A5082209312- Advanced Electron Microscopy Techniques and Applications
- Electron and X-Ray Spectroscopy Techniques
- Integrated Circuits and Semiconductor Failure Analysis
- Ion-surface interactions and analysis
- Cardiomyopathy and Myosin Studies
- Advanced X-ray Imaging Techniques
- Muscle Physiology and Disorders
- RNA regulation and disease
- Cardiovascular Effects of Exercise
- Cellular Mechanics and Interactions
- Microtubule and mitosis dynamics
- RNA and protein synthesis mechanisms
- Cytomegalovirus and herpesvirus research
- Nuclear Structure and Function
- Photosynthetic Processes and Mechanisms
- RNA Research and Splicing
- Herpesvirus Infections and Treatments
- Laser-induced spectroscopy and plasma
- Advanced Fluorescence Microscopy Techniques
- Interdisciplinary Research and Collaboration
- Healthcare and Venom Research
- Parvovirus B19 Infection Studies
- Sports Analytics and Performance
- Genetics and Physical Performance
- Advanced Materials Characterization Techniques
Centre for Human Genetics
2015-2025
University of Oxford
2015-2025
Rosalind Franklin Institute
2021-2025
Max Planck Institute of Molecular Physiology
2020-2023
Northumbria University
2010
Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo recruited, enveloped at inner membrane (INM), and delivered by fusion outer membrane. To understand structural underpinning this trafficking, we investigated egress progeny herpesvirus capsids where capsid envelopment mediated two viral proteins, forming complex (NEC). Using multi-modal imaging approach, visualized NEC in situ...
Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture how sarcomeres built underpins understanding their role in health disease. Here, we determine the architecture native vertebrate skeletal by electron cryo-tomography. Our reconstruction reveals details three-dimensional organization interaction actin myosin A-band, I-band, Z-disc demonstrates that α-actinin cross-links antiparallel filaments forming doublets with 6-nm spacing. Structures myosin,...
In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but underlying mechanism remains nebulous. this work, we used cryo–electron tomography subtomogram averaging to reveal structures native bound filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details interaction between actin, demonstrating stabilizing role nebulin. Myosin exhibited different conformations neck domain, highlighting its inherent structural variability...
Abstract The thick filament is a key component of sarcomeres, the basic units striated muscle 1 . Alterations in proteins are associated with familial hypertrophic cardiomyopathy and other heart diseases 2 Despite central importance filament, its molecular organization remains unclear. Here we present architecture native cardiac sarcomeres relaxed state, determined by cryo-electron tomography. Our reconstruction reveals three-dimensional myosin, titin myosin-binding protein C (MyBP-C)....
Abstract Structural biology studies inside cells and tissues require methods to thin vitrified specimens electron transparency. Until now, focused ion beams based on gallium have been used. However, implantation, changes surface chemistry an inability access high currents limit application. Here, we show that plasma-coupled sources can produce cryogenic lamellae of human in a robust automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced...
Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and structure of proteins at high resolution in situ. Most biological specimens are too thick be directly investigated therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure prone contaminations, which makes it tedious process, often leading suboptimal results. Here, we present new hardware that overcomes current limitations. We developed glove...
Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied vitrified samples, serial FIB/SEM is also a means target specific in cells tissues while maintaining constituents’ hydration shells for situ structural biology downstream. However, application non-stained cryogenic biological samples limited due low contrast, curtaining, charging artefacts. We address these challenges using...
Abstract The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit striated muscle 1 . Mutations in proteins are associated with familial hypertrophic cardiomyopathy other heart diseases 2, 3 Despite this central importance for sarcomere force generation, it remains unclear how filaments structurally organized its components interact each thin to enable highly regulated contraction. Here, we present molecular architecture native cardiac sarcomeres...
Electron cryo-tomography (cryoET) is currently the only technique that allows direct observation of proteins in their native cellular environment. Sub-volume averaging electron tomograms offers a route to increase signal-to-noise repetitive biological structures, such improving information content and interpretability tomograms. We discuss potential for sub-volume highlighting investigating specific processes situ, focusing on microtubule structure viral infection. show (i) situ from single...
Abstract Cryo focused ion beam lamella preparation is a potent tool for in situ structural biology, enabling the study of macromolecules their native cellular environments. However, throughput currently limited, especially thicker, more biologically complex samples. We describe how xenon plasma milling can be used routine bulk high-pressure frozen samples during lamellae with high success rate and determine 4.0 Å structure Escherichia coli ribosome on these using sub volume averaging....
Abstract Cellular cryo-electron tomography (cryoET) enables the capture of detailed structural information within a biologically relevant environment. However, in more complex samples, such as tissues, is lacking. Importantly, these observations need to be set context populations; imaging on molecular scale to-date limited few in-situ that struggle generalised. This due limitations throughput and versatility employed by current instrumentation. Here, we utilise plasma focused ion beam...
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...
Abstract Cryo-electron tomography is an emerging technique to study the cellular architecture and structure of proteins at high resolution in situ . Most biological specimens are too thick be directly investigated therefore thinned by milling with a focused ion beam under cryogenic conditions. This procedure prone frost amorphous ice depositions which makes it tedious process, leading suboptimal results especially when larger batches milled. Here, we present new hardware that overcomes...
Abstract An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of samples at mesoscale resolution. This is achieved collecting consecutive SEM images after successive rounds FIB that expose new surface each step. Due to instrumental limitations, some image processing necessary before 3D visualization and analysis the data possible. are...
For cryo-electron tomography (cryo-ET) of beam-sensitive biological specimens, a planar sample geometry is typically used. As the tilted, effective thickness along direction electron beam increases and signal-to-noise ratio concomitantly decreases, limiting transfer information at high tilt angles. In addition, range where data can be collected limited by combination various sample-environment constraints, including space in objective lens pole piece possible use fixed conductive braids to...
Abstract Structural biology inside cells and tissues requires methods able to thin vitrified specimens electron transparent thicknesses. Until now, focused ions beams based on gallium have been used. However, ion implantation, changes surface chemistry an inability access high currents limit Gallium as beam source. Here, we show that plasma-coupled sources can produce cryogenic lamella of human in a robust automated manner, with quality sufficient for pseudo-atomic structure determination....
Abstract Nebulin is a major structural protein of skeletal sarcomeres and essential for proper assembly contraction muscle 1 . It stabilises regulates the length thin filaments, 2 but mechanism remains nebulous. Using electron cryotomography sub-tomogram averaging, we present first structure native nebulin bound to filaments within A-band I-band intact sarcomeres. This in-situ reconstruction reveals unprecedented detail interaction at pseudo-atomic resolution between actin, providing basis...
Abstract Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 μm). When applied vitrified samples, serial FIB/SEM is also a means target specific in cells tissues while maintaining constituents’ hydration shells for in-situ structural biology downstream. However, application non-stained cryogenic biological samples limited due low contrast, curtaining charging artefacts. We address these...
Abstract Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging subcellular structures at the mesoscale in their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional picture cells and tissues. The correlation specific regions interest cryo-electron (cryoEM) additionally enable subsequent high-resolution analysis. However, adoption imaging-based methods is limited due artefacts arising from insulating...
Abstract The nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without breaking envelope barrier. It assembles into a lattice on inner membrane enveloping newly assembled nucleocapsids, which bud perinuclear space. primary virion subsequently fuses with outer membrane, releasing capsid cytosol. Here we interrogated NEC in context of intact cells infected pseudorabies or herpes simplex virus using focused-ion beam milling and electron cryo- tomography. We...