Figures and data
Datasets and acquisition parameters used in this study.
Representative tilt images and tomograms at varying tilt increment.
A Single projection images of the sample at effective zero-tilt from a tilt series acquired with a tilt increment of: 1 degree (i); 2 degrees (ii); 3 degrees (iii); 5 degrees (iv); and 10 degrees (v). B Central XY slices through the tomograms reconstructed from the tilt series shown in A. C Central XZ slices through the tomograms shown in B. White arrowheads point to membranes that appear to extend outside of the lamella. D Power spectra of representative tomograms for all five conditions. Yellow circles represent the Crowther criterion for the tomograms shown in B and C, with the numerical value shown as text in yellow. Scalebar is 200 nm and applies to all relevant panels.
Assessment of signal-to-noise ratio (SNR), tilt-alignment quality and CTF fitting parameters at different tilt increments.
A SNR measured from projection images of the sample at effective zero-tilt position. SNR was defined as the ratio of the squared mean to the squared standard deviation of each image. B SNR measured from the sample volume that represents the lamellae. C Residual alignment error after tilt-series alignment in Etomo. D-E CTF estimation parameters. (D) Figure of merit, which indicates the confidence level to which the CTF was fitted, and (E) the final resolution to which the CTF was fitted; both estimated with Gctf. F Scatter plot showing the final resolution as shown in (E) against the local lamella thickness for all conditions, with coefficients of determination of R2 = 0.30, 0.38, 0.66, 0.61 and 0.60 for the respective datasets.
The effect of tilt increment on template matching.
A Maximum F1-score for tomograms used for TM and STA. B Area under the precision-recall curves. See corresponding SFig. 7
The effect of tilt increment on STA.
A Rosenthal–Henderson plots of particle subsets when refined and averaged in RELION 3.1. B Rosenthal–Henderson plots of tomogram subsets when refined and averaged in M. Inset: ribosome density maps using all particles, coloured according to the local resolution. C Resolution progression for each M refinement, considering all available particles. The grid subdivision shown on the horizontal axis is used for both the Image warp and the Volume warp grids. For more details, see the Methods section. D Resolution gain for each refinement round, also shown in panel C.
