Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

Abstract

With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

Antibody-induced uncoating of human rhinovirus B14

“…After 2D classification, a dataset of 46,684 full particles (set-afull) and a dataset of 9,521 empty particles (set-a-empty) were selected from set-a; also, 23,090 full particles (set-b-full) were selected from set-b, and 36,866 particles (set-c-full) were selected from set-c. A "gold standard" single particle icosahedral reconstruction strategy was independently applied to each of these four sets of particle images using the program jspr (19,20). High-resolution refinements involved parameters that define particle orientation, particle center, phase error due to beam tilt, astigmatism, defocus, magnification, and anisotropic magnification distortion.…”

“…This produced four datasets: set-a-full (46,684 full particles from set-a), set-a-empty (9,521 empty particles from set-a), set-b-full (23,090 full particles from set-b), and set-c-full (36,866 full particles from set-c). A gold-standard 3D reconstruction strategy was applied to each of these four datasets using the program jspr as previously described (19,52). Icosahedral symmetry was imposed in this process.…”

Structure of faustovirus, a large dsDNA virus

“…Particles were boxed from micrographs using e2boxer.py from the EMAN2 software package (39). Reconstruction was performed using the jspr software suite (40). The final reconstruction used 1,188 and 10,915 particles for the reconstruction of the small and large virus capsids, respectively.…”

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity

“…Contrast levels of micrographs were corrected using the ctfit program in EMAN (43). For the reconstructions of VLP-4J21 and VLP-5M16, initial orientations of the particles were assigned with a "random start" model (44), and iterative refinement cycles then were performed to convergence using the jspr program (44). A total of 3,149 and 3,090 particles were used for the reconstruction of the VLP-4J21 and VLP-5M16 complexes, respectively.…”