A5004122762- Advanced Electron Microscopy Techniques and Applications
- Electron and X-Ray Spectroscopy Techniques
- Advanced Fluorescence Microscopy Techniques
- Biosensors and Analytical Detection
- Microfluidic and Capillary Electrophoresis Applications
- Enzyme Structure and Function
- Advanced Proteomics Techniques and Applications
- Advanced X-ray Imaging Techniques
- Viral gastroenteritis research and epidemiology
- Biochemical and Molecular Research
- Animal Virus Infections Studies
- Force Microscopy Techniques and Applications
- Viral Infections and Immunology Research
- Electrochemical sensors and biosensors
- Erythrocyte Function and Pathophysiology
- Cytomegalovirus and herpesvirus research
- Heat shock proteins research
- Vector-Borne Animal Diseases
- Bacteriophages and microbial interactions
- Herpesvirus Infections and Treatments
- Biomedical Text Mining and Ontologies
- Viral Infections and Outbreaks Research
- RNA Research and Splicing
- Polyomavirus and related diseases
- Cell Image Analysis Techniques
SLAC National Accelerator Laboratory
2020-2022
Stanford Synchrotron Radiation Lightsource
2022
Diamond Light Source
2017-2021
Stanford University
2021
Baylor College of Medicine
2012-2017
INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in membrane of eukaryotic cells that regulates import and export biomolecules between nucleus cytosol, with vertebrate NPCs ~110 to 125 MDa mass ~120 nm diameter. are organized into four main rings: cytoplasmic ring (CR) at cytosolic side, inner luminal on plane membrane, facing nucleus. Each possesses an approximate eightfold symmetry composed multiple copies different nucleoporins. have been implicated numerous biological...
We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice near-atomic resolution. This provides a vehicle for structure determination, bridging size gap between nanometer scale conventional electron diffraction and micron synchrotron microfocus beamlines. The demonstration atomic information can be retained suggests could provide such detail on sections cut from vitrified cells.
Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low images cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography cellular lamellae. Particle classification averaging yields to 5.6 Å resolution, sufficient identify secondary structural elements produce atomic model...
The integration of cellular and molecular structural data is key to understanding the function macromolecular assemblies complexes in their vivo context. Here we report on outcomes a workshop that discussed how integrate from range public archives. identified two main priorities: development tools file formats support segmentation (that is, decomposition three-dimensional volume into regions can be associated with defined objects), annotation biological structures.
Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of genome packaging release machinery in capsids these large complex double-stranded DNA viruses. However, such is tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In exits from virus particle through portal transferred into host cell by apparatus (i.e. tail) located at vertex. Here we use electron cryo-tomography herpes...
Abstract Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used answer structural questions create a “ground truth” for evaluation automated segmentation algorithms. Validation such lacking, but critical understanding the reproducibility annotations. Here, we voxel-based similarity scores variety specimens, ranging in complexity segmented by several annotators, quantify variation among their In addition, have identified...
MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique been largely limited to protein nanocrystals which grow either needles or plates measuring only few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized handling compound effects are unique electron diffraction data. Here, we present an integrated workflow microED,...
The recent resolution revolution in cryo-EM has led to a massive increase demand for both time on high-end cryo-electron microscopes and access microscopy expertise. In anticipation of this demand, eBIC was set up at Diamond Light Source collaboration with Birkbeck College London the University Oxford, funded by Wellcome Trust, UK Medical Research Council (MRC) Biotechnology Biological Sciences (BBSRC) provide equipment through peer review. is currently its start-up phase began offering...
Effects of polyomavirus SV40 microRNA on pathogenesis viral infections in vivo are not known. Syrian golden hamsters the small animal model for studies SV40. We report here effects and influence structure regulatory region dynamics DNA levels vivo. Outbred young adult were inoculated by intracardiac route with 1×107 plaque-forming units four different variants Infected animals sacrificed from 3 to 270 days postinfection loads tissues determined quantitative real-time polymerase chain...
Foot-and-mouth disease virus (FMDV) is highly contagious and infects cloven-hoofed domestic livestock leading to foot-and-mouth (FMD). FMD outbreaks have severe economic impact due production losses associated control measures. FMDV found as seven distinct serotypes, but there are numerous subtypes within each serotype, effective vaccines must match the circulating in field. In addition, O Southern African Territories (SAT) relatively more thermolabile their viral capsids readily dissociate...
SUMMARY Three-dimensional (3D) visualization of vitrified cells can uncover structures subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed 3D distribution organelles in whole...
Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted other biological samples. The basic principles samples, milling, and viewing lamellae are common to all instruments the can followed as general guide on-grid cryo-lamella preparation cryo-electron microscopy (cryoEM) tomography (cryoET). Electron supporting cells into liquid nitrogen-cooled ethane using manual or automated...
Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted other biological samples. The basic principles samples, milling, and viewing lamellae are common to all instruments the can followed as general guide on-grid cryo-lamella preparation cryo-electron microscopy (cryoEM) tomography (cryoET). Electron supporting cells into liquid nitrogen-cooled ethane using manual or automated...
Abstract Micro-Electron Diffraction (MicroED) has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique been largely limited to protein nanocrystals which grow either needles or plates measuring only few hundred nanometres in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimised handling compound effects are unique electron diffraction data. Here, we...
Traditionally molecular assembly pathways for viruses have been inferred from high resolution structures of stable intermediates purified in vitro , and low images cell sections as well genetic approaches including conditionally lethal mutants. Here, we directly visualise a previously unsuspected ‘single shelled’ icosahedral intermediate mammalian orthoreovirus, addition to the expected virions, cryo-preserved infected cells by cryo-electron tomography cellular lamellae 1,2 . Particle...