Abstract
The comparison of a pair of electron microscope images recorded at different specimen tilt angles provides a powerful approach for evaluating the quality of images, image-processing procedures, or three-dimensional structures. Here, we analyze tilt-pair images recorded from a range of specimens with different symmetries and molecular masses and show how the analysis can produce valuable information not easily obtained otherwise. We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass. The angular uncertainty is less than 1° for particles of high molecular mass (∼50 MDa), several degrees for particles in the range 1–5 MDa, and tens of degrees for particles below 1 MDa. Orientational uncertainty may be the major contributor to the effective temperature factor (B-factor) describing contrast loss and therefore the maximum resolution of a structure determination. We also made two unexpected observations. Single particles that are known to be flexible showed a wider spread in orientation accuracy, and the orientations of the largest particles examined changed by several degrees during typical low-dose exposures. Smaller particles presumably also reorient during the exposure; hence, specimen movement is a second major factor that limits resolution. Tilt pairs thus enable assessment of orientation accuracy, map quality, specimen motion, and conformational heterogeneity. A convincing tilt-pair parameter plot, where 60% of the particles show a single cluster around the expected tilt axis and tilt angle, provides confidence in a structure determined using electron cryomicroscopy.
Highlights
Single-particle electron microscopy (EM), whether carried out on negatively stained or ice-embedded specimens, is growing in popularity and productivity as a result of steady technical improvements
We show that the accuracy of orientation determination of individual single particles depends on molecular mass, as expected theoretically since the information in each particle image increases with molecular mass
We chose the rotavirus double-layered particles (DLPs) that Zhang et al had shown could produce a 3D map at 3.8 Å resolution.[36]
Summary
Single-particle electron microscopy (EM), whether carried out on negatively stained or ice-embedded specimens, is growing in popularity and productivity as a result of steady technical improvements. Single-particle EM is one aspect of three-dimensional (3D) EM of biological macromolecules It followed early applications of 3D reconstruction to helical,[1] icosahedral,[2] and two-dimensional crystalline arrays.[3] The first reliable application of singleparticle EM to particles with low symmetry was the negatively stained 50S ribosomal subunit.[4] The potential impact of single-particle EM in structural biology was greatly expanded in the 1980s when Dubochet et al developed their plunge-freeze method of embedding a solution of single particles in a thin film of vitreous ice.[5] This simple procedure led to maps arising from images of the intrinsic molecular structure itself[6] rather than the structure of a hollow shell of heavy-metal stain that outlined the surface contour of the macromolecule. Since following many near-atomicresolution structures from two-dimensional crystalline or helical arrays,[7] the resolution of maps from large unstained single particles in favorable cases has reached near-atomic resolution,8 3.3 Å in the best case,[9] where it is possible to trace the path of the polypeptide backbone and assign side-chain densities
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