Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography.
Pedram MehrabiSihyun SungDavid von StettenAndreas PresterCaitlin E HattonStephan Kleine-DöpkeAlexander BerkesGargi GoreJan-Philipp LeimkohlHendrik SchikoraMartin KolleweHolger RohdeMatthias WilmannsFriedjof TellkampEike-Christian SchulzPublished in: Nature communications (2023)
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.
Keyphrases
- high throughput
- high resolution
- klebsiella pneumoniae
- electron microscopy
- room temperature
- ionic liquid
- single cell
- escherichia coli
- protein protein
- binding protein
- single molecule
- gram negative
- multidrug resistant
- molecular dynamics
- amino acid
- small molecule
- metabolic syndrome
- convolutional neural network
- blood glucose
- mass spectrometry
- electron transfer
- insulin resistance
- glycemic control