Journal Article

PUBDB-2023-02014

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Mehrabi, P. (Corresponding author)CFEL*MPG*Extern* ; Sung, S.Extern*XFEL.EU* ; Stetten, D. v.EMBL* ; Prester, A. ; Hatton, C. E. ; Kleine-Döpke, S. ; Berkes, A. ; Gore, G. ; Leimkohl, J.-P.CFEL*MPG* ; Schikora, H.CFEL*MPG* ; Kollewe, M.CFEL*MPG* ; Rohde, H. ; Wilmanns, M. ; Tellkamp, F. (Corresponding author)CFEL*MPG* ; Schulz, E. (Corresponding author)Extern*

2023
Nature Publishing Group UK [London]

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Please use a persistent id in citations: doi:10.1038/s41467-023-37834-w  doi:10.3204/PUBDB-2023-02014

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.

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Contributing Institute(s):

1. Forschungsgruppe für strukturelle Dynamik (MPSD)
2. MPSD (CFEL-SSUMP)
3. EMBL (EMBL)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P13 (PETRA III)
2. PETRA Beamline P14 (PETRA III)

Appears in the scientific report 2023

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