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Instruments / Technical Area	Publication Details	Published	Actions
B24-Cryo Soft X-ray Tomography Krios III-Titan Krios III at Diamond	Using light and electron microscopy to understand the replication of aphthoviruses Pui Y. A. Lai Diamond Proposal Number(s): [26538, 21005, 27932] Show Abstract ▼ Abstract: Aphthovirus is a genus of the family Picornaviridae which includes Foot-and- mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly contagious pathogen infecting cloven-hoofed animals and is hence economically important. FMDV replication takes place in the cytoplasm and induces massive rearrangement of the host cell membranes to facilitate virus replication. Rearranged membranes form structures providing the site of viral genome replication known as the replication organelle (RO). The understanding of the RO, viral proteins and site of virus assembly is not well established. This project applies various microscopy approaches to investigate details of aphthovirus replication in cells. FMDV 3A protein is known to play a key role in viral replication machinery. We generated recombinant viruses of FMDV with various tags fused to this protein, subsequently allowing 3A to be detected in confocal microscopy. We developed a split-GFP system to study the dynamics of 3A protein in vitro. We showed that 3A signals appeared contiguous to the Golgi membrane signals suggesting that it potentially serve as a main source of membrane associated with viral replication. This approach was taken with the aim of facilitating the development of a correlative light electron microscopy (CLEM) system to unravel the localisation of virus proteins and their link to RO in cells. ERAV was used as a surrogate model to study FMDV replication in a lower containment laboratory using cryo-electron vi tomography (cryo-ET). Virus particles were observed associated with membrane structures with single membrane vesicles being more predominant than double membrane vesicles in infected cells. By sub-tomogram averaging, we reconstructed 3-dimensional (3D) models of intracellular ERAV full and empty particles which were compared with structures obtained for the virus purified from tissue culture and crystallized. Additional density was identified in the ERAV empty particles potentially corresponding to RNA contact sites inside the capsid.	Aug 2023
	Cryo-plasma FIB/SEM volume imaging of biological specimens Maud Dumoux , Thomas Glen , Jake L. R. Smith , Elaine M. L. Ho , Luis Ma Perdigão , Avery Pennington , Sven Klumpe , Neville B. Y. Yee , David A. Farmer , Pui Y. A. Lai , William Bowles , Ron Kelley , Jürgen M Plitzko , Liang Wu , Mark Basham , Daniel K. Clare , C. Alistair Siebert , Michele C. Darrow , James H. Naismith , Michael Grange Elife 12 DOI: 10.7554/eLife.83623 Open Access Show Abstract ▼ 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 to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.	Feb 2023

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