Immunology and Infection
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User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy
Chapters
Summary July 29th, 2021
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.
Transcript
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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