Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Pedram Mehrabi (), Sihyun Sung, David Stetten, Andreas Prester, Caitlin E. Hatton, Stephan Kleine-Döpke, Alexander Berkes, Gargi Gore, Jan-Philipp Leimkohl, Hendrik Schikora, Martin Kollewe, Holger Rohde, Matthias Wilmanns, Friedjof Tellkamp () and Eike C. Schulz ()
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Pedram Mehrabi: Institute for Nanostructure and Solid-State Physics, Universität Hamburg
Sihyun Sung: European Molecular Biology Laboratory, Hamburg Unit
David Stetten: European Molecular Biology Laboratory, Hamburg Unit
Andreas Prester: University Medical Center Hamburg-Eppendorf (UKE)
Caitlin E. Hatton: Institute for Nanostructure and Solid-State Physics, Universität Hamburg
Stephan Kleine-Döpke: Institute for Nanostructure and Solid-State Physics, Universität Hamburg
Alexander Berkes: Institute for Nanostructure and Solid-State Physics, Universität Hamburg
Gargi Gore: Institute for Nanostructure and Solid-State Physics, Universität Hamburg
Jan-Philipp Leimkohl: Max Planck Institute for the Structure and Dynamics of Matter
Hendrik Schikora: Max Planck Institute for the Structure and Dynamics of Matter
Martin Kollewe: Max Planck Institute for the Structure and Dynamics of Matter
Holger Rohde: University Medical Center Hamburg-Eppendorf (UKE)
Matthias Wilmanns: European Molecular Biology Laboratory, Hamburg Unit
Friedjof Tellkamp: Max Planck Institute for the Structure and Dynamics of Matter
Eike C. Schulz: Institute for Nanostructure and Solid-State Physics, Universität Hamburg

Nature Communications, 2023, vol. 14, issue 1, 1-9

Abstract: Abstract We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

Date: 2023
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DOI: 10.1038/s41467-023-37834-w

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