| 1 RAMC September 2013, Bischenberg

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

Alexey Rak Structure-Design-Informatics Paris LGCR France Sanofi R&D

Sanofi.com

Some of Sanofi’s brands

NO-SPA

Probiotics

Thanks to SB team at Sanofi R&D France

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Alain Dupuy

Maryse Lowinski Jean-Pierre Marquette

Annick Parent

Valerie Steier

Stephanie Pouzieux

Alexey Rak

François Vallee Thomas Bertrand

Magali Mathieu

Laure Delarbre

Alain Dupuy Jacques Houtmann

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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The presentation plan

● Introduction to MMS technology - microseeding

● MMS at Sanofi R&D in France

● Experimental details on microseeding plates setup

● Examples on small molecules-protein complexes and protein - protein complexes crystallization

● Conclusions about time- and cost – effectiveness to use MMS in ambitious structural biology labs

Introduction to MMS technology and the principles of microseeding ● Protein crystallization is a major bottleneck for protein structure

determination

● Protein crystallization still remains the least standard part of the protein structure determination process by X-Ray crystallography

● Nucleation is the first and critical step to succeed with protein crystallization

● Crystals nuclei (seeds) can be transferred from drop to drop to increase success with 3D growth of crystals

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Introduction to MMS technology and the principles of microseeding ● The seeding technique was introduced a few decades ago

● On page 77 of protein Crystallography by Blundell and Johnson (1979): • « This method (Vapour diffusion) is particularly effective for growing large

crystals , especially if the drop is seeded » • No reference given

● Successive 10-fold dilution was the standard from crushed crystals for

micro-seeding

● Enrico Stura introduced streak seeding and perfected macroseeding for sitting drops and the concepts of how to use seeding to understand where you may be in the phase diagram without actually calculating a phase diagram. This method revolutionized the protein crystallization process

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Introduction to MMS technology and the principles of microseeding ● Microseed Matrix Seeding – MMS

● First introduced by C.G. Ireton and B. Stoddard • C.G. Ireton and B. Stoddard (2004) Microseed matrix screening to improve

crystals of yeast cytosine deaminase. Acta Cryst. D60, 601-605

● Allan D’Arcy and coworkers expanded this method by automating the procedure and seeding directly into crystallization screens

• D'Arcy A, Villard F, Marsh M. An automated microseed matrix-screening method for protein crystallization. Acta Cryst. D63, 550-4. 2007

● A more detailed presentation on MMS can be accessed at http://hamptonresearch.com/ramc_2007.aspx?id=1 ● Allan D’Arcy’s presentation at Recent Advances in Macromolecular

Crystallization (RAMC) 2007 San Diego, CA USA

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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http://hamptonresearch.com/ramc_2007.aspx?id=1

Introduction to MMS technology and the principles of microseeding ● MMS – seeding - transfer of very small crystals – seeds to another

crystallization drop can give rise to improve crystal quality

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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● The method has been successfully demonstrated to be applicable for general use

● Showed success on different classes of proteins ● In generating new space groups ● Improving diffraction quality ● Finding useful hits when there were

none before

Why Mosquito is highly effective for MMS ● The Mosquito crystallization robot is very accurate at liquid handling of low

volume solutions 20-100 nL

● Mosquito - extremely reliable and robust instrument, more than 10 years personal experience

● Disposable tips – no cross-contamination and contact dispensing which are both very important instrument features for seeding – MMS

● Availability of the multi-aspirate dispensing mode for Mosquito pipetting ● Makes MMS by Mosquito – quick and efficient

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Why Mosquito is highly effective for MMS Multi-aspirate dispensing

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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http://www.youtube.com/watch?v=UFDq64vfPFY&list=PLJSU0gmdN4qJFeluG0KiowKZMwUs4NBSM&index=1

Experimental details on Mosquito assisted microseeding plates setup

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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● Multi-aspirate dispensing enables the pipetting of two (and more) solutions in conjunction

● Solution 1 is a seed stock, solution 2 is a crystallization solution reservoir ● Put together but not mixed inside of the Mosquito disposable tip

• « Shaken, not stirred! » ● Solution 1&2 pipetted at destination – crystallization drop pedestal which is

preoccupied by protein solution drop

● Routinely: (20nL of seeds + 80nL of reservoir) + 100nL of protein

● 1’ 33’’ for single protein 96 well plate - MMS using Mosquito ● < 5 minutes for three proteins 96 well plate - MMS using Mosquito

● 288 individual seeded crystallization conditions in less than 5 minutes

Experimental details on Mosquito assisted microseeding plates setup

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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http://www.youtube.com/watch?v=UFDq64vfPFY&list=PLJSU0gmdN4qJFeluG0KiowKZMwUs4NBSM&index=1

MMS success rate

● Crystallization optimization by seeding enforced screening is shown to be extremely successful by generating new crystal forms during MMS

● The number of hits generated by MMS is much grater than in crystallization screening without seeding

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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D'Arcy A, Villard F, Marsh M. An automated microseed matrix-screening method for protein crystallization. Acta Cryst. D63, 550-4. 2007

Sparse matrix screening

The same Sparse matrix screening but with seeding by MMS

MMS applications and its usefulness ● MMS is still good for :

● Generating new space groups ● Improving diffraction quality ● Finding useful hits when there were none before

● MMS is also good for : ● Generating crystals of otherwise non-crystallisable targets:

• new shorter or extended construct of the crystallized protein • Example will be given

• new ligand bound crystal forms of the crystallized protein • new proteins using similar but not identical protein crystals

• protein family cross seeding • protein complex using crystal seeds of one of the complex

component/protein • Example will be given

• individual proteins using crystals of protein complex where the aimed protein was complexed with another partner

• Example will be given

The benefits gained from a redesigned crystallisation

strategy focused on a high throughput seeding technique

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Statistics on small molecules-protein complexes and Ab-Ag complexes crystallization ● Crystallization optimization by MMS takes the same or less time than the

classical manual crystallization optimization but consumes much less protein ● Vapor diffusion rate for 100nl + 100nl drops is proportionally higher than

manually performed 1µl + 1µl ● 96 conditions by MMS require ~14µl of protein solution

• (20nL of seeds + 80nL of reservoir) + 100nL of protein ● 14µL of protein is enough to set up manually ~14 crystallization

conditions with micro-seeding ● It is quicker to set up 96 conditions MMS experiments by Mosquito than

14 crystallization/seeding drops performed manually ● The most expensive part of crystallization/optimization of medically

relevant human proteins is the cost of the protein itself

● Crystallization optimization by MMS is more time- and cost- effective than classical crystallization optimization

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Crystallization optimization, real example

● The only crystallization hit obtained

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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● Classical manual optimization ● ~ 6 weeks 0.3 FTE ● 4.5 mg protein ● The best result 3.2Å

resolution ● MMS in 4 x 96 condition

● Two sparse matrix and two grid screens used

● 26 new conditions found resulting crystals of better than initial quality

● ~ 1 day 1 FTE ● 0.6 mg protein ● 1.7 - 2.2Å resolution

Crystallization of different construct representing the same multi-domains protein ● PI3K beta – oncology target

● No high resolution structure • Despite many efforts

● >180 constructs of the heterodimer were expressed ● 32 constructs submitted to crystallization trials

• 2 constructs crystallized in classical crystallization screening • 19 constructs crystallized in MMS assisted crystallization screening

• Seeds of different construct(s) were used to fertilize new construct drops ● Crystallization optimization was achieved most successfully by MMS

• Seeds of the crystallization hits were used to generate new crystals of the same construct

• MMS was applied sometimes in several cycles • The better seeds result the better crystals

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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150um 150um 50um 50um

MMS MMS

2.6A diffracting Crystals !

Crystallization of protein complexes, MMS assisted combinatorial approach ● Need to crystallize multi-domain protein alone and in complex with its

interacting partners

● Classical crystallization screening: ● No crystals for the multi-domain protein alone X ● No crystals for complex with two partners X ● No crystals for complex with “red” partner X ● Crystals for complex with “green” partner V

● MMS using seeds of the “green” partner complex crystals to crystallize other combinations of the interest ● No crystals for complex with two partners X ● Crystals for multi-domain protein alone V ● No Crystals for complex with “red” partner X

● MMS using seeds of the multi-domain protein alone crystals to crystallize other combinations of the interest ● No crystals for complex with two partners X ● Crystals for complex with “red” partner V

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Conclusions

● Seeding by MMS is very effective method to optimize protein crystals quality and to generate crystals of otherwise non-crystallizable proteins

● Miniaturizing MMS is both time- and cost-effective

● Automating the process gives speed and reliability

● key features needed: ● multiple aspirations then direct dispense to combine both seed stock and

screen, then add them to the protein ● no cross-contamination ● accurate handling of very low volumes

● Mosquito has proven to be an essential tool for MMS in Sanofi R&D France labs

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique

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Merci

NOM DE LA PRESENTATION | 21

The benefits gained from a redesigned crystallisation strategy focused on a high throughput seeding technique�Sanofi.comSlide Number 3Thanks to�SB team at �Sanofi R&D�FranceThe presentation planIntroduction to MMS technology and the principles of microseeding�Introduction to MMS technology and the principles of microseedingIntroduction to MMS technology and the principles of microseedingIntroduction to MMS technology and the principles of microseedingWhy Mosquito is highly effective for MMS�Why Mosquito is highly effective for MMS�Multi-aspirate dispensingExperimental details on Mosquito assisted microseeding plates setupExperimental details on Mosquito assisted microseeding plates setupMMS success rate MMS applications and its usefulness��Statistics on small molecules-protein complexes and Ab-Ag complexes crystallization�Crystallization optimization, real exampleCrystallization of different construct representing the same multi-domains proteinCrystallization of protein complexes, MMS assisted combinatorial approachConclusionsMerci