Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography
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Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography. / Mehrabi, Pedram; Sung, Sihyun; von Stetten, David; Prester, Andreas; Hatton, Caitlin E; Kleine-Döpke, Stephan; Berkes, Alexander; Gore, Gargi; Leimkohl, Jan-Philipp; Schikora, Hendrik; Kollewe, Martin; Rohde, Holger; Wilmanns, Matthias; Tellkamp, Friedjof; Schulz, Eike C.
in: NAT COMMUN, Jahrgang 14, Nr. 1, 25.04.2023, S. 2365.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography
AU - Mehrabi, Pedram
AU - Sung, Sihyun
AU - von Stetten, David
AU - Prester, Andreas
AU - Hatton, Caitlin E
AU - Kleine-Döpke, Stephan
AU - Berkes, Alexander
AU - Gore, Gargi
AU - Leimkohl, Jan-Philipp
AU - Schikora, Hendrik
AU - Kollewe, Martin
AU - Rohde, Holger
AU - Wilmanns, Matthias
AU - Tellkamp, Friedjof
AU - Schulz, Eike C
N1 - © 2023. The Author(s).
PY - 2023/4/25
Y1 - 2023/4/25
N2 - 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.
AB - 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.
KW - Crystallography/methods
KW - Proteins/chemistry
KW - Temperature
KW - Humidity
KW - Crystallography, X-Ray
U2 - 10.1038/s41467-023-37834-w
DO - 10.1038/s41467-023-37834-w
M3 - SCORING: Journal article
C2 - 37185266
VL - 14
SP - 2365
JO - NAT COMMUN
JF - NAT COMMUN
SN - 2041-1723
IS - 1
ER -