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Protein Crystallisation

 

Crystal Screening - Nanoliter volumes

  • The Facility has access to a high-throughput macromolecular crystallisation platform. Liquid handling robots are available for preparation of nanoliter crystallisation drops in 96-well format, including a 96-well pipetting robot (Hydra II) for filling up reservoir wells and two Mosquito's for creating nanoliter crystallisation drops. Preparation of a single 96-well plate takes generally about 5 minutes.

  • Six different crystallisation screens - 96 conditions each - can be used to search for crystallization conditions.

  • About 20 microliter of a solution containing at least 5 mg/ml protein is sufficient to set up a single 96-well plate.

  • Crystallisation plates can be stored either at 4 C in the RockImager or at 20 in the CrystalFarm. Both systems have a capacity to store > 200 plates. Images of all single drops are taken according to a specified time schedule and can be inspected through a web application, which allows easy monitoring of the experiments by the users from any computer with internet access.

HydraII Mosquito's Crystal Farm Crystal Farm software Crystals!

 

Crystal Screening - Microfluidics

  • The crystallisation platform also comprises a TOPAZ microfluidics system (Fluidigm) for screening crystallization conditions in 96-well format. The system is based on free interface diffusion (FID) between protein solution and crystallant solution in chambers at nano scale.

  • Due to the small size of the chambers, only 2.0 microliter of protein solution (> 10 mg/ml) is sufficient for screening 96 conditions. Four proteins can be tested within one 4.96 chip. The experiment is finished after 7 days.

  • Images are taken by the AutoInspex station (Fluidigm) and are ranked according to the presence of crystalline objects by the analysis software.

TOPAZ Crystallizer
TOPAZ 4.96-microfluidics chip

Crystal Optimisation

  • Initial crystallisation conditions can be optimized in nanoliter or microliter drops, to improve crystals in terms of size, quantity etc. A Formulator (Formulatrix) has been acquired to prepare optimisation screens in 96-well format. The Formulator is a microfluidics-based device that is capable of dispensing microliter volumes in 96-well plates from 16 different solutions in an automated fashion. This facilitates the preparation and accuracy of optimisation of crystallisation conditions. 

  • When suitable crystals are obtained for x-ray diffraction analysis, the applicant can decide to collaborate with any crystallography group to collect diffraction data and solve the structure. Solving crystal structures is beyond the scope of the facility.

NKI Protein Facility                                                                                                                                                                                                         Last updated: 18 April 2012

NKI-AvL

Plesmanlaan 121

1066 CX Amsterdam

The Netherlands