Figure 1.

Preferred orientation results in artefacts that can be overcome by tilting. (a–b) Shown from left to right: Euler angle distribution; side view of the reconstruction (in grey, the direction of preferred orientation is indicated by the red arrow) superimposed onto a projection of the envelope of the HA trimer crystal structure (in pink), displayed alongside a top view thresholded at a lower value and a close-up of a particular region; half-map and map-to-model 3D FSCs; plots of the global half-map FSC (blue line) and map-to-model FSC (purple line), together with the spread of directional resolution values defined by +/− 1σ from the mean (green area encompassed by green dotted lines, left axis) and a histogram of one hundred such values evenly sampled over the 3D FSC (yellow bars, right axis). Dotted lines indicate 0.5 and 0.143 thresholds. Arrows (i, iv) indicate presence of false side views, arrows (ii, v) indicate streaking in the unsharpened maps, and arrows (iii, vi) indicate bumps in the global FSC, all of which result from overfitting. (a) Comparison of HA trimer reconstructions refined independently from untilted images (130,000 particles) or from 40°-tilted images (130,000 particles). While the dataset from 0° images produces a visually poor map that is characterized by artefactual density (red dotted lined circle in the inset) and low map-to-model resolution, the dataset from 40° tilted images readily produces a mid-4Å reconstruction using both half-map and map-to-model resolution evaluations. (b) HA trimer reconstruction refined from 260,000 particles combined from both datasets. Notably, the same set of particles from 0° images in (a) produces a different Euler angle distribution profile (more “top” views) when refined in combination with particles from 40° images.