Cryo-electron tomography of HIV-1 GagdeltaMASP1T8I assemblies
Ontology highlight
ABSTRACT:
SUBMITTER: Luiza Montenegro Mendonca
PROVIDER: EMPIAR-10643 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.
Ni Tao T Frosio Thomas T Mendonça Luiza L Sheng Yuewen Y Clare Daniel D Himes Benjamin A BA Zhang Peijun P
Nature protocols 20220112 2
Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography ST ...[more]
Similar Datasets
Project description:The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.
| S-EPMC2991257 | biostudies-literature
Project description:HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.
| S-EPMC2999899 | biostudies-literature
Project description:The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4-6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brick-shaped viral particles with slightly rounded edges and dimensions of approximately 360 x 270 x 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of approximately 18-19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.
| S-EPMC549483 | biostudies-literature
Project description:Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.
| S-EPMC3217200 | biostudies-literature
Project description:Centrosomes are cellular organelles that have a major role in the spatial organisation of the microtubule network. The centrosome is comprised of two centrioles that duplicate only once during the cell cycle, generating a procentriole from each mature centriole. Despite the essential roles of centrosomes, the detailed structural mechanisms involved in centriole duplication remain largely unknown. Here, we describe human procentriole assembly using cryo-electron tomography. In centrosomes, isolated from human lymphoblasts, we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A-microtubule is capped by a conical structure and the B- and C-microtubules elongate bidirectionally from its wall. These observations suggest that the gamma tubulin ring complex (gamma-TuRC) has a fundamental role in procentriole formation by nucleating the A-microtubule that acts as a template for B-microtubule elongation that, in turn, supports C-microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.
| S-EPMC2876950 | biostudies-literature
Project description:Developments in cryo-electron microscopy (cryo-EM) have been interwoven with the study of viruses ever since its first applications to biological systems. Following the success of single particle cryo-EM in the last decade, cryo-electron tomography (cryo-ET) is now rapidly maturing as a technology and catalysing great advancement in structural virology as its application broadens. In this review, we provide an overview of the use of cryo-ET to study viral infection biology, discussing the key workflows and strategies used in the field. We highlight the vast body of studies performed on purified viruses and virus-like particles (VLPs), as well as discussing how cryo-ET can characterise host-virus interactions and membrane fusion events. We further discuss the importance of in situ cellular imaging in revealing previously unattainable details of infection and highlight the need for validation of high-resolution findings from purified ex situ systems. We give perspectives for future developments to achieve the full potential of cryo-ET to characterise the molecular processes of viral infection.
| S-EPMC10578967 | biostudies-literature
Project description: Not available
| EMPIAR-10164 | biostudies-other
Project description:Modern methods of cryo electron microscopy and tomography allow visualization of protein nanomachines in their native state at the nanometer scale. Image processing methods including sub-volume averaging applied to repeating macromolecular elements within tomograms allow exploring their structures within the native context of the cell, avoiding the need for protein isolation and purification. Today, many different data acquisition protocols and software solutions are available to researchers to determine average structures of macromolecular complexes and potentially to classify structural intermediates. Here, we list the density maps reported in the literature, and analyze each structure for the chosen instrumental settings, sample conditions, main processing steps, and obtained resolution. We present conclusions that identify factors currently limiting the resolution gained by this approach.
| S-EPMC3962116 | biostudies-literature
Project description:The Bsoft package [Heymann, J.B., Belnap, D.M., 2007. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3-18] has been enhanced by adding utilities for processing electron tomographic (ET) data; in particular, cryo-ET data characterized by low contrast and high noise. To handle the high computational load efficiently, a workflow was developed, based on the database-like parameter handling in Bsoft, aimed at minimizing user interaction and facilitating automation. To the same end, scripting elements distribute the processing among multiple processors on the same or different computers. The resolution of a tomogram depends on the precision of projection alignment, which is usually based on pinpointing fiducial markers (electron-dense gold particles). Alignment requires accurate specification of the tilt axis, and our protocol includes a procedure for determining it to adequate accuracy. Refinement of projection alignment provides information that allows assessment of its precision, as well as projection quality control. We implemented a reciprocal space algorithm that affords an alternative to back-projection or real space algorithms for calculating tomograms. Resources are also included that allow resolution assessment by cross-validation (NLOO2D); denoising and interpretation; and the extraction, mutual alignment, and averaging of tomographic sub-volumes.
| S-EPMC2409064 | biostudies-literature
Project description:The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.
| S-EPMC7835367 | biostudies-literature