Protein Secondary Structure Determination by Constrained Single-Particle Cryo-Electron Tomography

Abstract

SUMMARY Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations necessary for 3D reconstruction. Here, we propose “constrained single-particle tomography” as a general strategy for 3D structure determination in cryo-EM. A key component of our approach is the effective use of images recorded in tilt series to extract high-resolution information and correct for the contrast transfer function. By incorporating geometric constraints into the refinement to improve orientational accuracy of images, we reduce model bias and overrefinement artifacts and demonstrate that protein structures can be determined at resolutions of ~8 Å starting from low-dose tomographic tilt series.

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

“…A different technique whose first pioneering works have recently appeared is called Single Molecule Tomography [105]. This technique is similar to Electron Tomography in its image acquisition scheme, but it differs in that, unlike Electron Tomography, a single molecule, not a cellular slice, is imaged.…”

Cellular structural biology as revealed by cryo-electron tomography

“…Such a resolution will be sufficient to unambiguously fit existing X-ray crystallography and NMR data into the EM densities of larger macromolecular assemblies, which will help build more realistic atomistic models of these macromolecules and provide a glimpse into their interorganization and function. Proof-of-concept studies on the GroEL chaperonin yielded the first subnanometer resolution structure that utilized cryo-ET (Bartesaghi et al, 2012). Subsequently, Schur et al resolved the highly symmetric immature HIV-1 capsid in intact virus particles by subtomograms averaging to 8.8 Å resolution and outlined an approach that might be helpful in determining the structures of other macromolecules within heterogeneous environments to subnanometer resolution (Schur et al, 2015).…”

Single Particle Tomography in EMAN2

“…The advantages of processing extracted per-particle subtiltseries rather than extracting subtomograms directly after reconstruction have been previously demonstrated (Iwasaki et al 2005;Zhang & Ren 2012;Bartesaghi et al 2012). In EMAN2, subtiltseries extraction from aligned tiltseries is automated through e2spt_subtilt.py, using the 3-D coordinates of subtomograms to geometrically estimate their position in each tilt image.…”

“…are incorporated into the algorithm. Extracted subtiltseries could be subsequently reconstructed into subtomograms with e2make3d.py using any of several available reconstruction methods (the default in EMAN2 is Fourier inversion), refined individually (Zhang, & Ren 2012), or used for novel SPT-SPA hybrid approaches (Bartesaghi et al 2012). Our subtiltseries extraction tool also allows keeping different subsets or ranges of particles.…”