A Case Study

In this session, we will work through one electron micrograph (cryo-EM), selecting particles and applying alignment, from which we can build an initial low-resolution density model. Before we start, create a working directory and move into it. This place will be the playground of this practice session. Download the dataset to your working directory. Then, at the command-line, type
tar
xfv demo.tar
Launch the program by the command
signature &

Particle Selection

  1. Define a new project space (Menu: Project: New) in the working directory and name it "TEST". A directory "TEST.ems" will be created, together with a set of sub-directories. The "Project" label at the upper right corner of the GUI window becomes "TEST".
  2. Load the micrograph "6605.mrc" (in "data/") from Menu: Data: Micrograph: Load MRC. The "Film" label at the upper right corner of the GUI window becomes "6605".
  3. The intensity histogram of the micrograph will be displayed in Image Quality Monitor. To observe the spectrum, activate the SPEC button and use the mouse cursor to select a region (left-click) for display. The size of the region can be customized at Box Size. Adjust the intensity and contrast dials to achieve the optimal visualization.
  4. In Micrograph Masking box, press Apply for automatic masking. When that is done, use the drawing tools to refine the mask manually.
  5. Take some notes at Keynote if you like. It will be saved into the project space automatically.
  6. Load the template images "templates.mrc" (in "data/") from Menu: Data: Template: Load MRC. In Circular Masking box, set LCF profile radius to 0.7, and SCF profile radius to 0.6, then click Apply.
  7. In Rotational Search box, set Interval to 5 degrees.
  8. Save this particle screening project from Menu: Project: Save: Screening PRJ. It will be internally named and managed by the system.
  9. Click the Particle button at the bottom of the GUI window, Particle OpPanel appears.
  10. In Particle Parameters box, set Resize to 4. In order to save time, we will skip the full-range screening (please see the user manual for the operational detail) and apply only the test-run. Click the T button, the system becomes busy, as indicated by the red light at the bottom right corner of the GUI window. Once the task is completed, the indicator becomes green. This should take less than one minute.
  11. The number of identified particles is displayed at Particle Count under the monitor. Change the LCF and SCF threshold slidebars at will and observe the effect. After each adjustment, press T again to update.
  12. Manual selection: CTRL+Left-click to select, SHIFT+Left-click to delete. More detail can be found here.
  13. This step is optional: to inspect the particle selection through the monitors, click the Accept (CTRL+a) and/or Reject (CTRL+r) buttons. Undo (CTRL+u) will take one step back. The Score bar displays the best matching score of the current selection with the template images. The automatically selected particles are marked by red crosses, and the accepted selections are marked by green crosses. Set the display scale and blurring to various levels to achieve the best visual perception. Remember to press ENTER to trigger the update. Toggle the cross-hair display in the monitors by the + button under the monitor. The distance between the two pairs of short-bars indicates the particle's diameter as specified at Size in Particle Parameters box. The distance display is also affected by the pixel size of the image, which is defined in Micrograph OpPanel.
  14. Save the particle selection into an MRC stack (Menu: Particle: Save Image Frames). The file will be named "TEST.ems/particles/6605.mrc". Then load the particle set into Image Stack Editor. (Alternatively, press the button , the particle set will be automatically saved and loaded into Image Stack Editor.)
  15. Apply Normalization function, set the proper display Scale, and browse the particle image set in the editor. Select unwanted particles (CTRL+left-click) and, at the end, press the Delete button. When prompted to synchronize with the micrograph, make sure to answer "Yes".