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Crystallization

 

 

 

The crystallization experiments are performed with a Cartesian PixSys 4200 crystallization robot (Genomic Solutions, U.K.).Cartesian RobotStandard drops set up is 100nl of the sample mixed with 100nl of crystallization condition. All our protocols are described below. Customized setup can be performed upon request . Please contact the HTX lab staff well in advance to discuss about feasibility and availability.


The facility protocols are also described in :
Dimasi N., Flot D., Dupeux F., & Márquez J.A., (2007) Acta Crystallogr Sect F. 63(Pt 3): 204–208.

 

 

Should the experiments produce results that lead directly or indirectly to publications, the High Throughput Crystallization Laboratory (HTX Lab) at the EMBL Grenoble should be acknowledged and the publication mentioned previously included in the list of references.

 

In addition to this BIOSTRUCT-X users should acknowledge the European Commission with the following phrase: "The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 227764".

 

Quick link to:

- Protocols available

- Volumes required for a crystallization trial

- Microseeding

- Additives screens

- Crystallization plates

- Crystal optimization

 

 

Protocols available


•   Sitting drops (setup per default)

Sitting drop of 100nl of sample plus 100nl of reservoir solution is the standard set up performed at the HTX platform. Special ratios (sample:reservoir) can be requested through the 2 ratios and 3 ratios protocols. Sitting drops allow setting up to 3 samples or ratios per plate.

 

Sitting drops protocols
Set up Standard 2 ratios
3 ratios
Ratio 1:1 1:1 2:1 1:3 1:1 3:1
Sample 100nl 100nl 100nl 50nl 100nl 150nl
Precipitant 100nl 100nl 50nl 150nl 100nl 50nl

 

 •   Hanging drops

Hanging drop is a special set up that can be requested at the HTX platform. Its preparation requires manual steps that make this protocol not fully adapted for high-throughput. Therefore, we kindly ask our users to request this set up only if it is compulsory. Hanging drops allow setting up to 2 samples or ratios per plate.

 

Hanging drops protocols
Set up Standard 2 ratios
Ratio 1:1 1:1 2:1
Sample 100nl 100nl 100nl
Precipitant 100nl 100nl 50nl

 

 

Volume of sample required for a crystallization trial


The volume of sample to provide is 15µl of sample per plate (and per ratio) for the first 3 plates, and then an additional 10µl is required per supplementary plate. See below the detailed volumes for our standard protocols. For any other protocols, please contact the HTX staff to check the volume needed. We also recommend providing a couple of microliters extra to compensate potential imprecision during pipetting (especially if you have not calibrated your pipettes recently).

 

Number of plate(s) requested
Protocols 1 2 3 4 5 6
Standard 20 µl 30 µl 45 µl 55 µl 65 µl 75 µl
2 ratios 30 µl 60 µl 90 µl 110 µl 130 µl 150 µl
3 ratios 45 µl 90 µl 135 µl 165 µl 195 µl 225 µl

 

 

Microseeding


Microseeding can be performed at the HTX platform. We recommend using the Seed Bead kit from Hampton Research to prepare your seeds stock. Please note the seeds must be thoroughly crushed to ensure a proper aspiration and dispensing by the tips of the Cartesian robot. Standard microseeding protocol consists of delivering 50nl of the seeds stock on top of a 100nl+100nl drop. Microseeding being more time-consuming than a normal setup, its usage is limited to a couple of plates per request.

 

 

Additives screens


The initial hit can also be optimized by addition of a new component which may improve the crystal quality. The HTX platform offers two additives screens (see screening) allowing to test 96 different additives in a single crystallization plates. In order to perform an additive screen, the user should provide, with the sample, at least 15ml of the base solution (initial crystallization condition). A crystallization plate will be prepared by the HTX staff mixing 90µl of the base solution with 10µl of the additives screen (10X concentrated) in each well. The standard drop setup (100nl+100nl) will then be applied.

 

 

Crystallization plates

 

Name

Company

Shelf bottom

Reservoir volume

Profile

UV compatibility

Special features

CrystalQuick

Greiner

Flat

80µl

High

Medium background

Hanging drops

CrystalDirect™

EMBL

Flat

45µl

Low

No background

In-situ characterization & automated crystal harvesting

Intelliplate

Art Robbins

Concave

65µl

High

Medium background

Recommended if sample contains detergent (extra cost 3.5€ per plate)

 

CrystalDirect™ plate is the standard plate type used at the HTX platform. However, the crystallization plate type can be changed upon request (please let us know in the comment field of the crystallization request form).

 

 

Crystal optimization

 

The HTX platform offers:

- Preparation of custom screens (see Refinement)

- Optimization of the crystal quality using a microfluidic chip (see MPCS)

- Optimization of the crystal growth using seeds (see above)

- Optimization of the crystal conditions using additives (see above)