Unbend, correction of local beam-induced sample motion in cryo-EM images using a 3D spline model

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur, software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant and Grigorieff, 2015). Here, we present Unbend, extending Unblur by accommodating more localized sample bending and distortions using a 3D cubic B-spline model. Unbend is integrated into our cisTEM software with a new local motion visualization panel. We processed movie frames from various in situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3–8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to ~300%. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.