In Situ Melting and Revitrification as an Approach to Microsecond Time-Resolved Cryo-Electron Microscopy

TL;DR

This paper introduces a novel microsecond time-resolved cryo-electron microscopy technique using in situ laser melting and rapid revitrification to observe protein dynamics in their native liquid state.

Contribution

It presents a new method combining laser-induced melting and quick revitrification to achieve microsecond temporal resolution in cryo-EM.

Findings

Demonstrated viability of the melting and revitrification approach

Achieved microsecond time resolution in cryo-EM

Captured transient protein configurations after damage

Abstract

Proteins typically undergo conformational dynamics on the microsecond to millisecond timescale as they perform their function, which is much faster than the time-resolution of cryo-electron microscopy and has thus prevented real-time observations. Here, we propose a novel approach for microsecond time-resolved cryo-electron microscopy that involves rapidly melting a cryo specimen in situ with a laser beam. The sample remains liquid for the duration of the laser pulse, offering a tunable time window in which the dynamics of embedded particles can be induced in their native liquid environment. After the laser pulse, the sample vitrifies in just a few microseconds, trapping particles in their transient configurations, so that they can subsequently be characterized with conventional cryo-electron microscopy. We demonstrate that our melting and revitrification approach is viable and affords microsecond time resolution. As a proof of principle, we study the disassembly of particles after they incur structural damage and trap them in partially unraveled configurations.