Devitrification reduces beam-induced movement in cryo-EM.

Jan-Philip Wieferig,Deryck J Mills,Werner Kühlbrandt

doi:10.1107/s2052252520016243

Jan-Philip Wieferig, Deryck J Mills + Show 1 more

Open Access

https://doi.org/10.1107/s2052252520016243

Copy DOI

Journal: IUCrJ	Publication Date: Mar 1, 2021
Citations: 18	License type: CC BY 4.0

Affiliation: Max Planck Institute of Biophysics

Abstract

As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å-2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.

Highlights

The new direct electron detectors and image-processing programs have resulted in a ‘resolution revolution’ in electron cryo-microscopy
Some motion is thought to be caused by the vitrified water itself (Russo & Passmore, 2014), but the Brownian-type movement transmitted to the protein by the surrounding water molecules in the highly viscous liquid film is not expected to contribute much to beam-induced motion (McMullan et al, 2015)
We examine the potential of devitrification for high-resolution single-particle cryo-EM, taking advantage of the speed and sensitivity of direct electron detectors that were not available at the time of the original experiments in 1994

Summary

Introduction

The new direct electron detectors and image-processing programs have resulted in a ‘resolution revolution’ in electron cryo-microscopy (cryo-EM; Kuhlbrandt, 2014). The enhanced detective quantum efficiency of direct detectors greatly improves the signal-to-noise ratio, and their high readout speed allows image acquisition as stacks of dose-fractionated movie frames (McMullan et al, 2014). Evaluation of movie stacks identified beam-induced specimen movement as a main limiting factor in cryo-EM (Henderson et al, 2011; Brilot et al, 2012; Campbell et al, 2012). Some motion is thought to be caused by the vitrified water itself (Russo & Passmore, 2014), but the Brownian-type movement transmitted to the protein by the surrounding water molecules in the highly viscous liquid film is not expected to contribute much to beam-induced motion (McMullan et al, 2015)

Methods

Results

Conclusion