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Instruments / Technical Area	Publication Details	Published
Krios III-Titan Krios III at Diamond	Cryo-EM structure of the bacterial divisome core complex and antibiotic target FtsWIQBL Lisa Kashammer , Fusinita Van Den Ent , Magnus Jeffery , Nicolas L. Jean , Victoria L. Hale , Jan Lowe Nature Microbiology 8, 1149 - 1159 DOI: 10.1038/s41564-023-01368-0 Diamond Proposal Number(s): [23268] Show Abstract ▼ Abstract: In most bacteria, cell division relies on the synthesis of new cell wall material by the multiprotein divisome complex. Thus, at the core of the divisome are the transglycosylase FtsW, which synthesises peptidoglycan strands from its substrate Lipid II, and the transpeptidase FtsI that cross-links these strands to form a mesh, shaping and protecting the bacterial cell. The FtsQ–FtsB–FtsL trimeric complex interacts with the FtsWI complex and is involved in regulating its enzymatic activities; however, the structure of this pentameric complex is unknown. Here, we present the cryogenic electron microscopy structure of the FtsWIQBL complex from Pseudomonas aeruginosa at 3.7 Å resolution. Our work reveals intricate structural details, including an extended coiled coil formed by FtsL and FtsB and the periplasmic interaction site between FtsL and FtsI. Our structure explains the consequences of previously reported mutations and we postulate a possible activation mechanism involving a large conformational change in the periplasmic domain. As FtsWIQBL is central to the divisome, our structure is foundational for the design of future experiments elucidating the precise mechanism of bacterial cell division, an important antibiotic target.	Jun 2023
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity Xian Deng , Andres Gonzalez Llamazares , James Wagstaff , Victoria L. Hale , Giuseppe Cannone , Stephen H. Mclaughlin , Danguole Kureisaite-Ciziene , Jan Lowe Nature Microbiology 16 DOI: 10.1038/s41564-019-0544-0 Show Abstract ▼ Abstract: Bactofilins are small β-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions, from cell stalk formation in Caulobacter crescentus to chromosome segregation and motility in Myxococcus xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here, sequence analysis and electron microscopy results reveal that, in addition to being widely distributed across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Electron cryomicroscopy analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerize through end-to-end association of the β-helical domains. Using a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerization-impairing mutation enabled crystallization and structure determination, while reaffirming the lack of polarity and the strength of the β-stacking interface. To confirm the generality of the lack of polarity, we performed coevolutionary analysis on a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in Escherichia coli cells. Membrane binding is probably a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.	Sep 2019
B24-Cryo Soft X-ray Tomography	Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress Victoria Hale , Jean M. Watermeyer , Fiona Hackett , Gema Vizcay-Barrena , Christiaan Van Ooij , James A. Thomas , Matthew C. Spink , Maria Harkiolaki , Elizabeth Duke , Roland A. Fleck , Michael J. Blackman , Helen Saibil Proceedings Of The National Academy Of Sciences DOI: 10.1073/pnas.1619441114 Diamond Proposal Number(s): [14410] Show Abstract ▼ Abstract: In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10–30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.	Mar 2017

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