Micrographs were binned twofold (yielding a pixel size of 1.30654 Å per pixel) and further motion-corrected with MotionCor2 (39). Defocus values were determined for each micrograph using CTFFIND3 (40). All 2D and 3D classification and refinement were performed using RELION v1.3 (41) or RELION v2.0 (42).

For the autopicking of 20S particles, 3000 20S particles were boxed from 100 micrographs using e2boxer.py in EMAN2 (43). Then, the boxed particles were extracted from micrographs and 2D classified using RELION 1.3. The generated 2D class averages were used as the templates for the subsequent autopicking of 20S particles. A total of 1,371,140 particles were picked up from 5912 micrographs. After 2D and 3D classifications, good classes containing 237,356 20S particles were then subjected to 3D autorefinement, resulting in a 3D reconstruction map of whole 20S particle with a resolution of 4.6 Å.

To further refine the map of the α-SNAP–SNARE subcomplex and the NSF part, which are flexible to each other, the particle segmentation procedure described in our previous work (30, 34) was applied to the 3D reconstruction of the 20S particles. Briefly, to segment the α-SNAP–SNARE subcomplex from 20S particles, the signals corresponding to the NSF-D1D2 part were first subtracted from the raw images. Then, the α-SNAP–SNARE subcomplex particles were reextracted from the raw particle images and served as a new dataset for 2D classification and 3D reconstruction. The N domain of NSF was too flexible and not subtracted from the raw images. Thus, the segmented particles actually contained both the N domains of NSF and the α-SNAP–SNARE subcomplex. The segmented α-SNAP–SNARE particles were further 2D and 3D classified. Good classes containing 97,910 α-SNAP–SNARE particles were selected and then subjected to 3D autorefinement without applying any symmetry, resulting in a final 3D reconstruction map with a resolution of 3.9 Å based on gold-standard Fourier shell correlation (FSC) 0.143 criteria after applying a soft mask around the α-SNAP–SNARE portion. Similar to the processing of the α-SNAP–SNARE subcomplex described above, the NSF part of 20S particles was also segmented from raw images by subtracting the signals of the α-SNAP–SNARE subcomplex from raw images. A total of 163,942 NSF particles were selected from 2D and 3D classification and then subjected to 3D autorefinement without imposing any symmetry, resulting in a final 3D reconstruction map with a resolution of 3.7 Å after applying a soft mask around the NSF-D1D2 portion. The local resolution map was estimated using ResMap (44). The workflow of data processing is also summarized in fig. S2.

The procedure of focused classification is summarized in fig. S5A. We first performed the whole 20S reconstruction with all of the 237,356 particles by applying the angles obtained from the refinement of the segmented D1D2 part to the whole particles. Then, the D1D2 signals were subtracted from each particle image. The subsequent 3D classification on the modified particles was carried out by applying a mask around the α-SNAP–SNARE subcomplex and the NSF N domain with all particle orientations fixed at the value determined in the whole 20S particle 3D refinement. Following the first round of 3D classification, one class showing poor density was discarded. The particles from the rest of the classes were combined and then reclassified into 12 3D classes. This classification produced six groups exhibiting distinct patterns of the N domains relative to the asymmetric D1 ring. The corresponding particles before density subtraction from each group were selected and refined, yielding 3D maps ranging from 7.3 to 8.2 Å.

To calculate the distributions of the residue of the SNARE complex relative to the NSF-D1D2 part, we first docked the crystal structure of the SNARE complex [protein data bank (PDB) accession code: 1SFC; (2)] into the unsharpened map of the segmented α-SNAP–SNARE subcomplex according to the registers obtained from the atomic model of the SNARE complex (see below) to identify the target residue at the very N terminus of the SNARE complex. Then, the coordinate of the target residue of the SNARE complex (here, we used atom Cα as representative) in the original micrograph could be determined on the basis of the 3D refinement results of the segmented α-SNAP–SNARE subcomplex. Similarly, the coordinate of any residue of NSF-D1D2 (here, we used atom Cα of K304 of NSF as representative) in the original micrograph could be identified on the basis of the data of the segmented NSF-D1D2 part refinement and its atomic model. Then, the shift from the coordinate of the D1D2 residue in the micrograph to that of the target residue of the SNARE complex can be calculated for each particle. Thus, on the basis of these shifts and the angles obtained from the refinement of the segmented D1D2 and using a Gaussian dot generated by EMAN2 as the image of the target residue, the map representing the distribution of the target residue of the SNARE complex relative to the NSF-D1D2 can be 3D reconstructed with the program relion_reconstruct from the RELION package.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.