LoTToR: An Algorithm for Missing-Wedge Correction of the Low-Tilt Tomographic 3D Reconstruction of a Single-Molecule Structure

Abstract

A single-molecule three-dimensional (3D) structure is essential for understanding the thermal vibrations and dynamics as well as the conformational changes during the chemical reaction of macromolecules. Individual-particle electron tomography (IPET) is an approach for obtaining a snap-shot 3D structure of an individual macromolecule particle by aligning the tilt series of electron tomographic (ET) images of a targeted particle through a focused iterative 3D reconstruction method. The method can reduce the influence on the 3D reconstruction from large-scale image distortion and deformation. Due to the mechanical tilt limitation, 3D reconstruction often contains missing-wedge artifacts, presented as elongation and an anisotropic resolution. Here, we report a post-processing method to correct the missing-wedge artifact. This low-tilt tomographic reconstruction (LoTToR) method contains a model-free iteration process under a set of constraints in real and reciprocal spaces. A proof of concept is conducted by using the LoTToR on a phantom, i.e., a simulated 3D reconstruction from a low-tilt series of images, including that within a tilt range of ±15°. The method is validated by using both negative-staining (NS) and cryo-electron tomography (cryo-ET) experimental data. A significantly reduced missing-wedge artifact verifies the capability of LoTToR, suggesting a new tool to support the future study of macromolecular dynamics, fluctuation and chemical activity from the viewpoint of single-molecule 3D structure determination.

HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes

“…The primary technique for macromolecular structural determination is so-called subtomogram averaging (StA), in which subtomograms are classified, rigid-body aligned and averaged into 3D density maps iteratively (Mahamid et al, 2016 ; Schur et al, 2016 ; Wan and Briggs, 2016 ; Albert et al, 2017 ; Böck et al, 2017 ; Bykov et al, 2017 ; Pfeffer et al, 2017 ; Riedel et al, 2017 ; Wan et al, 2017 ; Davies et al, 2018 ; Guo et al, 2018 ; Hutchings et al, 2018 ; Kovtun et al, 2018 ; Mosalaganti et al, 2018 ; Park et al, 2018 ; Kaplan et al, 2019 ; Rapisarda et al, 2019 ). However, with recent instrumentation and software development, more research moves in the direction of studying single-particle subtomograms individually (with no or a minimum of averaging) by developing new methods for denoising, missing wedge correction, and 3D reconstruction (Zhang and Ren, 2012 ; Moebel and Kervrann, 2020 ; Zhai et al, 2020 ).…”

Designed and biologically active protein lattices

“…3e, f ) revealed encapsulated ferritins in the 2D lattice by the additional globular density. High-resolution 3D reconstruction was achieved by the individual particle electron tomography (IPET) 63 with missing-wedge correction techniques 64 , by focusing on small selected areas of a lattice to avoid image distortion at a larger scale 65 . Here, an area of the ferritin/Octa lattice was selected for serial refinement to obtain a final IPET 3D density map (Fig.…”

Molecular Organization of the Early Stages of Nucleosome Phase Separation Visualized by Cryo-Electron Tomography

“…To regain NCP molecular details and the connectivity between NCPs in the nucleosome arrays provided by the DNA, the initial and the coarse labelling 3D maps were superimposed, and the later was used as marker to select surrounding connected map density within the former. The resulting maps were then low-pass filtered and served as a mask for local missing wedge correction (Zhai et al, 2020). This procedure yielded the final denoised 3D map depicting NCPs isolated or in condensates, as well as their flanking DNA (Fig.…”

“…To reduce the tilt limitation caused the elongation artifact in the 3D reconstruction, the missing wedge correction was performed by the low-tilt tomographic reconstruction (LoTToR) method (Zhai et al, 2020), which is a model-free iterative process, under a set of constraints in the real and reciprocal spaces, aiming to fill the missing wedge zone in Fourier space. To implement the algorithm, a soft boundary 3D mask was generated using the previously denoised density map after 30 Å low-pass filtering.…”