Tutorial: Manually Curate Exposures

Manually Curate Exposures is an interactive job in cryoSPARC that enables a user to visually inspect and curate a set of micrographs or movies, using either population-level statistics and adjustable thresholds, or individual inspection of diagnostic plots.

This interactive job allows you to:

  • Load a stack of (i) micrographs, (ii) movies or (iii) micrographs and particles
  • View and sort the stack by certain metrics associated with the data you loaded, e.g., average defocus, astigmatism, CTF fit, motion trajectories and power scores relating to picked particles
  • Create and visualize interactive plots to view the distribution of micrographs/movies over different parameters, and identify outliers
  • Adjust thresholds to bulk-exclude micrographs based on the statistic of interest
  • View available CTF diagram (to inspect Thon rings), CTF fit, and rigid and local motion trajectory plots associated with an individual micrograph, all in one place
  • Accept or reject micrographs/movies from a stack, for further processing

When to use this job

The overall goal of this interactive job is to aid in cleaning up your data so that low-quality micrographs, and their associated particles, do not detract from achieving the highest possible resolution refined structures.

After pre-processing: After Full-Frame Motion Correction and CTF estimation, you can use Manually Curate Exposures to help you identify and exclude micrographs with sub-optimal characteristics, e.g., broken or thick ice, carbon edges, too much motion, etc.

After particle picking and local motion correction: Once you have picked particles and estimated location motion trajectories, you can use this information in Manually Curate Exposures to identify and exclude micrographs associated with those picks whose local motion trajectories deviate from the norm for your dataset. In this case, the job will output a filtered set of micrographs as well as particles.

Build and launch job

  1. Select Manually Curate Exposures from the Job Builder:

  1. Drag and drop the desired inputs from previous jobs. You can connect (a) micrographs only, (b) movies only, or (c) micrographs/movies and particles. In this example, we have connected micrographs that were already processed using Full-Frame Motion Correction and CTF Estimation, and particles from the output of a previously run Local Motion Correction job.
  2. There are no parameters to adjust for this interactive job, so simply click Queue to launch the job.

Overview mode: visualize and create plots for all micrographs

Overview and Individual mode: The default view is Overview. An explanation of the Individual view can be found below.

Table of exposures: On the left, exposures are listed in a table with index values, and summary statistics are available at the top of the table. In this case, we are looking at a total of 1536 micrographs.

Selected: At the start of the job, all loaded micrographs are 'Selected', as indicated at the top of the table and their values in blue in the table. Once we begin adjusting the interactive plot and thresholds, micrographs whose statistics do not fall within the selected thresholds will become un-selected, and their values will turn grey in the table.

Available columns: A number of columns are displayed depending on the type of input. For example, because we loaded micrographs and particles with local motion trajectories, we see columns displaying not only those statistics associated with micrographs (e.g., average defocus, astigmatism, CTF fit, etc.) but also those corresponding to the picks and local motion trajectories (pick CC, pick power, local motion distribution, etc.) Click on a statistic to sort the table.

Create plots: The remainder of the window is dedicated to an interactive plot. All micrographs in the table are displayed in the plot. The X-Axis and Y-Axis drop-down menus directly below allow you to select different statistics by which to plot the micrographs. Selecting a different value from the drop-down will automatically refresh the plot. Common plots include:

Average defocus (A)Astigmatism (A)General range of CTF parameters in the dataset
CTF Fit resolution (A)Total full-frame motion distance (pixels)Two most common metrics for filtering bad data - large motions or poor CTF resolution indicate problem during exposure
IndexPhase Shift (deg)Plot showing change of phase shift over time during data collection (NB: micrographs should be imported in chronological order for this to be correct)

Interact with plots: Hover over the plot area and a toolbar will appear at the top. These tools allow you to: download the plot as a PNG file, zoom, pan, and reset the axes. Note: Selections in the plot currently do NOT affect selection or acception/rejection of micrographs. Use the threshold sliders below the plot to make a selection and then the buttons above the plot to accept/reject/invert your selection.

Adjust thresholds to select micrographs: Once you have explored the distribution of micrographs on one or more plots and have identified outliers, you can adjust the various thresholds below the plot to filter the micrographs. The corresponding values in the table will turn grey. (Note: The plot does not currently reflect your selection - all micrographs will be shown on the plot.) For example, here we plotted CTF fit resolution (A) on both the x-axis and y-axis, and then adjusted the CTF fit resolution (A) slider to keep only those micrographs with a value of less than 8.48 A. In the table, 3 micrographs are greyed out, indicating they do not fall within the threshold values.

Accept selection: To keep all micrographs with CTF values falling within the desired range, click 'Accept Selection' at the top of the plot. Corresponding micrographs will then be highlighted green in the table.

Reject selection: Alternatively, you can reject micrographs with statistics falling within a certain range, following the same process as above, but click 'Reject selection' above the plot. Rejected micrographs will turn red in the table.

When you have finished inspecting and curating exposures, click Done to exit and complete the job. Note that all micrographs in green will be included in the micrographs_accepted output of the job, micrographs in red will be included in the micrographs_rejected output, but a selection (blue highlight) will not affect the output unless the selection is accepted or rejected.

Individual mode: visualize plots for one micrograph at a time

To switch to `Individual' mode, click on the button at the top, or by clicking on an individual row in the table:

Select a row to view details for that micrograph:

You can then view CTF plots, motion trajectories and other available plots corresponding to the selected exposure. It is also possible to move to the next exposure in the table (using the left/right arrows), as well as Accept or Reject the individual micrograph from this view. You can also click on any individual plot to enlarge it.

When you have finished inspecting and curating exposures, click Done to exit and complete the job. Note that all micrographs in green will be included in the micrographs_accepted output of the job, micrographs in red will be included in the micrographs_rejected output, but a selection (blue highlight) will not affect the output unless the selection is accepted or rejected.

View outputs and continue processing

After clicking Done, the job will finish running and a list of outputs will become available. These can be used for further processing as required.

Common next steps include:

  • Runningparticles_accepted through a 2D Classification job, and/or starting an Ab-Initio Reconstruction using only these particles, followed by Refinement
  • Using micrographs_accepted to continue processing particle picking and local motion correction using only the good data

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