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Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton
Published on: February 25, 2021
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Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography
Pedram Mehrabi1,2, Sihyun Sung3, David von Stetten3
1Institute for Nanostructure and Solid-State Physics, Universität Hamburg, Hamburg, Germany. pedram.mehrabi@uni-hamburg.de.
Nature Communications
|May 15, 2023
Summary
The spitrobot enables millisecond-resolution cryo-trapping of protein reactions, allowing researchers to capture fleeting intermediate states. This new tool provides unprecedented insight into crucial catalytic events in enzymes.
Area of Science:
- Structural Biology
- Biochemistry
- Crystallography
Background:
- Understanding enzyme mechanisms requires observing transient intermediates.
- Current methods often lack the temporal resolution to capture these states.
Purpose of the Study:
- Introduce the spitrobot, a novel device for rapid reaction quenching in protein crystals.
- Enable millisecond-timescale cryo-trapping of reaction intermediates.
Main Methods:
- The spitrobot uses an electropneumatic piston to plunge protein crystals into liquid nitrogen after reaction initiation.
- Reactions are initiated using the liquid application method (LAMA) within a controlled environment.
- Automated high-magnification imaging captures states before and after reaction initiation.
Main Results:
- Demonstrated successful cryo-trapping of ligand binding (glucose, 2,3-butanediol, avibactam, ampicillin) in enzyme microcrystals.
- Captured reaction intermediates and conformational changes in tryptophan synthase crystals.
- Achieved millisecond-time resolution for trapping catalytic events.
Conclusions:
- The spitrobot facilitates the study of dynamic processes in protein crystals with millisecond resolution.
- Enables detailed mechanistic investigations of enzyme catalysis and ligand interactions.
- Compatible with high-throughput structural biology workflows.
