Cytoplasmic polyhedrosis virus (CPV) data was collected using Leginon on DE-12 camera (4k×3k, 6μm/pixel, operated at 25 fps, Direct Electron, LP, San Diego) installed on a 300kV FEI Titan Krios electron microscope in Z. Hong Zhou’s lab at UCLA. The images were taken with a dose rate of 12.5e/Å2/s and 2-second exposure time. From 1,708 micrographs 28,824 particles with the box size of 864x864 pixels were selected for further processing using the JSPR software via Cryo-EM Tool on Diagrid.
The movie-mode data was both drift and damage corrected based on individual particles using the free Python script of DE_process_frames.py developed by Direct Electron.
The particle images were divided into two subsets in the beginning in order to independently process the data in the gold-standard way. All the particle images were first shrunk 4 times to the box size of 216×216 to accelerate the data processing at low resolutions. About 3,000 shrunk particle images with larger defocuses were selected from each subset to build de novo initial model. The first refinement for the two independent subsets was performed at the resolution range of 200 ~ 50Å. The two resulted maps, one for each subset, were seeded as the initial models for the next iteration. The subsequent refinements were performed using data up to a resolution slightly lower than the resolution assessed by the gold-standard FSC=0.143 criterion in the previous iteration. The refinements continued until no further improvement can be achieved based on the gold-standard FSC. The orientations and centers for the shrunk data were then migrated to the full-size (864×864) particle images for more refinements. The resolution was calculated to be 3.5 Å (Figure 1) from the most recent two density maps (Figure 2 based on gold-standard FSC=0.143 criterion. No defocus, astigmatism or scale was refined during this test. Subsequent refinements of these additional parameters improved the resolution to 2.93 Å resolution (Figure 3).
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