8awv

Publication Abstract from PubMed

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.

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography.,Mehrabi P, Sung S, von Stetten D, Prester A, Hatton CE, Kleine-Dopke S, Berkes A, Gore G, Leimkohl JP, Schikora H, Kollewe M, Rohde H, Wilmanns M, Tellkamp F, Schulz EC Nat Commun. 2023 Apr 25;14(1):2365. doi: 10.1038/s41467-023-37834-w. PMID:37185266^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.