Name: user-friendly, high-throughput, and fully automated data acquisition software for single-particle cryo-electron microscopy
Uploaded: 2021-07-29T14:45:00
Description: Watch this Scientific Journal Video about User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy at JoVE.com

We acknowledge Department of Biotechnology, Department of Science and Technology (DST) and Science, and Ministry of Human Resource Development (MHRD), India, for funding and the cryo-EM facility at IISc-Bangalore. We acknowledge DBT-BUILDER Program (BT/INF/22/SP22844/2017) and DST-FIST (SR/FST/LSII-039/2015) for the National Cryo-EM facility at IISc, Bangalore. We acknowledge financial support from the Science and Engineering Research Board (SERB) (Grant No.-SB/S2/RJN-145/2015, SERB-EMR/2016/000608 and SERB-IPA/2020/000094), DBT (Grant No. BT/PR25580/BRB/10/1619/2017). We thank Ms. Ishika Pramanick for preparing cryo-EM grids, cryo-EM data collection, and preparing the Table of Materials. We also thank Mr. Suman Mishra for cryo-EM image processing and for helping us to prepare the figures. We thank Prof. Raghavan Varadarajan for helping us to obtain the purified spike protein sample for this study.

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 f...

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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...

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 macromolecule2,3. Before the development of DEDs, the resolution achieved from single-particle reconstruction ranged between 4 and 30 &#197;4,5. Recently, the achievable resolution from single-particle cryo-EM has reached beyond 1.8 &#197;6. DED and automated data acquisition software have been major contributors to this resolution revolution7, where human intervention for data collection is minimal. Generally, cryo-EM imaging is performed at low electron dose rates (20-100 e/&#197;2) to minimize electron beam-induced radiation damage of biological samples, which contributes to the low signal-to-noise ratio (SNR) in the image. This low SNR impedes the characterization of the high-resolution structures of biological macromolecules using single-particle analysis.
The new generation electron detectors are CMOS (complementary metal-oxide-semiconductor)-based detectors, which can overcome these low SNR-related obstacles. These direct detection CMOS cameras allow fast readout of the signal, due to which the camera contributes better point spread function, suitable SNR, and excellent detective quantum efficiency (DQE) for biological macromolecules. Direct detection cameras offer high SNR8 and low noise in the recorded images, resulting in a quantitative increase in the detective quantum efficiency (DQE)-a measure of how much noise a detector adds to an image. These cameras also record movies at the speed of hundreds of frames per second, which enables fast data acquisition9,10. All these characteristics make fast direct detection cameras suitable for low-dose applications.
Motion-corrected stack images are used for data processing to calculate 2D classification and reconstruct a 3D density map of macromolecules by using various software packages such as RELION11, FREALIGN12, cryoSPARC13, cisTEM14, and EMAN215. However, for single-particle analysis, an enormous dataset is required to achieve a high-resolution structure. Therefore, automatic data acquisition tolls are highly essential for data collection. To record large cryo-EM data sets, several software packages have been used over the past decade. Dedicated software packages, such as AutoEM16, AutoEMation17, Leginon18, SerialEM19, UCSF-Image420, TOM221, SAM22, JAMES23, JADAS24, EM-TOOLS, and EPU, have been developed for automated data acquisition.
These software packages use routine tasks to find hole positions automatically by correlating the low-magnification images to high-magnification images, which assists in identifying holes with vitreous ice of appropriative ice thickness for image acquisition under low-dose conditions. These software packages have reduced the number of repetitive tasks and increased the throughput of the cryo-EM data collection by acquiring a vast amount of good-quality data for several days continuously, without any interruption and the physical presence of the operator. Latitude-S is a similar software package, which is used for automatic data acquisition for single-particle analysis. However, this software package is only suitable for K2/K3 DEDs and is provided with these detectors.
This protocol demonstrates the potential of Latitude-S in the automated image acquisition of SARS-CoV-2 spike protein with a direct electron detector equipped with a 200 keV cryo-EM (see the Table of Materials). Using this data collection tool, 3,000 movie files of SARS-CoV-2 spike protein are automatically acquired, and further data processing is carried out to obtain a 3.9-4.4 &#197; resolution spike protein structure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blotting paper</td>
			<td>Ted Pella, INC.</td>
			<td>47000-100</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>Capsule</td>
			<td>Thermo Fisher Scientific</td>
			<td>9432 909 97591</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>1020863</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>C-Clip</td>
			<td>Thermo Fisher Scientific</td>
			<td>1036171</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>C-Clip Insertion Tool</td>
			<td>Thermo Fisher Scientific</td>
			<td>9432 909 97571</td>
			<td>EM specimen preparation tool</td>
		</tr>
		<tr>
			<td>C-Clip Ring</td>
			<td>Thermo Fisher Scientific</td>
			<td>1036173</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>EM grid (Quantifoil)</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q3100AR1.3</td>
			<td>R 1.2/1.3 300 Mesh, Gold</td>
		</tr>
		<tr>
			<td>Glow discharge Machine</td>
			<td>Quorum</td>
			<td>N/A</td>
			<td>Quorum GlowQube glow discharge machine</td>
		</tr>
		<tr>
			<td>K2 DED</td>
			<td>Gatan Inc.</td>
			<td>N/A</td>
			<td>Cryo-EM data collection device (Camera)</td>
		</tr>
		<tr>
			<td>Latitude S Software</td>
			<td>Gatan Inc.</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Loading station</td>
			<td>Thermo Fisher Scientific</td>
			<td>1130698</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>Talos 200 kV Arctica</td>
			<td>Thermo Scientific&#8482;</td>
			<td>N/A</td>
			<td>Cryo-Electron Microscope</td>
		</tr>
		<tr>
			<td>Vitrobot Mark IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>EM specimen preparation unit</td>
		</tr>
	</tbody>
</table>

user-friendly, high-throughput, and fully automated data acquisition software for single-particle cryo-electron microscopy

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.

In the current pandemic situation, cryo-EM plays a key role in characterizing the structures of various proteins from SARS-CoV-226,27,28,29, 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...

Watch this Scientific Journal Video about User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy at JoVE.com

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

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.

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for ...

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