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.
Project description:The gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike, making it flexible and disordered. These data reveal information on the position of the V3 loop and its relative flexibility and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the coreceptor and altering the structure of the envelope spike.Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved ?-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. We show here that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.
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.
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.
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.
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.
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.
Project description:Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.
Project description:Human infectious disease is classified into five etiologies: bacterial, viral, parasitic, fungal, and prion. Viral infections are unique in that they recruit human cellular machinery to replicate themselves and spread infection. The number of viruses causing human disease is vast, and viruses can be broadly categorized by their structures. Many viruses, such as influenza, appear to be amorphous particles, whereas others, such as herpes simplex virus, rhinovirus, dengue virus, and adenovirus, have roughly symmetric structural components. Icosahedral viruses have been a target of electron microscopists for years, and they were some of the first objects to be reconstructed three-dimensionally from electron micrographs. The ease with which highly purified and conformationally uniform virus samples can be produced makes them an ideal target structural studies. Apart from their biological significance, these virus samples have played a pivotal role in the development of new methodologies in the field of molecular biology as well as in cryo-electron microscopy and cryo-electron tomography.