scholarly journals Hierarchical assembly governs TRIM5α recognition of HIV-1 and retroviral capsids

2019 ◽  
Vol 5 (11) ◽  
pp. eaaw3631 ◽  
Author(s):  
Katarzyna A. Skorupka ◽  
Marcin D. Roganowicz ◽  
Devin E. Christensen ◽  
Yueping Wan ◽  
Owen Pornillos ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Hexagonal Lattice ◽  
Hierarchical Assembly ◽  
Virus Core ◽  
Capsid Shell ◽  
Cryo Electron Tomography ◽  
Nonself Recognition ◽  
Subtomogram Averaging ◽  
Dependent Processes ◽  
Hiv 1

TRIM5α is a restriction factor that senses incoming retrovirus cores through an unprecedented mechanism of nonself recognition. TRIM5α assembles a hexagonal lattice that avidly binds the capsid shell, which surrounds and protects the virus core. The extent to which the TRIM lattice can cover the capsid and how TRIM5α directly contacts the capsid surface have not been established. Here, we apply cryo–electron tomography and subtomogram averaging to determine structures of TRIM5α bound to recombinant HIV-1 capsid assemblies. Our data support a mechanism of hierarchical assembly, in which a limited number of basal interaction modes are successively organized in increasingly higher-order structures that culminate in a TRIM5α cage surrounding a retroviral capsid. We further propose that cage formation explains the mechanism of restriction and provides the structural context that links capsid recognition to ubiquitin-dependent processes that disable the retrovirus.

scholarly journals A flexible framework for multi-particle refinement in cryo-electron tomography

PLoS Biology ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. e3001319
Author(s):  
Alister Burt ◽  
Lorenzo Gaifas ◽  
Tom Dendooven ◽  
Irina Gutsche
Keyword(s):  
Software Package ◽  
Electron Tomography ◽  
Macromolecular Structure ◽  
Computational Tools ◽  
Cryo Electron Tomography ◽  
Flexible Framework ◽  
Subtomogram Averaging ◽  
Particle Refinement ◽  
Hiv 1

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

scholarly journals Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martin Obr ◽  
Clifton L. Ricana ◽  
Nadia Nikulin ◽  
Jon-Philip R. Feathers ◽  
Marco Klanschnig ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Electron Tomography ◽  
Structural Mechanism ◽  
Rous Sarcoma ◽  
Cryo Electron Tomography ◽  
Sarcoma Virus ◽  
Subtomogram Averaging ◽  
Opposing Effects ◽  
Hiv 1

AbstractInositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

scholarly journals Tools enabling flexible approaches to high-resolution subtomogram averaging

2021 ◽  
Author(s):  
Alister Burt ◽  
Lorenzo Gaifas ◽  
Tom Dendooven ◽  
Irina Gutsche
Keyword(s):  
Software Package ◽  
State Of The Art ◽  
Electron Tomography ◽  
Macromolecular Structure ◽  
Computational Tools ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Tomography Data ◽  
Hiv 1

AbstractCryo-electron tomography and subtomogram averaging are increasingly used for macromolecular structure determination in situ. Here we introduce a set of computational tools and resources designed to enable flexible approaches to subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

scholarly journals Benchmarking tomographic acquisition schemes for high-resolution structural biology

2019 ◽  
Author(s):  
Beata Turoňová ◽  
Wim J. H. Hagen ◽  
Martin Obr ◽  
Hans-Georg Kräusslich ◽  
Martin Beck
Keyword(s):  
Structural Biology ◽  
Electron Tomography ◽  
Atomic Resolution ◽  
Phase Plate ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography ◽  
Experimental Parameters ◽  
Native Context ◽  
Subtomogram Averaging ◽  
Hiv 1

AbstractCryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution, in principle, can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available at EMPIAR-10277 as well as EMD-10207 and might be used to further develop processing routines.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals The Dynamo Software Package for Cryo-electron Tomography and Subtomogram Averaging

2020 ◽  
Vol 26 (S2) ◽  
pp. 3142-3145
Author(s):  
Paula Navarro ◽  
Stefano Scaramuzza ◽  
Henning Stahlberg ◽  
Daniel Castaño-Díez
Keyword(s):  
Software Package ◽  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

scholarly journals Cryo Electron Tomography of Native HIV-1 Budding Sites

2010 ◽  
Vol 6 (11) ◽  
pp. e1001173 ◽  
Author(s):  
Lars-Anders Carlson ◽  
Alex de Marco ◽  
Heike Oberwinkler ◽  
Anja Habermann ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography ◽  
Hiv 1

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

scholarly journals RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

2015 ◽  
Vol 89 (19) ◽  
pp. 9739-9747 ◽  
Author(s):  
Simone Mattei ◽  
Annica Flemming ◽  
Maria Anders-Össwein ◽  
Hans-Georg Kräusslich ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Electron Tomography ◽  
Significant Proportion ◽  
Dimensional Structure ◽  
Capsid Assembly ◽  
Interaction Domain ◽  
Cryo Electron Tomography ◽  
Rna Genome ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.IMPORTANCEFormation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.

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