Computational protocol: Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer

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Protocol publication

[…] Three microliters of MHV spike at 1.85 mg/mL were applied to a 1.2/1.3 C-flat grid (Protochips), which had been glow-discharged for 30s at 20mA. Thereafter, grids were plunge-frozen in liquid ethane using a Gatan CP3 and a blotting time of 3.5 s. Data was acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector. Coma-free alignment was performed using the Leginon software. Automated data collection was carried out using Leginon to control both the FEI Titan Krios (used in microprobe mode at a nominal magnification of 22,500 ×) and the Gatan K2 Summit operated in counted mode (pixel size: 1.315 Å) at a dose rate of ~9 counts/physical pixel/s which corresponds to ~12 electrons/physical pixels/s (when accounting for coincidence loss). Each movie had a total accumulated exposure of 53 e/Å2 fractionated in 38 frames of 200 ms (yielding movies of 7.6 s). A dataset of ~1600 micrographs was acquired in a single session using a defocus range comprised between 2.0 and 5.0 μm. [...] Whole frame alignment was carried out using the software developed by Li et al, which is integrated into the Appion pipeline, to account for stage drift and beam-induced motion. The parameters of the microscope contrast transfer function were estimated for each micrograph using ctffind3. Micrographs were manually masked using Appion to exclude the visible carbon supporting film for further processing. Particles were automatically picked in a reference-free manner using DogPicker. Extraction of particle images was performed using Relion 1.4 with a box size of 320 pixels and applying a windowing operation in Fourier space to yield a final box size of 288 pixels (corresponding to a pixel size of 1.46 Å). From the 1.2 million particles initially picked, a subset of 50,000 particles were randomly selected to generate class averages using RELION. An initial 3D model was generated using OPTIMOD within the Appion pipeline. The entire data set was subjected to 2D alignment and clustering using RELION and particles belonging to the best-defined class averages were retained (~500,000 particles). These ~500,000 particles were then subjected to RELION 3D classification with 4 classes (using c1 symmetry) starting with our initial model low-pass filtered to 40Å resolution. We subsequently used the ~230,000 best particles (selected from the 3D classification) and the map corresponding to the best 3D class (low-pass filtered at 40 Å resolution) to run Relion 3D auto-refine (c3 symmetry) which led to a reconstruction at 4.4 Å resolution. We utilized the particle polishing procedure in RELION 1.4 to correct for individual particle movement and radiation damage,. A second round of 3D classification with 6 classes (c3 symmetry) was performed using the polished particles resulting in the selection of 82,000 particles. A new 3D auto-refine run (c3 symmetry) using the selected 82,000 particles and the map corresponding to the best 3D class (low-pass filtered at 40 Å resolution) yielded a map at 4.0 Å resolution following post processing in RELION. The final map was sharpened with an empirically determined B factor of −220 Å2 using Relion post processing. Reported resolutions are based on the gold-standard FSC=0.143 criterion and Fourier shell correction curves were corrected for the effects of soft masking by high-resolution noise substitution. The soft mask used for FSC calculation had a 10 pixel cosine edge fall-off. The overall shape and dimensions of our reconstruction agree with previous data although the HR2 stem connecting to the membrane is not resolved. [...] Fitting of atomic models into cryoEM maps was performed using UCSF Chimera and Coot,. We initially docked the MHV domain A structure (PDB 3R4D) and used a crystal structure of a bovine coronavirus domain A (PDB 4H14) to model the three-stranded β-sheet and the α-helix present on the viral membrane proximal side of the galectin-like domain. Next, the MERS-CoV domain B crystal structure (PDB 4KQZ) was also fit into the density, and rebuilt and refined using RosettaCM. Although we could accurately align the sequences corresponding to the core β-sheet of the MHV and MERS-CoV B domains, the ~100 residues forming the β-motif extension (residues 453-535, MERS-CoV/SARS-CoV receptor-binding moiety) could not be aligned with confidence. We used RosettaCM to build models of each of the 945 possible disulfide patterns into the density for domain B. For each disulfide arrangement, 50 models were generated, and there was a very clear energy signal for a single such arrangement (). Then, 1000 models with this disulfide arrangement were sampled, and the lowest energy model (using the Rosetta force field augmented with a fit-to-density score term) was selected. Due to the poor quality of the reconstruction at the apex of the S trimer, the confidence of the model is lowest for the segment corresponding to residues 453-535, as homology-modeling was used to fill in details missing in the map.A backbone model was then manually built for the rest of the S polypeptide using Coot. Sequence register was assigned by visual inspection where side chain density was clearly visible. This initial hand built model was used as an initial model for Rosetta de novo. The Rosetta-derived model largely agreed with the hand-built model. Rosetta de novo successfully identified fragments allowing to anchor the sequence register for domains C and D as well as for helices α21-α25. Given these anchoring positions, RosettaCM augmented with a novel density-guided model-growing protocol was able to rebuild domains C and D in full. The final model was refined by applying strict non-crystallographic symmetry constraints using Rosetta. Model refinement was performed using a training map corresponding to one of the two maps generated by the gold-standard refinement procedure in Relion. The second map (testing map) was used only for calculation of the FSC compared to the atomic model and preventing overfitting. The quality of the final model was analyzed with Molprobity. Structure analysis was assisted by the PISA and DALI servers. The sequence alignment was generated using MultAlin and colored with ESPript. All figures were generated with UCSF Chimera. […]

Pipeline specifications

Software tools Leginon, Appion, CTFFIND, RELION, UCSF Chimera, Coot, MolProbity, DALI, MultAlin
Applications cryo-EM, Protein structure analysis
Organisms Mus musculus, Homo sapiens
Diseases Pneumonia