Generalized single-particle cryo-EM – a historical perspective

Abstract

This is a brief account of the earlier history of single-particle cryo-EM of biological molecules lacking internal symmetry, which goes back to the mid-seventies. The emphasis of this review is on the mathematical concepts and computational approaches. It is written as the field experiences a turning point in the wake of the introduction of digital cameras capable of single electron counting, and near-atomic resolution can be reached even for smaller molecules.Key words: 3D reconstruction, image processing, ribosome, molecular structureThe spectacular, fast-paced advances in single-particle cryogenic electron microscopy (cryo-EM) in the past 3 years, following the introduction of novel direct detection device (DDD) cameras with superior signal-to-noise ratio and resolution [1,2], are currently the subject of many commentaries and news and views articles conveying the general excitement of the structural biology community and the scientific community at large. As someone who has been in the field since the early days, I would like to contribute by a recollection of how the field developed.Single-particle EM, as a novel approach to structural biology, required a heretical concept going against the grain of wisdom which held that quantitative structure determination by 3D reconstruction from EM projections [3] is not feasible unless molecules are arranged in crystalline order. These ordered structures include those with helical symmetry, as in DeRosier and Klug's reconstruction of a bacteriophage tail [3], arranged in a two-dimensional crystal [4,5], or with high symmetry as in viruses [6]. Viruses, although classifiable as single particles, contain structural information in a highly redundant form. For instance, the projection of a virus with icosahedral symmetry contains 60 projections of its asymmetric unit. Not only is the spatial arrangement of these units fixed and recoverable from the Fourier transform, but the signal is also retrieved with an instant bonus of nearly 8-fold reduction in the power of noise. Crowther and coworkers, in developing the common lines approach for recovering the structure of an icosahedral virus from its projection, did state that 'there is in principle no reason why the method should not be extended to systems with lower symmetry' [6], but it was not apparent how this could be practically accomplished for objects lacking symmetry altogether.At the time, the idea of single-particle averaging of entirely asymmetric molecules [7] therefore created some excitement: If such, [i.e. asymmetric single-particle] methods were to be perfected, then, in the words of one scientist, the sky would be the limit [8]. Thus, Robinson's Research News article started with an appreciation of Unwin and Henderson's seminal work, the 3D reconstruction of bacteriorhodopsin

Ryanodine Receptor Structure and Function in Health and Disease

“…For very large molecules such as RyR, several advantages of the single-particle cryogenic electron microscopy (cryo-EM) approach (Frank 2016) over X-ray crystallography come to bear (also see separate section below): first, high-resolution 3D information can be obtained from the 2D projection images of the molecule flash-frozen in solution, obviating the need for ordered crystals; and second, structural heterogeneity inherent to the function of the molecule may be studied as multiple conformers can be retrieved through the use of image classification.…”

“…The single-particle approach in cryo-EM (see Frank et al 2002; Frank 2016; Nogales 2016) is distinct from approaches involving crystals (with two-dimensional or helical symmetry) in that the molecules to be visualized are suspended freely in the aqueous buffer, without contacts with neighboring molecules. Thus, there are no constraints on the conformations they can assume, and, provided that the processes of purification and freezing themselves do not significantly alter their structure, the molecules are depicted in their native range of conformations and binding states.…”

Toward High-Resolution Cryo-Electron Microscopy: Technical Review on Microcrystal-Electron Diffraction

“…It is an important analytical technique that is used for gaining insight into the functional and molecular mechanisms of biomolecules involved in several physiological processes (Dubochet, 2012;Frank, 2016;Henderson, 2004). Cryo-EM can be subdivided into the following three groups according to the analytic purpose and the features of the biological samples: cryo-electron tomography (cryo-ET), cryo-singleparticle reconstruction, and cryo-electron crystallography (Asano et al, 2016;Dubochet, 2012;Frank, 2009;Henderson, 2004;Nannenga & Gonen, 2014).…”

“…Cryo-ET is a unique EM technique that is used to study intact biomolecular complexes within their native environments; it can provide mechanistic insights that are challenging for other EM-methods (Asano et al, 2016). However, the resolution of reconstructed threedimensional (3D) models generated by cryo-ET is low, while single-particle reconstruction can reproduce biomolecular structures having near-atomic resolution without the need for crystallization unless the samples are large (>200 kDa) and highly symmetrical (Frank, 2016). Cryo-electron crystallography is subdivided into the following two categories according to the types of samples: one category that deals with two-dimensional (2D) crystalline arrays (2D Cryo-electron microscopy (cryo-EM) is arguably the most powerful tool used in structural biology.…”

While the revolution will not be crystallized, biochemistry reigns supreme

“…In many ways, this very rapid improvement in achievable resolutions seems like the spontaneous generation of a new field of structural biology; 1 however, cryo-EM's current success grows directly from deep theoretical and practical roots (reviewed in Ref. [2][3][4][5]. For singleparticle EM, the practical applications of the approach have finally started to catch up with its theoretical potential.…”