ANOXTAL : Automated Crystallization of Proteins under Anoxic Condition

Presentation

An installation allowing crystallization of proteins under anoxic conditions for their structural analysis. Automation allows high throughput screening of standard conditions of crystallization

Typical Samples :

• Oxygen-sensitive metallo-proteins
• Proteins that can form disulfide bonds by oxidation
• Radical chemistry-based proteins

The AnoXtal platform consists of two main instruments :

• An automated crystallization robot : Gryphon (ArtRobbins Inst.) ,
• A semi-automatic device to visualize crystallization plates : CrysCam (ArtRobbins Inst.) .

This system is installed in a temperature-regulated (20.5°C) glove box (Jacomex), under an anoxic atmosphere.
As member of the “Partnership for Structural Biology” (PSB), we work in collaboration with the HTX lab from EMBL-Grenoble, France.

Keywords

Glove box, Anaerobe, Crystallization, Robot, Nanovolume

Dedicated staff

Juan C. Fontecilla-Camps : manager,
Claudine Darnault, Lydie Martin, Xavier Vernède : management of the platform, technical and scientifical expertise, quality control, users ‘contact.

Temporary staff

Laura Zeppieri

Specific equipment

• The Jacomex glove box :
  The Jacomex glove box is an enclosure of 2m3 which over-pressured atmosphere(between 0.6 to 5 mBar) is controlled in oxygen concentration (<10ppm), hygrometry (>45%) and temperature (20.5°C +/-0.2°C). It allows the crystallization of oxygen-sensitive proteins.

• GRYPHON, the crystallization robot :
  The crystallization robot automatically performs the preparation of crystallization nano-drops in 96-well plates, using the vapour-diffusion technique with sitting-drops,
  A total of 1250 different conditions of crystallization (13 plates of 96 wells) from commercial screens are proposed,
  The protein is dispensed using a nano-needle delivering volumes between 200 and 400 nL,
  Three protein samples can be tested per crystallization plate,
  Setting up a plate (96 wells) with 3 protein samples (288 drops) takes about 10 minutes.

• CrysCam, the device for visualization of crystallization plates CrysCam :
  The crystallization plates are stocked in a glove box at 20.5°C. The CrysCam carries out a semi-automatic reading of the drops in the crystallization plates.
  Reading the plates holds at day (D) 1(+1), D3 (+1), D7 (+/-2), D15(+/-3), D32 (+/-4), then 2 to 3 months after drops setting. Others reading times can be envisaged upon request, at ordering time.
  The images at day (D)3 (+1), D7 (+/-2), D15(+/-3), D32 (+/-4), then 2 to 3 months after the drops formation will be sent by a secured system of sending large files (to be downloaded within days after reception of the link by mail).
  Plates will be kept in a glove box 2 to 3 months after drop setting. The images will not be kept by the platform.

Proposed crystallization conditions :

Crystallization screens available on the plateform :

• 1- Crystal Screen I / Crystal Screen II (HAMPTON)
• 2- Crystal Screen Lite / Crystal Screen Peg-ion (HAMPTON)
• 3- Crystal Screen MembFac / Crystal Screen Natrix (HAMPTON)
• 4- Crystal Screen Ammonium Sulfate/Screen Malonate/ Quick Screen/ NaCl (HAMPTON)
• 5- PEG 6000 / et MPD / BASIC 3 / PEG LiCl (HAMPTON et JENABIOSCIENCE POUR BASIC3)
• 6- INDEX (HAMPTON)
• 7- SALT RX I /II (HAMPTON)
• 8- WIZARD I / II (JENA BIOSCIENCE)
• 9- THE PEG (PEG SUITE) (QIAGEN)
• 10- JCSG + (QIAGEN)
• 11- PACT (QIAGEN)
• 12- ANION SUITE (QIAGEN)
• 13- CATION SUITE (QIAGEN)

More details on the distribution of conditions in the plates are available :

• For screens 1 to 6 (click on the name of the plate or the number of the well to see the details) .
• For screen 7 to 13, click on numbers 7, 8, 9, 10, 11, 12 et 13.

Cost :

Please, contact Juan C. Fontecilla-Camps

Localisation

Institut de Biologie Structurale , room 272

How to make a request ?

Please use the order form that can be downloaded here.

Samples (to be provided by the applicant)

All information concerning your samples has to be mentioned in the order form.
The quality of the samples will be checked by the operator.
Each order should include :

• A picture of SDS/PAGE with colored protein bands,
• A chromatographic profile of the last purification step.

Sample specifications :

One to 3 samples can be tested per crystallization plate.
Samples to be tested have to be :

• Soluble ( not precipitated, not aggregated),
• delivered in a diluted buffer (50mM maximum) and low ionic strength in case of salt requirement,
• clearly identified :
  « Name of the applicant, name of the sample, protein concentration (mg/ml), date of sending, volume »

Filtration

Unless requested otherwise, the samples will be filtered by us (Millipore Ultrafree 0.45 micron) before crystallization. Please mention in the order form if your sample should not be filtered.

Volumes of samples required :

In addition to the theoretical volume necessary for the setting of a plate, an extra 20µl of protein solution will be needed, both for sample filtration and as a safety margin.

_These volumes are for 1 sample per plate.
Example : for one sample, 1 plate and 200nL volume drops, 50µL of proteins solution will be needed.

Sample storage

Upon reception, samples will be stored according to the conditions defined in the order form :

• In the glove box under an anoxic atmosphere at either 20°C or 4°C,
• Frozen at -20°C,
• Frozen at -80°C,
• Frozen in liquid nitrogen.
  No more protein than requested for the experiments should be sent. Any excedent will not be sent back to the user but will be destroyed instead.

Address where samples have to be sent

Juan C. FONTECILLA-CAMPS
PLATEFORME ANOXTAL
IBS/METALLO
EPN Campus
71, avenue des Martyrs
38044 GRENOBLE CEDEX 9
FRANCE

For sample shipping, the platform does not recommend the use of dry ice. A dry-shipper should be used instead in order to assure proper delivery of the protein sample. For a better reception, please inform Juan Fontecilla (link to the email address) of the day of the sending.

Reporting of results

• Service n°1 : a file containing raw images of the drops will be sent after each visualization.
• Service n°2 : service n° 1 + higher magnification images available on request.
• Service n°3 : Service n°2 + data reading assistance, interpretation and report.
  If crystals are obtained, the platform will provide all the information concerning their growth. However, we cannot guarantee that the crystal will be reproduced elsewhere.

Average time between application and experiment :

From two weeks to 3 months depending on the requests, to be specified when ordering.

Acknowledgments :

In the general case, users must aknowledge the platform used in their publications as follow :
"This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003)."
We thank C. Darnault, J.-C. Fontecilla, L. Martin, X. Vernede and Laura Zeppieri from the IBS platform "AnoXtal" for the “anaerobic crystallization assay experiments”.