Near-atomic resolution reconstructions from in situ revitrified cryo samples

被引：7

作者：

Bongiovanni, Gabriele ^{[1
]}

Harder, Oliver F. ^{[1
]}

Voss, Jonathan M. ^{[1
]}

Drabbels, Marcel ^{[1
]}

Lorenz, Ulrich J. ^{[1
]}

机构：

[1] Ecole Polytechn Fed Lausanne EPFL, Lab Mol Nanodynam, CH-1015 Lausanne, Switzerland

来源：

ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY | 2023年 / 79卷

基金：

瑞士国家科学基金会; 欧洲研究理事会;

关键词：

microsecond melting and revitrification; microsecond time-resolved cryo-EM; protein dynamics; time-resolvedelectron microscopy; preferential orientation; DYNAMICS; EM;

D O I：

10.1107/S2059798323003431

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in the liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. Two alternative implementations of the technique have previously been described, using either an optical microscope or performing revitrification experiments in situ. Here, it is shown that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within the spatial resolution. Interestingly, it is observed that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.

引用

页码：473 / 478

页数：6