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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The authors have no competing or financial conflicts of interest to declare.

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 ...

Automated Data Acquisition Software is extremely necessary for collecting huge amount of cryo data for structural characterization of biological macromoleculess. Automated Data Acquisition Software package was used here to characterize the structure of various biological macromoleculess, which are directly associated with pathogen biology. For example, mycobacterium, HIV and SASCO 2.To begin with, start Latitude-S Automated Data Acquisition Software by clicking on DigitalMicrograph from the start menu. Next, select the Technique Manager, and then the Latitude-S icon for Single-Particle Automated Data Collection. To create a session based on the previous session settings, check the based on prior session checkbox in the palette, and then select the new button or to continue an existing session, press the continue button.To start an entirely new session, click on the new tab in the palette. Choose the folder containing the session to be continued and select the folder to save the data. Next, click on the setting icon and in the managed state explore box that appears, add state, set the TEM condition, camera condition, and image or stock option.And then, name the state or open the state setup summary to open the save settings. Next, configure the Atlas state by clicking on the Atlas state palette and set the parameters for magnification, illumination conditions, and camera exposure time, and click on next to move to the next state. Then configure the grid state by clicking on the grid state palette, set the parameters for microscope imaging optics, illumination conditions, and camera exposure time, and click on next.Configure the hole state by clicking on the hole palette and set the imaging optics, illumination conditions, binning and camera exposure time parameters. After clicking on next, configure the next focus state by clicking on the focus palette and set the microscope settings for imaging optics, illumination conditions, binning and camera exposure time. Then focus on the amorphous carbon area near the hole and click next to move to the next state.Configure the data state by clicking on the data palette and set the parameters for microscope settings, then click on next. Click on the focus configuration palette specifying the range of defocus values and the step size in the given tab and press the next button to move to the next step. Focus on required features on the grid and click on the capture button.Then position the red cross mark on the same feature on each image of different states. Start with Focus, Data and Hole states, because the field of view is bigger than the Atlas and Grid states. Next, zoom on Atlas and Grid states to position the red cross mark on the same feature.To calculate the positions and offset between each of five different states and reflect the positions and offsets to the output window, click the calculate button. Click on capture palette and choose the size of the Atlas to cover the entire grid or part of the grid based on the requirement. To capture the Atlas, navigate on the Atlas and select the grid square based on the ice thickness.Once the desired grid squares are selected, click on the schedule button and observe the tiles in the grid square fill up as each grid square is captured. Once the schedule button is clicked, select a representative hole in the grid square by adding the hole's position. Once the hole image is acquired, define the data and focus positions and save the layout as a template.Click on auto find, enter the hole size, and click on the find button in the program to automatically find the holes based on the diameter. Set up the intensity to remove the holes from the grid square and ice contamination. And after adding the selected and yellow mark tools through auto find, click on the schedule button in latitude tasks.As per the initial manual screening of motion corrected micrographs using cisTEM software, most of the data were found to be within the desired signal range. Additionally, images collected at defocus ranges, were also manually checked by cisTEM. The spike particles were manually picked to calculate the 2D class averages for structural visualization, which strongly suggested, but high-resolution structural characterization was possible using the respective data set.The 3D classification indicated that Spike Protein has 1RBD in up open confirmation and all other RBD in down close confirmation. The 1RBD up open confirmations of the Spike Protein were reconstructed using C1 symmetry. The Spike Protein map is represented in the side top and bottom views.And the EM map is fitted with atomic structure for better visualization of the side chains. The RBD down close confirmations were refined with C3 symmetry and the spike 3D refined model and EM map, fitted with atomic structure as shown. And intermediate confirmation of the Spike Protein identified as the S2 sub domain indicated the side chains of individual amino acid residues.The results suggested that Latitude-S can collect high resolution cryo EM data of biological macromolecules. This Automated Data Collection Software could be used to any other electron microscopy system to collect data automatically.

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JoVE Journal

Immunology and Infection

3.2K Görüntüleme.  Indian Institute of Science. Otomatik Veri Toplama Yazılımı, biyolojik makromoleculess'ın yapısal karakterizasyonu için büyük miktarda kriyo verisi toplamak için son derece gereklidir. Otomatik Veri Toplama Yazılımı paketi, patojen biyolojisi ile doğrudan ilişkili olan çeşitli biyolojik makromoleculess'in yapısını karakterize etmek için kullanılmıştır. Örneğin, mikobakteri, HIV ve SASCO 2.Başlangıç olarak, başlat menüsünden DigitalMicrograph'a tıklayarak Latitude-S Otomatik Veri To...