2.2. Data acquisition and analysis  

The cryo-EM data set for GI was collected using a 200 kV Talos Arctica microscope equipped with a K2 Gatan camera, with a physical pixel size of 0.91 Å. A phase plate was not used and the objective aperture was not inserted. A total of 202 movies with an exposure time of 100 s per movie were collected. Each movie contains 200 frames with an exposure time of 0.5 s per frame and an electron dose of 120 e Å⁻² per movie (Table 1 ▸).

Both the HemQ-57K and HemQ-45K data sets were also collected in the same alignment conditions on a 200 kV Talos Arctica microscope with a K2 Gatan camera run in super-resolution mode, with physical pixel sizes of 0.72 Å for HemQ-57K and 0.91 Å for HemQ-45K. For HemQ-57K, 268 movies were collected with an exposure time of 40 s per movie. Each movie contains 100 frames with an exposure time of 0.4 s per frame and an electron dose of 90 e Å⁻² per movie. For HemQ-45 K, 257 movies were collected with an exposure time of 40 s per movie. Each movie contains 100 frames with an exposure time of 0.4 s per frame and an electron dose of 90 e Å⁻² per movie.

Complete data sets for EMPIAR depositions 10204, 10185 and 10186 were processed as examples of data sets collected at 200 kV. EMPIAR 10185 and EMPIAR 10186 were collected consecutively on the same instrument, with EMPIAR 10185 collected with a traditional setup by moving only the stage and EMPIAR 10186 collected with the beam-image shift method. We performed image-specific correction for coma in both data sets, producing a material improvement in resolution (Table 2 ▸). EMPIAR 10263, data set III, served as an example of a data set collected at 300 kV with a large coma value that was partially corrected by alternative methods.

We processed all data sets with cisTEM (Grant et al., 2018 ▸). We modified the cisTEM pipeline by adding reference-based refinement of aberrations, including coma and trefoil, as in JSPR and RELION (Li et al., 2019 ▸; Zivanov et al., 2018 ▸). As discussed in Sections 3 and 4, we performed multiple cycles that included aberration refinement, orientation refinement and creation of a new reference (Grant et al., 2018 ▸). In these cycles, the resolution limit of the data used for the orientation refinement was selected based on manual assessment of the signal to noise (SNR) estimate in cisTEM exceeding a threshold value, typically around 4. The data-collection and analysis statistics are summarized in Table 1 ▸. The cryo-EM movies, maps and models used in the data analysis of GI and HemQ proteins have been deposited in the PDB, the EMDB and EMPIAR under codes 6vrs, EMD-21371 and EMPIAR-10360 for GI; 6vsa, EMPIAR-10363 and EMD-21373 for HemQ-57K; and 6vsc, EMPIAR-10362 and EMD-21376 for HemQ-45K. We solved all of the structures using MOLREP (Vagin & Teplyakov, 2010 ▸) and refined them with REFMAC (Murshudov et al., 2011 ▸) used within CCPEM (Wood et al., 2015 ▸; Burnley et al., 2017 ▸; Nicholls et al., 2018 ▸) with manual inspection using Coot (Emsley & Cowtan, 2004 ▸; Emsley et al., 2010 ▸).

In the process of modifying cisTEM, we introduced many changes in the underlying data structures, which resulted in the development of a separate software package. The package does not rely on the particle-stack concept which is used in other cryo-EM packages. We plan to release it in the future, and it will include the implementation of aberration refinement described above. The module is, however, not ready yet for standalone release owing to its reliance on unusual data structures.