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      Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

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      Journal of Visualized Experiments : JoVE
      MyJove Corporation

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          Abstract

          Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy 1 and two-dimensional (2D) electron crystallography 2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method 3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments 4, microtubules 5, amyloid fibers 6, tobacco mosaic viruses 7, and bacteria flagella 8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.

          In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid 9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

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          Single-particle electron cryo-microscopy: towards atomic resolution.

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            Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function.

            Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.
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              The iterative helical real space reconstruction method: surmounting the problems posed by real polymers.

              Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pili, and filamentous bacteriophage. The first application of three-dimensional reconstruction from electron microscopic images was to a helical polymer, and a number of laboratories today are using helical tubes of integral membrane proteins for solving the structure of these proteins in the electron microscope at near atomic resolution. We have developed a method to analyze and reconstruct electron microscopic images of macromolecular helical polymers, the iterative helical real space reconstruction (IHRSR) algorithm. We can show that when there is disorder or heterogeneity, when the specimens diffract weakly, or when Bessel functions overlap, we can do far better with our method than can be done using traditional Fourier-Bessel approaches. In many cases, structures that were not even amenable to analysis can be solved at fairly high resolution using our method. The problems inherent in the traditional approach are discussed, and examples are presented illustrating how the IHRSR approach surmounts these problems.
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                Author and article information

                Journal
                J Vis Exp
                J Vis Exp
                JoVE
                Journal of Visualized Experiments : JoVE
                MyJove Corporation
                1940-087X
                2011
                9 August 2011
                9 August 2011
                : 54
                : 3041
                Affiliations
                Department of Structural Biology, University of Pittsburgh School of Medicine
                Author notes

                Correspondence to: Peijun Zhang at pez7@ 123456pitt.edu

                Article
                3041
                10.3791/3041
                3211131
                21860371
                f5c897b1-dc2c-45a8-ba5a-1f492c406d51
                Copyright © 2011, Journal of Visualized Experiments

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Immunology

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