Title

Development and Optimization of the Protocol for the SSRL Plate for In-situ Crystallization and Automated Diffraction

Abstract

In the discipline of protein crystallography it is common for crystals to be cryogenically flash frozen in liquid nitrogen for shipping, storage and data collection. But cryogenically cooling crystals with liquid nitrogen can sometimes change the conformation of proteins and can damage the crystal structure. Therefore the Plate for In-situ Crystallization and Automated Diffraction Data Collection (SSRL-ICP) was developed to ship, store and collect data from protein crystals at ambient temperatures. Experiments were performed in order to develop a protocol for proper use and optimization of the SSRL-ICP. When using the SSRL-ICP for shipping the crystallization solution should be mixed with agarose to obtain a final concentration of 0.6% agarose in order to gel the liquid in the plate to prevent spillage and sloshing. Crystals should be stored in loops or on grids and the SSRL-ICP should be sealed with tape in order to prevent drying. Some crystals can last for up to 6 days*, others have lasted up to 3 days* when stored in the plate with agarose and the crystallization solution. The SSRL-ICP can be used to grow, using grids, and store crystals harvested from other crystallization plates at ambient temperatures.The SSRL-ICP enables crystallization and remote access diffraction data collection using the same sample storage container, avoiding manual manipulation of delicate crystals.The SSRL-ICP is compatible with robotic sample mounting using the Stanford Automated Mounter at SSRL crystallography beamlines for data collection at ambient temperatures and controlled humidity. Testing for the custom engineered shipping container is still ongoing and is scheduled to be completed by the end of the year.

*Crystals may have lasted longer but they could not be stored/tested

further due to limited time and materials shortage.

Mentor

Silvia Russi, Aina Cohen

Lab site

SLAC National Accelerator Laboratory (SLAC)

Funding Acknowledgement

The 2018 STEM Teacher and Researcher Program and this project have been made possible through support from Chevron (www.chevron.com), the National Marine Sanctuary Foundation (www.marinesanctuary.org), the National Science Foundation through the Robert Noyce Program under Grant #1836335 and 1340110, the California State University Office of the Chancellor, and California Polytechnic State University in partnership with SLAC National Accelerator Laboratory. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the funders.

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URL: https://digitalcommons.calpoly.edu/star/506