Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Summary

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

Abstract

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

Introduction

The advent of direct electron detectors over the past decade^1,2,3 has spurred an exponential increase in the number of high-resolution structures of macromolecules and macromolecular assemblies solved using single-particle cryo-EM^4,5,6. Almost all purified macromolecular species are expected to be amenable to structure determination using cryo-EM, except for the smallest proteins ~10 kDa in size or below⁷. The amount of starting material ....

Protocol

1. Sample preparation

1. Use grids containing gold foil and gold grid support¹⁶ (see Table of Materials) because tilted data collection can accentuate beam-induced motion¹⁷.
   NOTE: For the present study, samples on grids were vitrified using the manual plunging and blotting technique¹⁸ in a humidified (greater than 80%) cold room (~4 °C).
2. Avoid using grids containing copper support and carbon foil or a continuous layer of amorphous carbon unless absolutely necessary, as these grids may lead to greater beam-induced motion

Results

DPS at 0.3 mg/mL was used to demonstrate imaging at 0°, 30°, and 60° tilts. Data from different tilt angles were collected on the same grid at different grid regions. CTF resolution fits for higher angle tilts tend to be poorer, as was the case when comparing the three datasets in this study. Figure 4 demonstrates comparative representative images and 2D classification averages. Although the protein concentration is unchanged across the different tilt angles, a higher tilt angle makes the im.......

Discussion

Preferred particle orientation caused by specimen adherence to the air-water interface is one of the last major bottlenecks to routine high-resolution structure determination using cryo-EM SPA^4,5,6. The data collection scheme presented here provides an easy-to-implement strategy for improving the orientation distribution of particles within a dataset. We note that the protocol requires no additional equipment or software and doe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryosparc Live v3.1.0+210216	Structura Biotechnology
DPS protein			Purification adapted from protocol described in K.Naydenova et al IUCrJ. 2019 Nov 1; 6(Pt 6): 1086–1098.
K2 Summit Direct Electron Detector	Gatan
Leginon software suite			C Suloway et al Journal of Structural Biology 151 (1): pp. 41-60.
Manual plunging device			Homemade guillotine-like device for vitrification of EM grids
Talos Arctica	FEI/Thermo Fisher
UltrAufoil R1.2/1.3 300 mesh grids	Quantifoil	N1-A14nAu30-01