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 "/gcdata1/jzchen/temp/demo/demo.tar" 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 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  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".


Particle Alignment

Assuming that you have collected enough particles. The next step is to alignment them. In order to achieve precise particle alignment, the projections from a low-resolution model will be used as the template images. With the micrograph "6605.mrc" and the particle stack "particles/6605.mrc" are in place (continuing from the previous section),
  1. Load the density "model.mrc" (in "data/") from Menu : Data : Density Map. The system will be automatically switched to the 3D Model OpPanel, and the density model will be displayed in the main display window.
  2. In Model Projection box, take all default values and press Apply. A set of 2D projections will be generated and saved to "modeling/projections.mrc" in the project space.
  3. Inspect these 2D projections in Image Stack Editor. There should be 25 frames. We notice that they are white particles, while those in the original micrograph are black. Apply the Intensity Scaling function with the Multiplier parameter set to "-1". Then save the new image stack to "TEST.ems/template/templates25.mrc" and load it as the template from Menu : Data : Template : LoadMRC.
  4. Activate Menu : Function : Particle Alignment. Then save this alignment project from Menu : Project : Save : Alignment PRJ. Now, if you press the Apply button, particle alignment will commence and might take quite a while (please see the user manual for the operational detail). We will have to skip this part and use a pre-processed dataset instead.
  5. Copy "data/6605.aln", "data/6605_stack.ptk" and "data/6605_particle.mrc" to "TEST.ems/particles". Rename "6605_particle.mrc" to "6605.mrc". And, copy "data/parameter.ctf" to "TEST.ems/micrograph".
  6. Open "TEST.ems/particles/6605.mrc" in Image Stack Editor. Make sure that the Precision value is set to "5" degrees (located in Menu : Function : Particle Alignment). Then save this alignment project from Menu : Project : Save : Alignment PRJ.
  7. Now go to Menu : Function : Particle Alignment and press the Apply button to start particle alignment. The pre-processed alignment parameters will be loaded and displayed in two charts: "Correlation Coefficient Distribution" and "Shift Distribution". Adjust the corresponding slidebars in the OpPanel to set the proper thresholds. Press Apply once again, those particles with higher C.C. and lower Shift values will be selected. The result of thresholding can be observed in Image Stack Editor.
  8. A Frealign-style PAR file can be exported for the selected particles: Menu : Particle : Export Frealign PAR. This parameter file can be read directly by the Frealign program for model reconstruction and model refinement.
  9. Clear the current particle selection from Menu : Particle : Clear Selection.  Then move back to Particle Alignment page, press the Sync button, the alignment shift of each particle will be compensated in its coordinates and the updated positions will be displayed in Particle OpPanel.
  10. Press the  button, a better centered particle stack will be appear in Image Stack Editor. This is the final particle stack from this micrograph.


Model Reconstruction

Now that the particle dataset has been aligned, we can directly build a new model using the Frealign program. Go to the directory "TEST.ems/frealign" and make copy the file "data/mparameters" and "data/mreconstruct.com". You will need to modify the file "mparameters" to define the working directory properly. Then type at the command-line

ln -s ../particles/6605.mrc .
mv 6605.par toyModel_1.par
mreconstruct.com 1 500 1

When the Frealign program terminates, a new model "toyModel_1.mrc" will be generated. Load this density map into Signature from Menu : Data : Density Map. Because of the limited number of particles used in this reconstruction, the model is naturally quite noisy. We can improve the visualization by applying a low-pass filtering:
  1. In Image Processing toolbox, choose Band-pass Filter function.
  2. Set Low-pass to 20.0 Å and High-pass to 100.0 Å, press Apply. A filtered density model shows up.
By the time tens of thousands of particles have been collected following the same procedure, the Frealign refinement can start directly from here.