CNRS - AIX MARSEILLE UNIV: UMR7257

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Structural Biology

Crystallogenesis and crystallography

Heads Gerlind SULZENBACHER, Véronique ZAMBONI

The crystallization facility offers a state-of-the-art automated infrastructure for the screening of hundreds of conditions with very small amounts of biological sample and ensures the observation of results issuing from crystallization trials. This service is equipped with a nano-volume crystallization robot for the screening and optimization steps, and two cabinets for visualization and storage. A second liquid handling robot is dedicated to the filling of reservoir solution from the screening kits and the custom design of optimization grids. Assessment of crystal quality and optimal cryo-conditions to be employed are carried out with a complete X-ray diffraction equipment. Data collection of full data sets is also possible.

Notice: The most crucial factor is the quality and purity of your protein sample. We strongly recommend that you check the quality of your protein sample before you plan for an experiment.

Accessibility

Academics and industrials

How to apply?

Complete the proposal from
http://frisbi.eu/Submit-a-proposal
and check “Marseille” for the “High throughput crystallization” item

Specific equipments

  • 1 Mosquito robot (TTP Labtech); 1 Genesis liquid handling robot (Tecan)
  • 2 RockImager (Formulatrix) cabinets at 20°C and 8°C, equipped with UV modules and the CRIMS software
  • 1 X-ray microfocus generator (Bruker AXS MICROSTAR) equipped with Osmic mirrors and two Mar345 image plates

Cost

To be defined based on the service requested.

Publications

  • Jabafi I, Selisko B, Coutard B, De Palma AM, Neyts J, Egloff MP, Grisel S, Dalle K, Campanacci V, Spinelli S, Cambillau C, Canard B, Gruez A (2007) Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase. Acta Crystallogr. F63, 495-8.
  • Vincentelli R, Bignon C, Gruez A, Canaan S, Sulzenbacher G, Tegoni M, Campanacci V, Cambillau C (2003) Medium-scale structural genomics: strategies for protein expression and crystallization. Acc. Chem. Res. 36, 165-72.
  • Sulzenbacher G, Gruez A, Roig-Zamboni V, Spinelli S, Valencia C, Pagot F, Vincentelli R, Bignon C, Salomoni A, Grisel S, Maurin D, Huyghe C, Johansson K, Grassick A, Roussel A, Bourne Y, Perrier S, Miallau L, Cantau P, Blanc E, Genevois M, Grossi A, Zenatti A, Campanacci V, Cambillau C (2002) A medium-throughput crystallization approach. Acta Crystallogr. D58, 2109-15.

For more information ...

For the preliminary screening step, we use a large range of commercial crystallization kits, including some specific kits, in 96-well plates. For each condition, 100-300 nl of protein solution are mixed with 100 nl of a 100-150 µl reservoir solution. Usually, we test 3 different protein concentrations (>3 mg/ml) in varying the volume (100, 200 and 300 nl) of the protein sample. For an initial screening we recommend to assay 2 to 3 different kits. This procedure requires at least 70 µl of protein sample per plate. Various plate types can be provided to users.

For optimization of the initial hits, we have designed a 8x8 grid screen which permits to vary two parameters (e.g., pH value versus precipitant concentration). Each grid requires 10 µl to 48 µl of protein sample for the screening of one or three different protein concentrations. In most cases, several rounds of optimization are required before identification of the best conditions. This step can also be performed manually in 24-well Linbro plates.

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