User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy

Summary

Single-particle cryo-electron microscopy demands a suitable software package and user-friendly pipeline for high-throughput automatic data acquisition. Here, we present the application of a fully automated image acquisition software package, Latitude-S, and a practical pipeline for data collection of vitrified biomolecules under low-dose conditions.

Abstract

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions.

It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

Introduction

Single-particle cryo-EM has become a mainstream structural biology technique for high-resolution structure determination of biological macromolecules¹. Single-particle reconstruction depends on acquiring a vast number of micrographs of vitrified samples to extract two-dimensional (2D) particle images, which are then used to reconstruct a three-dimensional (3D) structure of a biological macromolecule^2,3. Before the development of DEDs, the resolution achieved from single-particle reconstruction ranged between 4 and 30 Å^4,5. ....

Protocol

NOTE: Three important steps are required for cryo-EM data collection: 1. cryo-EM grid preparation, 2. calibration and alignment of the microscope, 3. automatic data collection (Figure 1). Furthermore, automated data collection is subdivided into a. suitable area selection, b. optimization of Latitude-S, c. start automatic hole selection, and d. start automatic data acquisition (Figure 1).

1. Cryo-EM grid preparation and sample loading for automatic data acquisition

1. Clean the grids using a glow discharger and vary the glow discharge parameters based on experimental requi....

Results

In the current pandemic situation, cryo-EM plays a key role in characterizing the structures of various proteins from SARS-CoV-2^26,27,28,29, which may help develop vaccines and drugs against the virus. There is an urgent need for fast-paced research efforts with limited human resources to combat the coronavirus disease of 2019. Data acquisition in single-particle cryo-EM is a time-consuming but.......

Discussion

Latitude-S is an intuitive user interface, which provides an environment to automatically set up and collect thousands of high-resolution micrographs or movie files in two days. It provides easy navigation across the grids and maintains the position of the microscope stage while it moves from low magnification to high magnification. Each step of data acquisition with Latitude-S is time-efficient, with features such as a simple user interface, fast streaming of data at up to 4.5 GB/s speed, and simultaneous display of dat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blotting paper	Ted Pella, INC.	47000-100	EM specimen preparation item
Capsule	Thermo Fisher Scientific	9432 909 97591	EM specimen preparation unit
Cassette	Thermo Fisher Scientific	1020863	EM specimen preparation unit
C-Clip	Thermo Fisher Scientific	1036171	EM specimen preparation item
C-Clip Insertion Tool	Thermo Fisher Scientific	9432 909 97571	EM specimen preparation tool
C-Clip Ring	Thermo Fisher Scientific	1036173	EM specimen preparation item
EM grid (Quantifoil)	Electron Microscopy Sciences	Q3100AR1.3	R 1.2/1.3 300 Mesh, Gold
Glow discharge Machine	Quorum	N/A	Quorum GlowQube glow discharge machine
K2 DED	Gatan Inc.	N/A	Cryo-EM data collection device (Camera)
Latitude S Software	Gatan Inc.		Imaging software
Loading station	Thermo Fisher Scientific	1130698	EM specimen preparation unit
Talos 200 kV Arctica	Thermo Scientific™	N/A	Cryo-Electron Microscope
Vitrobot Mark IV	Thermo Fisher Scientific	N/A	EM specimen preparation unit