak seeding. 2. To observe the effects of decreasing protein concentration on the nucleation rate.

100 mg/ml

Materials needed: • A crystal wand – you can make your own from a cat whisker, human hair or horse tail hair, super glue or wax to affix the whisker to a yellow pipette tip, and use a razor blade to cut the whisker. • lysozyme 100 mg/mL stock solution. • A dilution series of lysozyme at 80, 60, 40 and 20 mg/mL. • 30% polyethylene glycol (PEG) 5000 or 6000 in 1 M NaCl, 50 mM Na acetate, pH 4.7 = the precipitant. • A surface for making the drops, e.g., a Petri dish lid, cover slip, etc.

Method: 1. On the Petri dish lid, pipette 10 µl of lysozyme (100 mg/mL). Now add an equal volume (i.e., 10 µl) of the precipitant (= 30% PEG 6000 in 1M NaCl, 50 mM Na acetate, pH 4.7). 2. Watch the drop under the microscope. The first thing you should see is a phase separation. After that, the crystals should nucleate in 5-15 minutes. The fresher the lysozyme the longer time it will take to nucleate, so the time will vary. It can take some practice to recognize the nucleation in its initial stages. Note: If the protein precipitates immediately, the protein concentration is too high. Redo with a slightly lower protein concentration; try 80 mg/mL.

28

4. Add 10 μl of the precipitant to each of these 4 new drops and stir. Important! These drops also need precipitant, not just protein, or the experiment does not work. Students often forget this step. 5. Now you can transfer the nuclei (from the parent drop) by streak seeding. Dip the seeding wand once into the drop of parent crystals to pick up the seeds, then streak the wand across the four new drops. You do not need to re-dip the wand into the parent drop for each new drop.

What to look for or think about: • See if you can get the crystals to grow along a streak line. Because of the high protein concentrations you will also get spontaneous nucleation but many crystals will form preferentially along the streak line. The crystals will be extremely small because they grow so rapidly. In this experiment the protocol has been optimized for speed, rather than crystal size. • You don't need to save these drops, but you can use them in the next experiment. In the current experiment we demonstrated the method and the principles for streak seeding. In the next experiment, a more refined version will be presented.

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 29

EXPERIMENT 2:

6. Repeat for MIXTURE B and PLATE B. 7. Row 1 will be the control.

Comparing the effects of adding different amounts of seeds

8. Row 2 will be jab seeded. Using the seed transfer tool, dip or stir it into the parent drop in the previous experiment (experiment 1) to pick up seeds.

Purpose of this experiment: 1. To illustrate the effect of adding different amounts of seeds. 2. To illustrate a very fast and easy way of creating a dilution series of seeds. In contrast to experiment 1 left, this experiment will be allowed to equilibrate overnight to permit slower growth of the crystals after seeds are added.Three rows of identical drops will be prepared. Seeds will be added by either jabbing the drops or streak seeding them. These drops will be compared to a control where no seeds have been added. An entire row of drops will be seeded sequentially to create a dilution of the seeds. Thus, the first drop will have the most seeds and the last drop in that row will have the least.

Materials required:

9. Now jab all the drops in row 2 with the seed transfer tool. To do so, barely touch the outermost edge of the drop with the tool = a quick jab. Rinse the tool in the reservoir solution between each jab to create a dilution series of the seeds. 10. Row 3 will be streak seeded. Rinse the seed transfer tool thoroughly and wipe it off with a tissue. Dip or stir it into the parent drop in the previous experiment (experiment 1) to pick up a new batch of seeds. 11. Now streak seed the drops in row 3 with the seed transfer tool. Rinse the tool in the reservoir solution between each streak to create a dilution series of the seeds. 12. Invert the lids of the Petri dishes over the correct, respective reservoirs and seal the rims with parafilm. 13. Wait 24 hours to observe the results.

• Two 20 mL Petri dishes (the large size) • 5 mL of 6% NaCl in 50-100 mM sodium acetate buffer, pH 4.7

Expected results:

• 5 mL of 12% NaCl in 50-100 mM sodium acetate buffer, pH 4.7

1. After 24 hours, the control row in PLATE A will probably be still clear (but crystals will grow there after 48 hours, so it is best to examine the plates before that). In PLATE B, you probably will see sea urchintype crystals by 24 hours.

• 100 microliters of 20 mg/mL lysozyme, dissolved in water • A seeding wand (you can use the same one from the previous experiment.) • Two Eppendorf tubes

Method: 1. In an eppendorf tube, mix 50 microliters of lysozyme (20 mg/mL) with 50 microliters of 6% NaCl in 50-100 mM sodium acetate buffer, pH 4.7 = MIXTURE A. 2. In another eppendorf tube (mark them, so you don’t mix them up!), mix 50 microliters of lysozyme (20 mg/mL) with 50 microliters of 12% NaCl in 50-100 mM sodium acetate buffer, pH 4.7 = MIXTURE B.

2. The rows with jab seeding and streak seeding will give different effects. Jab seeding works best if the drop is very large, because there is a dilution effect across the surface of the drop. The drops here are only 5 microliters. Once back home, try the effect on large sitting drops (40 microliters) to make it more pronounced. 3. Jab and streak seeding several drops in a row (serial seeding) is a fast and easy way of making a dilution series of seeds in the new drops. The drops seeded last (by either method) should show the least number of crystals compared to the first drops in the row. See an example in Figure 3.

3. In one Petri dish, pour approximately 5 mL of the buffered 6% NaCl solution in the bottom = PLATE A. 4. Repeat for the other Petri dish, using buffered 12% NaCl instead = PLATE B. 5. Using MIXTURE A, pipette 3 rows of 6 drops each onto the lid of PLATE A. Each drop will contain 5 microliters. See picture:

control row

jab seed

streak seed

Figure 3. Comparison of drops that have been serially seeded with a control. These results were photographed 48 hours after setup. Row 1 is a control and has not been seeded. The lysozyme crystallizes in the form of sea urchins or spherulites. Row 2 has been jab seeded. The needle inoculated with seeds was jabbed into the center of the drops. Row 3 has been streak seeded. Notice that crystals tend to form along the seed line, but there is much spontaneous nucleation even outside the streak line in the first drops. However, the final drop in the row (drop 5 in the series) has nice, large crystals. This effect was achieved because the number of seeds added to the drop was diluted by passing the streak wand through the first drops in the row. (Photos by Terese Bergfors).

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MD Journal Build_Layout 1 6/5/13 10:10 AM Page 30

Optimization

48 & 96 REAGENT CUSTOM SCREEN

Over the years Molecular Dimensions have gained extensive experience of producing custom crystallization screens for its many customers. (Some of these screens have since gone on to become some of our best-selling crystallization screens, (e.g. Morpheus®, Clear Strategy & MemGold™). We can now provide custom crystallization screens & solutions designed by you. Simply provide the specifications; molarity, pH etc. and we’ll do the rest; from small-scale (mL) to large-scale (L). All solutions are manufactured to our exacting standards using ultra-pure 18 MegaOhm water. All custom reagents & screens are treated as strictly confidential and are provided with full traceability.

CONTACT MOLECULAR DIMENSIONS FOR YOUR TAILORED CUSTOM SCREEN QUOTATION 30



I have been ordering custom screens from Molecular Dimensions since I started my crystallography project 3 years ago. Very quick and convenient !!! MRC Prion Unit, London, UK.



ORDER INFORMATION Description

Code

48 reagent Custom screen Set-up + 1 kit (10 mL or HT96)

MD1-CUSTOM48

48 reagent custom screen 10 mL kit ( 1 box)

MD1-CUSTOM48-10ML

48 reagent custom screen HT-96 kit

MD1-CUSTOM48-HT96

96 reagent Custom screen Set-up + 1 kit (10 mL or HT96)

MD1-CUSTOM96

96 condition custom screen 10 mL kit (2 box kit)

MD1-CUSTOM96-10ML

96 condition custom screen HT-96 kit MD1-CUSTOM96-HT96

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MD Journal Build_Layout 1 6/5/13 10:11 AM Page 31

SEEDING

NAOMI’S NUCLEANT

MICROSEED BEADS™

NAOMI’S NUCLEANT HAS FACILITATED THE CRYSTALLIZATION OF THE HIGHEST NUMBER OF PROTEINS REPORTED FOR ANY SINGLE NUCLEANT. MANY OF THESE PROTEINS HAVE PROVEN DIFFICULT TO CRYSTALLIZE AND SOME OF THESE, INCLUDING MEMBRANE PROTEINS, HAVE ONLY BEEN CRYSTALLIZED IN THE PRESENCE OF NAOMI’S NUCLEANT.

FOR THE PRODUCTION OF MICROSEEDS FOR OPTIMIZATION EXPERIMENTS.

Optimization

NUCLEANT

MicroSeed Beads are used to generate microseeds (submicroscopic crystals that have been ‘crushed-up’ into fragments). Crystals are crushed using PTFE balls and suspended into serial dilutions of mother liquor.

In addition to test proteins those proteins that can be named are: multi drug resistance protein (a membrane protein), modified cyclodextrine, oxyntomodulin, myosin binding protein C, lobster shell α-crustacyanin, c-phycocyanin, α-actinin actin binding protein. Several other proteins have also been crystallized but cannot be named at this time. Often the crystals obtained were of increased diffracting quality compared to those resulting from standard techniques. For example myosin binding protein C diffracted to 1.6Å compared to 3Å.

Features of MicroSeed Beads: n Improve the size of crystals or control the number of crystals. n Get more consistent results. n Speed up results if spontaneous nucleation is slow. n Avoid cross-contamination unlike glass beads. n Obtain a wider range of crystal forms (polymorphs) by seeding into totally different precipitants.

Features of Naomi’s Nucleant: n Simply add a single grain to a crystallization drop. n Easy to place with fine tweezers. n Use in screening or optimization to nucleate supersaturated conditions. n Use in optimization where excessive nucleation occurs (i.e. lots of tiny crystals). Back off the precipitant concentration to the metastable zone and then use a grain to nucleate. n Negates twinning. n Protein crystals are easily detached from the nucleant using a microprobe or a cryo loop.

Find out more about MicroSeed Beads here

ORDER INFORMATION Description

Pack

Code

MicroSeed Beads

24 x PTFE MicroSeed Beads In individual 1.5 mL microfuge tubes

MD2-14

PHASING

I3C PHASING KIT Crystals (arrowed) of β-lactamase grown on a grain of Naomi’s Nucleant by Rosalida Leone at Imperial College, London.

ORDER INFORMATION

FOR HEAVY ATOM DERIVITIZATION OF BIOLOGICAL MACROMOLECULES FOR SUBSEQUENT SINGLE WAVELENGTH ANOMALOUS DISPERSION (SAD) OR SINGLE ISOMORPHOUS REPLACEMENT WITH ANOMALOUS SCATTERING (SIRAS)

Description

Pack

Naomi’s Nucleant

1 vial MD2-07 (approx 3mg) (approx. 300 grains)

Code

Features of I3C Phasing Kit:

Microspatula

1

MD9-08

n Use in co-crystallization.

Fine tweezers

1

MD9-25

n Pre-weighed aliquots.

Microprobe sampler kit

1 kit

MD9-01

Developed and patented at Imperial College London, the most effective nucleant of any material tested. References 1. Chayen, N.E., Saridakis, E. and Sear R. Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium. PNAS (2006) 103, 597-601. 2. Saridakis, E. and Chayen N.E., Towards a ‘universal’ nucleant for protein crystallization. Trends in Biotechnology (2009) 27, 99-106 3. Eisenstein, M. The shape of things. Nature Methods (2007) 4, 95-101.

n Use in heavy atom soaking experiments.

ORDER INFORMATION Description

Pack

Code

I3C Phasing Kit

12 aliquots of I3C (280 mg each) + 12 aliquots of 2.0 M LiOH (650 µL each)

MD2-11

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TRACE FLUORESCENT LABELLING USING VISIBLE FLUORESCENCE FOR CRYSTALLIZATION SCREENING Dr Marc Pusey

INTRODUCTION:

O

btaining sufficiently well diffracting crystals is the bottleneck process in macromolecule structure determination. Finding these conditions depends upon determining successful outcomes in screening trials. Current trends are to utilize ever smaller crystallization trial volumes, to enable setting up more trials. This results in large numbers of experiments to be set up, maintained, and periodically monitored, leading in turn to the increased use of robotics for all aspects of the procedure. Robotic analysis of white light images is not simple, and is liable to be confounded by, e.g., masses of crystals or crystals buried in precipitate. The latter outcome can also present problems to a manual visual analysis of crystallization trials, a process that can result in missing many leads and also a mind-numbingly tedious task for even small scale screening trials and the results thereby negatively affected. These considerations led to development of the trace fluorescent labeling approach for crystallization screening (Forsythe et al., 2006). The basis of this method is that 1.8 M

Ammonium sulphate concentration. The lower set of images are of the initial drops. Images provided courtesy of University of Hamburg.

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PRODUCT INDEX Description

1,2,3-Heptanetriol 1,2-Propanediol 1,2-Propanediol 1,2-Propanediol 1,3-Propanediol 1,4-Butanediol 1,4-Dioxane 1,6-hexanediol 1,6-hexanediol 1.3-Propanediol 1.4-Butanediol 10cc Air Adapter Assembly 10 ml barrels (10cc clear syringes) - White PE Piston 1,2,3-heptanetriol 18 mm round no. 2 glass cover slips 18 mm round plain coverslips No 3 glass 1-Butanol 1-Butanol 1-Propanol 22mm round coverslips No 2 glass 22mm round plain glass coverslips No 3 glass 22mm square coverslips No 2 glass 2-Mercaptoethanol 2-methyl-2,4-pentanediol (MPD) 2-methyl-2,4-pentanediol (MPD) 2-Propanol 2-Propanol 30cc Air Adapter Assembly 30 ml barrels (30cc clear syringes) - White PE Piston 3D Structure Screen 5 shelf rack for CXR500 Dry Shipper 96 Square Well Storage Block Mat 96 Well Flat Bottom Plates AAB buffer pH4 and pH9 Acrylic acid/maleic acid copolymer (50:50) Na salt Acrylic acid/maleic acid copolymer (50:50) Na salt ADA Additional Shelf for 180L Incubator Additional Shelf for 250L Incubator Additional Shelf for 390L Incubator Additive Screen Air Compressor Unit Ammonium acetate Ammonium acetate Ammonium acetate Ammonium chloride Ammonium citrate Ammonium dihydrogen phosphate Ammonium formate

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49 39 43 46 39 39 39 40 46 46 46 117 117 40 62 62 39 46 39 62 62 62 50 40 43 39 46 117 117 7 111 73 72 45 38 48 44 122 122 122 36 117 41 44 47 41 41 41 41

Description

Page

Ammonium hydrogen citrate -dibasic Ammonium hydrogen phosphate -dibasic Ammonium nitrate Ammonium sulfate Ammonium sulfate Anzergent 3-12 APF LB Broth Lennox APF LB Broth Luria APF LB Broth Miller APF Media Optimization Kit Assorted wicks Atholate Augmedia powder Bacteriorhodopsin (inc. free turntable base) Barium chloride dihydrate Bench Top Incubator 100 L Programmable Bench Top Incubator 100 L Non-Programmable Bench Top Incubator 27.5L Non-Programmable Bench Top Incubator 27.5L Programmable Bench Top Incubator 55L Non- Programmable Bench Top Incubator 55L Programmable Bench Top Incubator Stacker Benzamidine HCl Bicine Biomolecular Crystallography Bis Tris Bis Tris Propane Bovine Pituitary Extract BRFF-BMZERO BRFF-EPM2 BRFF-HPC-1 serum free medium BRFF-P4-8F Cadmium chloride dihydrate Cadmium sulfate 8/3 hydrate Calcium acetate Calcium chloride dihydrate Calcium chloride dihydrate Calixar Additive Kit Canister for XT-20 7 XT-10 dewar Canned Air CAPS CAPSO Cesium chloride Cetyltrimethylammonium bromide (CTAB) CHAPS CHC buffer pH4 and pH10 C-Hega-11 CHES Chromium (III) chloride hexahydrate

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

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Description

Citrate Classic 25 (LD Series 25L capacity) Classic Block (inc. free turntable base) Clear Sealing tape (50mm x 66M) Clear Strategy Screen Combination Clear Strategy Screen Combination pH Premixed Clear Strategy Screen I Clear Strategy Screen II ClearVue Sheets Coarse wicks Cobalt chloride Conc. HCl for titration Containerless Crystallization oils Cooled Crystallization Incubator 100 litre Cooled Crystallization Incubator 180 litre Cooled Crystallization Incubator 250 litre Cooled Crystallization Incubator 390 litre Copper(II) Chloride Coverslide Combi (vacuum pen and Performus V) Cryocane Coders Cryocane for 5 vials Cryocane for 6 vials CryoCap Data Matrix Reader with Vial Rack CryoCaps CryoCaps with Data Matrix Cryogenic vial closure colour coders Cryogenic vial holder CryoMount Elliptical LithoLoop Set CryoMount LithoLoop Set CryoMount LithoLoop Set Sampler Pack CryoMount Mesh LithoLoop Set CryoPins 18mm CryoProtX CryoProtX CryoProtX Mix Reagents Cryosleeve CryoTongs 24 mm CryoTongs for SPINE caps Cryotool Set Cryoware labels Cryoware Marker Set/4 Cols Crystal & Life Abad-Zapatero Crystal Quick X-ray plates Crystal X2 CrystalClene Slips 18mm round CrystalClene Slips 18mm square CrystalClene Slips 22mm square CrystalClene Slips 22mm round CrystalHarp

Page

44 111 119 73 8 8 8 8 73 97 41 43 51 122 122 122 122 41 117 114 114 114 105 104 104 114 114 103 103 103 103 104 43 109 109 114 63 112 112 114 114 115 69 126 62 62 62 62 63

Description

Page

CrystalHarp Capillary Cutting Tool CrystalHarp Capillary Tweezers- rubber tipped CrystalHarp Starter Pack Crystallization of Nucleic Acids and Proteins Crystallography Made Crystal Clear Cube block 10x10x10 inc free turntable Custom LithoLoop CryoMount Set Custom screen CX100 Dewar lid CX100 Dry shipper CX100 Shipping Case CXR100 Cryogenic Shipper with Replaceable Absorbant CXR100 Replaceable adsorbent set CXR500 Dry Shipper CXR500 Shipping Case Cyclodextrin Screening Kit Cymal 1 Cymal 2 Cymal 4 Cymal 5 Cymal 6 Cymal 7 Data Logger for CX100 Dry shipper Data logger for CXR500 Taylor-Wharton dry shippers David Blow: Outline of Cryst. for Biologists DDM n-Dodecyl-ß-D-maltoside Deoxy-BigCHAP Detergent Screening Kit Deuterium Oxide Dewar Full Base Low Form Full Aluminum Dewar Full Base Low Form Full Aluminum hemispherical Dewar Full Base w/Metal Handle Dewar low form full base cylindrical D-galactose D-glucose Di-ethylene glycol Di-ethylene glycol Di-ethylene glycol Dimethyl sulfoxide Dimethyl sulfoxide DL-alanine DL-glutamic acid DL-serine DM (n-decyl-beta-D-maltopyranoside) D-mannose DMEM / F12 Serum-Free Medium DMG (n-dodecyl-NN-dimethylglycine) DNA Double helix (inc. free turntable base) DNA Protein Interactions - paperback

North & South America orders: +1 877 479 4339 email: [email protected]

63 63 63 115 115 119 103 30 110 111 110 111 111 111 111 96 49 49 49 49 49 49 110 111 115 49 49 95 50 110 110 110 110 46 46 40 43 46 40 43 47 47 47 49 47 92 50 119 115

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PRODUCT INDEX Description

DNG (decyl maltose neopentyl glycol) DSM (n-decyl-beta-D-thiomaltopyranoside) DTT D-xylose EasySeal Sheets EDTA EGTA Empty 96-deep well blocks EPPS ESRF Sample Changer Basket ESRF Sample Changer Basket tongs ESRF Sample Changer Canister ESRF Sample Changer Canister Plug ESRF Sample Changer COMPLETE Kit ESRF Sample Changer COMPLETE Kit & CryoCaps and CryoVials ESRF Sample Changer STARTER Kit ESRF Sample Changer STARTER Kit with CryoCaps & Vials ESRF Sample Changer Transfer Support ESRF Sample Changer Vial Tongs 90 deg Ethanol Ethylene glycol Ethylene glycol ExpressMAX Media Screening Kit F24 X-Chip Set F24 X-Chip Starter Kit F6 X-Chip Set F6 X-Chip Starter Kit Fine Tweezers Fine Tweezers Fine wicks Finger Switch- Electric Fluorescent 'Green Screen' Dye 20X stock FNC Coating Mix Foam Dewar Value Pack (1 small 1 std 1 large dewar) Foam Dewar Value Pack Large Foscarnet (phosphoformic acid) Foscarnet (phosphoformic acid) Fos-Choline 12 Fos-Choline 9 Freezing Media Pair Fundamentals of Crystallography- Giacvazzo paperback Gadolinium(III) chloride hexahydrate GeBAflex tubes Gelled Surface Kit Giant block (inc. free turntable base) 20 x 10 x 10 Glass Capillary Tubes Borosilicate Glazed Panel Door for 180L Incubator Glazed Panel Door for 250L incubator

136

Page

49 49 50 47 73 49 49 73 44 112 112 112 112 112 112 112 112 112 112 39 40 43 86 69 69 69 69 31 116 97 117 43 91 110 110 40 50 49 49 91 115 50 97 51 119 97 122 122

Description

Page

Glazed Panel Door for 390L incubator Glucose free nutrient mix for SelenoMet media Glucose M9Y Glucose Nutrient Mix Glutaric acid Glutaric acid Glycerol Glycerol Glycerol ethoxylate Glycerol ethoxylate Glycine Glycine Gly-Gly-Gly GPCR Molecular Pharmacology and Drug Targeting GPCR Signalling Complexes Grease Applicator - Performus I F/pedal Control Dispenser Greiner pre-greased 24 well Combo Plate with Lid Greiner 24 well Combo Plate with Lid Hanging-drop Plate & Seal Pack Hanging-drop Starter Kit + JCSG+ HT-96 Hanging-drop Starter Kit + MemGold HT-96 Hanging-drop Starter Kit + MemStart & MemSys HT-96 Hanging-drop Starter Kit + PACT HT-96 Hanging-drop Starter Kit + ProPlex HT-96 Hanging-drop Starter Kit + Structure Screen I & II HT-96 HC35 roller base HC20 High Capacity Dewar HC34 High Capacity Liquid Nitrogen Storage Dewar HC35 ESRF Basket Storage System HC35 ESRF Canister Storage Canister HC35 High Capacity Liquid Nitrogen Storage Dewar Heamoglobin (inc. free turntable base) Heavy & Light Twin Pack Single Reagent Heavy + Light Twin pack HT-96 Hega 10 Hega 11 HELIX HEPES HEPES Buffered Saline Heterologous Expression of Membrane Proteins Methods and Pro Hexaminecobalt(III) chloride HTG (n-heptyl-beta-D-thioglucopyranoside) Humidifier - evaporative Hyper Broth I3C Phasing Kit IMDM Serum-Free Medium Imidazole Imidazole / DL Malic acid

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

122 87 84 84 40 50 40 43 38 48 40 44 50 115 115 117 61 61 72 72 72 72 72 72 72 111 111 111 111 111 111 119 7 7 49 49 25 44 92 115 41 50 122 83 31 93 44 45

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MD Journal Build_Layout 1 6/5/13 10:14 AM Page 137

Description

Page

Immunoglobulin G (inc. free turntable base) 119 Introduction to Macromolecular Crystallography 2nd Edition115 JCSG plus 9 JCSG plus Screen Single Reagent 9 Jeffamine D2000 39 Jeffamine ED-2001 39 Jeffamine ED2003 39 Jeffamine ED900 39 Jeffamine M2005 39 Jeffamine M2070 39 Jeffamine M600 39 Jeffamine SD2001 39 Jeffamine T403 39 KMES 44 L-glutathiione 40 Laminex Film Cover 73 Laminex Frames 73 Laminex Glass Base 100 micron 73 Laminex Glass Base 200 micron 73 Laminex Glass Base SBS 100 micron 73 Laminex Glass Base SBS 200 micron 73 Laminex Glass Cover 73 Laminex Plastic Base 100 micron 73 Laminex Plastic Base 200 micron 73 Laminex Plastic Cover 73 Laminex Starter Kit 100 micron 73 Laminex Starter Kit 200 micron 73 LAPAO (3-laurylamido-NN'-dimethylpropyl amino oxide) 50 Large Foam Dewar 110 L-arginine 40 LB Booster 85 LB Broth Lennox 81 LB Luria Broth 81 LB Miller Broth 81 LD10 cryogenic dewar (20 L capacity] 111 LD4 cryogenic dewar (4L capacity) 111 LDAO (n-dodecyl-NN-dimethylamine-N-oxide) 49 LED Turntable Light Base 110 V 119 LED Turntable Light Base 220V 119 L-fucose 46 L-glutathione reduced 50 Lighting 1 x 20 Watt for 250L incubator 122 Lighting 1 x 20 Watt for 390L incubator 122 Lipidic-Cubic Phase Mixing Adapter 97 Lipidic-Sponge Phase Reservoir Solution 22 Lipidic-Sponge Phase Screen 22 Lipidic-Sponge Phase Single Reagent 22 Lithium acetate 41 Lithium chloride 41

Description

Page

Lithium citrate tetrahydrate Lithium formate Lithium sulfate monohydrate LNG (lauryl maltose neopentyl glycol) Low Level Alarm for HC34 dewar Low Liquid Alarm for HC35 VHC35 XT20 Low Profile Laminex Frames L-proline LS Series 35L Liq N2 storage system for racks (750 samples) Lysine HCl Lysine HCl Macromolecular Crystallography- Conventional and HT methods MacroSol MacroSol Screen Single Reagent Magnesium acetate Magnesium chloride hexahydrate Magnesium chloride hexahydrate Magnesium formate dihydrate Magnesium nitrate hexahydrate Magnesium sulphate Magnetic 45 degree bent angle Cryo Wand for SPINE caps Magnetic Cryo Wand for SPINE caps with Suregrip handle Magnetic CryoVial Magnetic CryoVial with CryoCaps Magnetic CryoVial with CryoCaps with data matrix Maltose Manganese(II) chloride tetrahydrate Media Optimization Kit Medium wicks MemAdvantage Membrane Protein Combination Membrane Protein Structure Determination Methods and Protocol MemGold MemGold Combo Value Pack MemGold Single Reagent MemGold2 MemGold2 Single Reagent MemMagic Bicelle Screen Kit MemPlus MemPlus Screen Single Reagent MemStart + MemSys MemStart MemStart Single Reagent MemSys MemSys Single Reagent MERPOL HCS

North & South America orders: +1 877 479 4339 email: [email protected]

41 41 41 49 111 111 73 40 111 40 47 115 14 14 41 41 46 41 41 41 112 112 104 104 104 43 50 81 97 24 17 115 19 19 19 19 19 23 18 18 17 17 17 18 18 49

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PRODUCT INDEX Description

MES MES/Bis Tris Methanol Methionine solution 250x Methods & Results in Cryst. of Membrane Proteins, So Iwata MIB pH4 and pH10 Micro Chisel Micro Knife Micro Measuring Grid Micro Needle Micro Probe Micro Scale Micro Scraper Micro Spade Micro Spatula Micro Tool Kit Microprobe Sampler Kit Microprobe Sampler Kit MicroSeed Beads Microspatula Microtool Handle MIDAS MIDAS OptiMax Kit MIDAS Single Reagent Mineral Oil Mini Screen Mini Screen Single Reagent MMT pH4 and pH9 Modelling of GPCRs - 2013 Monoolein MOPS Morpheus Morpheus - Buffer System 1 Morpheus - Buffer System 2 Morpheus - Buffer System 3 Morpheus Additive OptiMax Kit Morpheus- Alcohols Mix Morpheus- Amino Acids Mix Morpheus- Buffer 1 Mix Morpheus- Buffer 2 Mix Morpheus- Buffer 3 Mix Morpheus- Carboxylic Acids Mix Morpheus- Divalents Mix Morpheus- EDO_P8K Mix Morpheus- Ethylene Glycols Mix Morpheus- GOL_P4K Mix Morpheus- Halogens Mix Morpheus- Monosaccharides Mix

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44 44 39 87 115 45 116 116 116 116 116 116 116 116 116 116 31 116 31 31 116 15 15 15 43 7 7 45 115 22 44 13 45 45 45 13 45 45 45 45 45 45 45 45 45 45 45 45

Description

Page

Morpheus- MPD_P1KP3350 Mix Morpheus- NPS Mix Morpheus- P550MME_P20K Mix Morpheus Screen Single Reagent Mounted Elliptical LithoLoops Mounted LithoLoop Sampler Pack (NEW) Mounted LithoLoop Sampler Pack-CUSTOM Mounted LithoLoops Mounted Mesh LithoLoops MRC Crystallisation Plate Polystyrene MRC Crystallisation Plate UV MRC MAXI Optimization Plate (Polystyrene) MRC MAXI Optimization Plate (UV) MRC Under Oil Crystallization Plate - UVP MultiXtal Na/K phosphate N-acetyl-D-glucosamine Naomi's Nucleants NDSB-201 NG (n-nonyl-beta-D-glycopyranoside) Nickel chloride Nickel II sulfate hexahydrate NM (n-nonyl-beta-D-maltopyranoside) Notched metal tubes Nozzle allignment tool NR-LBD + NR-LBD Extension Screen Single Reagent NR-LBD Extension Screen NR-LBD Extension Screen Single Reagent NR-LBD Screen NR-LBD Screen Single Reagent Nuclear Receptor Combination Octaethylene glycol monododecyl ether (C12E8) OG (n-octyl-β-D-glucoside) OM-fluorinated (octyl maltoside flourinated) ONG (octyl glucose neopentyl glycol) OptiClear Seals for MRC Crystallization Plates DLS Optimizer Osmium(III) chloride hydrate PACT premier PACT premier Screen Single Reagent Paraffin oil Parrafin oil PCTP pH4 and pH9.5 PEG 1000 PEG 10000 PEG 12000 PEG 1500 PEG 200 PEG 2000

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

45 45 45 13 102 102 102 102 102 68 68 68 68 69 7 44 46 31 43 49 41 41 49 104 105 16 16 16 16 16 16 50 49 49 49 73 125 50 9 9 51 43 45 37 37 37 37 37 37

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Description

PEG 2000 MME PEG 20000 PEG 300 PEG 3000 PEG 3350 PEG 350 MME PEG 400 PEG 4000 PEG 500 MME PEG 5000 MME PEG 600 PEG 6000 PEG 750 MME PEG 8000 Penta erythritol propoxylate (5/4 PO/OH) Pentaerythritol ethoxylate (15/4 EO/OH) Pentaerythritol ethoxylate (15/4 EO/OH) Pentaerythritol ethoxylate (3/4 EO/OH) Pentaerythritol ethoxylate (3/4 EO OH) Pentaerythritol propoxylate (17/8 PO/OH) Pentaerythritol propoxylate (17/8 PO/OH) Pentaerythritol propoxylate (5/4 PO/OH) Penta-ethylene glycol Penta-ethylene glycol Performus V PERK - Protein Expression Rescue Kit PET: Cell Dissociation Formula PGA Screen PGA screen Single Reagent PGA-HM Phosphate/citrate buffer PIPES Piston U 30/55cc WH Wiper 20 Poly Carb Box for CXR500 Dry Shipper Polyacrylate 2 sodium salt Polyacrylate 2100 Polyacrylate 5 sodium salt Polyacrylate 5100 Polyacrylic acid 5100 Polyethyleneimine Polyethyleneimine Poly-glutamic acid (PGA-LM) Polypropylene glycol 400 Polypropylene glycol 400 Polyvinyl alcohol type II Polyvinyl alcohol type II Polyvinylpyrrolidone (PVP) Polyvinylpyrrolidone (PVP) Potassium acetate

Page

37 37 37 37 37 37 37 37 37 37 37 37 37 37 48 38 48 48 38 38 48 38 40 46 117 85 90 22 22 38 44 44 117 111 48 38 48 38 38 38 48 38 38 48 38 48 38 40 41

Description

Page

Potassium bromide Potassium chloride Potassium citrate Potassium di-hydrogen phosphate (monobasic) Potassium fluoride Potassium fluoride Potassium formate Potassium hydrogen phosphate (dibasic) Potassium iodide Potassium nitrate Potassium phosphate Potassium silicate Potassium silicate Potassium sodium tartrate Potassium thiocyanate Power Broth Power Combo Value Pack (Morpheus & MIDAS) Power Prime Broth Practical Protein Crystallography Prime-olate media optimization kit Principles of Biochemsitry -2012 Principles of Protein X-Ray Crystallography 3rd Ed Production of Membrane Proteins: Strategies for Expression ProPlex ProPlex Single Reagent Protein Crystallisation Techniques Strategies & Tips Protein Crystallization Strategies for Structural Genomics Protein Crystallography: A Concise Guide Protein Methods 2nd Ed 1996 QuickFold Protein Refolding Kit Really Useful Buffer Kit Really Useful Buffer Kit Roller Roller Base for Classic 25 (LD Series 25L capacity) Roller Base for XT-20 Liquid Nitrogen Storage Dewar Roller for LS Series 35L Liq N2 Storage system for racks Rotating Light Base (LUMINORUM) Rubidium chloride Rubidium chloride Samarium(III) chloride Sealing grease Sealing Sheet Applicator SelenoMet base media and nutrients SelenoMet base medium and Nutients Glucose Free SelenoMet Medium Base SelenoMet Medium Complete SelenoMet Medium Complete Glucose-Free SelenoMethionine Solution

North & South America orders: +1 877 479 4339 email: [email protected]

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PRODUCT INDEX Description

SeMet Nutrient Mix Serum-Free Medium Screening Kit Si coverslips 18mm round Si coverslips 22mm round Si coverslips 22mm square SILAC- L-Arg SILAC- L-Leu SILAC- DMEM high glucose media SILAC- DMEM low glucose media SILAC- DMEM/F12 SILAC- Harm's F12 SILAC- L-Lys SILAC- L-Met SILAC- McCoy's 5A SILAC- MEM SILAC- RPMI 1640 Silicone oil Single reagents Sitting drop bridges Small Foam Dewar Sodium acetate Sodium acetate Sodium azide Sodium azide Sodium borate Sodium bromide Sodium bromide Sodium cacodylate Sodium chloride Sodium citrate Sodium citrate Sodium citrate Sodium fluoride Sodium fluoride Sodium formate Sodium formate Sodium Hepes Sodium hydrogen phosphate dibasic Sodium hydrogen phosphate dibasic Sodium hydroxide Sodium iodide Sodium iodide Sodium malonate Sodium metaborate Sodium nitrate Sodium nitrate Sodium oxamate

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87 94 62 62 62 87 87 87 87 87 87 87 87 87 87 87 51 36 61 110 42 44 42 50 44 42 46 44 42 42 47 44 42 46 42 47 44 42 46 43 42 46 42 42 42 46 42

Description

Page

Sodium oxamate Sodium phosphate Sodium phosphate dibasic Sodium sulfate Sodium tartrate Sodium thiocyanate Sodium/Potassium phosphate (PACT) Sokalan CP 42 Sokalan CP 5 Sokalan CP 5 Sokalan CP 7 Sokalan CP 7 Sokalan HP 56 Sokalan HP 56 Solubility Tool Kit - Precipitant Screen Box 2 Solubility Tool Kit - Solubility Screen Box 1 Solubility Tool Kit (Box 1 + 2) Solubility Tool Kit Single Reagent SpectroLight 600 scanning DLS and UV/White light imaging system SpectroLight 600 scanning DLS and white light imaging system SpectroSize 300 Cuvette based DLS instrument Spermidine Spermidine Spermine HCl SPG pH4 and pH10 Standard Foam Dewar Strontium chloride Strontium chloride Structure Screen 1 Structure Screen 1 + 2 Single Reagent Structure Screen 1 Single Reagent Structure Screen 2 Structure Screen 2 Single Reagent Structure Screen Combination Structure Screen 1 + 2 Stura Footprint Combination Stura Footprint Screen Single Reagent Stura Footprint screens Stura MacroSol Single Reagent Succinic acid Sucrose monodecanoate (sucrose monocaprate) Sucrosse monododecanoate Super size block (inc. free turntable) Super2 Combo Value Pack (JCSG+ & PACT) Superglue for CryoPins

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

47 44 42 42 42 42 42 38 38 48 38 48 38 48 8 8 8 8 130 130 130 40 50 40 45 110 42 50 5 5 5 6 6 5 5 6 6 6 14 40 49 50 119 9 104

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MD Journal Build_Layout 1 6/5/13 10:14 AM Page 141

Description

Page

Superior Broth Superior Prime Broth SureSeal DWB Swissci Hanging Drop Plate -UVP Swissci LCP Kit Swissci Triple Drop Plate Polystyrene Swissci Triple Drop Plate UV Polymer Syringe Loader (922BL)- 2.5 OZ Cartridge Filling Station Tall Foam Dewar Tapered Tips (nozzles)- size 18 guage Taurine Taurine TBG buffer pH4 and pH9 TCEP Terasaki Microbatch Plate 72-well Tertiary-butanol Tetra-ethylene glycol Tetra-ethylene glycol Tetraethylene glycol monooctyl ether (C8E4) Tetrahydrofuran Textbook of Structural Biology Trace Fluorescence Dual-Labelling Kit Trace Fluorescence Labelling Kit Trehalose Trehalose Tri DM (n-tridecyl-beta-D-maltoside) Trial Pack 10 MRC Crystallization Plates 10 ClearVue Sheets Tricine Triethylammonium phosphate Triethylene glycol (TEG) Triethylene glycol (TEG) Trimethylamine N-oxide (TMAO) Tripao Tris Tris hydrochloride Tungsten Carbide Scriber Turbo Broth Turbo Prime Broth UDM (n-undecyl-beta-D-maltoside) UDTM (n-undecyl-B-D-thiomaltopyranoside) Understanding Diseases by Understanding Proteins UV Hanging Drop Sheets Vacuum Pen- Air Powered F/Pedal Control Vacuum Pen Vacuum Tweezers- Hand Held Vacuum Pick Up Tool VHC35 High capacity Liquid Nitrogen Storage Dewar VHC35 roller base Vial Tongs 45 Degrees

82 82 73 72 73 68 68 117 110 117 40 50 45 50 69 39 40 46 50 39 115 35 35 40 43 49 68 44 50 40 46 38 49 44 44 73 82 82 50 50 115 72 117 117 111 111 112

Description

Page

Volatile Oil for MRC Under Oil Plate Water 18 MegaOhm purified/filtered 1 Liter XRL Plate XRL Plates incl. CrystalClene Slips XT-10 Extended Time Cryogenic dewar (10 L) XT-20 Extended Time Cryogenic dewar (20 L) X-taLight 100 X-taLight 200 White light imaging system X-taLight 210 UV and white light imaging system XTL3 Extended Time Cryogenic dewar (3 L) Xylitol Xylitol Zinc acetate Zinc chloride Zinc nitrate hexahydrate Zinc nitrate hexahydrate Zinc sulfate heptahydrate

51 43 61 61 111 111 129 128 128 111 40 43 42 42 42 50 42

We kindly acknowledge the following people for their crystal images used throughout this publication: Alexey Rak, Karolien Van Belle, Mareike Kurz, Isabell Wingartz, Simon Newstead, Marek Brzozowsky, Gary Parkinson, Stephen Jameison, Annabelle Varrot, Natacha Perebaskine, Anwar Ullah, Karolina Mierzejewska, Albert Guskov, Daljit Sangar and Jane Potter.

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Notes

142

Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

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MD Journal Build_Layout 1 6/5/13 10:14 AM Page 143

North & South America orders: +1 877 479 4339 email: [email protected]

visit: www.moleculardimensions.com

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Notes

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Europe and rest of world orders: +44 (0)1638 561 051 email: [email protected]

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02

03

The Meaning of Quality

Welcome

ARTICLES

How to order You can place your order online, by fax, telephone or via e-mail. Our site includes a full e-commerce facility for selecting and ordering your product. Credit card orders can be placed securely on-line.

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Towards High Efficiency Screening

Exploring More Crystallization Space

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The MemGold Family: the science behind the screens

Seeding Lab Exercises

Making Use of Counter Diffusion

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67 Choosing the right Crystallization Plate

Advanced Methods

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Molecular Dimensions Ltd. Unit 6, Goodwin Business Park, Willie Snaith Road, Newmarket, Suffolk, CB8 7SQ, UK Telephone: +44 (0)1638561051 Fax: +44 (0)1638660674

Molecular Dimensions Inc. 849 Sunshine Lane, Altamonte Springs, Florida, 32714 USA Telephone: +1 877 479 4339 or +1 407 886 6901 Fax: +1 321 972-8896

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32 Identifying hits with Trace Fluorescent Labelling

Media Composition influences Recombinant Protein accumulation in E. Coli

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99 Understanding Radiation Damage

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108 Systematic Approaches to Crystallization and Data Collection

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MD Journal Cover with Spine_MD Journal Build Cover with Spine 6/5/13 10:29 AM Page 1

MOLECULAR DIMENSIONS Providing you with the latest innovations in protein crystallography

Inside

For Protein Crystallography

Molecular Dimensions Ltd. Unit 6, Goodwin Business Park, Willie Snaith Road, Newmarket, Suffolk, CB8 7SQ, UK Telephone: +44 (0)1638561051 Fax: +44 (0)1638660674

Molecular Dimensions Inc. 849 Sunshine Lane, Altamonte Springs, Florida, 32714 USA Telephone: +1 877 479 4339 or +1 407 886 6901 Fax: +1 321 972-8896

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