For the influenza hemagglutinin trimer benchmark, raw movie data and pre-extracted particles from EMPIAR-10097 were downloaded. The movies were processed with the full Warp pipeline using the following settings: motion correction with a temporal resolution of 40 for the global motion, and 5x5 spatial resolution for the local motion, using the 0.03–0.25 Nyquist range and a B-factor of -400 A2; CTF estimation with 6x6 spatial resolution, using the 0.1–0.35 Nyquist range; particle picking with a BoxNet model retrained on particles from 3 micrographs, using the default 0.95 threshold. Quality filters were applied in Warp as follows: defocus between 0.3 and 5.0 µm, resolution better than 8 Å, intra-frame motion of at most 1.5 Å, particle count above 120. Particles were extracted from the micrographs meeting these filters and subjected to processing in cryoSPARC: no 2D classification was performed; ab initio refinement was performed with 6 classes and no symmetry; the 6 classes were then refined heterogeneously, with no symmetry imposed; the only class showing the expected Hemagglutinin structure was refined with C3 symmetry. The original particle set from EMPIAR-10097 was subjected to 3 different processing strategies. First, the full set was refined in cryoSPARC with C3 symmetry using the original CTF estimates. Second, the full set was subjected to the same classification and refinement as the particles from Warp, using the original CTF estimates. Third, particles from the Hemagglutinin class obtained in the second processing branch were updated with local CTF estimates from Warp, and refined again with C3 symmetry. Resolution estimates were obtained for all maps using the respective masks automatically generated by cryoSPARC.
For the β-galactosidase benchmarking studies, raw data from EMPIAR-10061 were downloaded. The movies were processed with the full Warp pipeline using the following settings: motion correction with a temporal resolution of 38 for the global motion, and a 5x5 spatial resolution for the local motion, using the 0.03–0.60 Nyquist range and a B-factor of -160 Å2; CTF estimation with 5x5 spatial resolution, using the 0.08–0.60 Nyquist range; particle picking with a BoxNet model retrained on particles from 5 micrographs, using a threshold of 0.30. No quality filters were used as the data already represent a high-quality subset curated for the initial publication. Picked and extracted particles were subjected to 2D and 3D classification with C1 symmetry in RELION 2.1 to remove incomplete particles. The remaining particles were refined with D2 symmetry. The final half-maps were then used to refine beam tilt and per-particle defocus in RELION 3.0. Global motion tracks for all movies were exported from Warp to RELION 3.0 to perform Bayesian particle polishing.
To assess the frame alignment accuracy in Warp independently of downstream map refinement, β-galactosidase movies were aligned in Warp as described above, and using the default settings in MotionCor2. CTF fitting was performed with 5x5 spatial resolution, using the 0.08–50 Nyquist range. Frequency-dependent fit quality was calculated as described in the ‘Resolution estimation’ section, and all resulting curves averaged. The resolution was then estimated at a cut-off value of 0.3.
For the β-galactosidase benchmarking studies, raw data from EMPIAR-10061 were downloaded. The movies were processed with the full Warp pipeline using the following settings: motion correction with a temporal resolution of 38 for the global motion, and a 5x5 spatial resolution for the local motion, using the 0.03–0.60 Nyquist range and a B-factor of -160 Å2; CTF estimation with 5x5 spatial resolution, using the 0.08–0.60 Nyquist range; particle picking with a BoxNet model retrained on particles from 5 micrographs, using a threshold of 0.30. No quality filters were used as the data already represent a high-quality subset curated for the initial publication. Picked and extracted particles were subjected to 2D and 3D classification with C1 symmetry in RELION 2.1 to remove incomplete particles. The remaining particles were refined with D2 symmetry. The final half-maps were then used to refine beam tilt and per-particle defocus in RELION 3.0. Global motion tracks for all movies were exported from Warp to RELION 3.0 to perform Bayesian particle polishing.
To assess the frame alignment accuracy in Warp independently of downstream map refinement, β-galactosidase movies were aligned in Warp as described above, and using the default settings in MotionCor2. CTF fitting was performed with 5x5 spatial resolution, using the 0.08–50 Nyquist range. Frequency-dependent fit quality was calculated as described in the ‘Resolution estimation’ section, and all resulting curves averaged. The resolution was then estimated at a cut-off value of 0.3.