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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair Souvik Naskar , Andrea Merino , Javier Espadas , Jayanti Singh , Aurelien Roux , Adai Colom , Harry H. Low Nature Structural & Molecular Biology 184 DOI: 10.1038/s41594-024-01401-8 Open Access Show Abstract ▼ Abstract: The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems.	Nov 2024
Krios IV-Titan Krios IV at Diamond	Bidirectional pilus processing in the Tad pilus system motor CpaF Michael Hohl , Emma J. Banks , Max P. Manley , Tung B. K. Le , Harry H. Low Nature Communications 15 DOI: 10.1038/s41467-024-50280-6 Open Access Show Abstract ▼ Abstract: The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.	Aug 2024
Krios I-Titan Krios I at Diamond	Assembly mechanism of a Tad secretion system secretin-pilotin complex Matteo Tassinari , Marta Rudzite , Alain Filloux , Harry Low Nature Communications 14 DOI: 10.1038/s41467-023-41200-1 Open Access Show Abstract ▼ Abstract: The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.	Sep 2023
Krios I-Titan Krios I at Diamond	Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily Jiwei Liu , Matteo Tassinari , Diorge P. De Souza , Souvik Naskar , Jeffrey K. Noel , Olga Bohuszewicz , Martin Buck , Tom A. Williams , Buzz Baum , Harry H. Low Cell 184, 3660 - 3673.e18 DOI: 10.1016/j.cell.2021.05.041 Diamond Proposal Number(s): [16942] Open Access Show Abstract ▼ Abstract: Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.	Jul 2021
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond	Structural basis for membrane tethering by a bacterial dynamin-like pair Jiwei Liu , Jeffrey K. Noel , Harry H. Low Nature Communications 9 DOI: 10.1038/s41467-018-05523-8 Diamond Proposal Number(s): [15597] Open Access Show Abstract ▼ Abstract: Dynamin-like proteins (DLPs) are large GTPases that restructure membrane. DLPs such as the mitofusins form heterotypic oligomers between isoform pairs that bridge and fuse opposing membranes. In bacteria, heterotypic oligomerisation may also be important for membrane remodelling as most DLP genes are paired within operons. How DLPs tether opposing membranes is unknown. Here we show the crystal structure of a DLP heterotypic pair from the pathogen Campylobacter jejuni. A 2:2 stoichiometric tetramer is observed where heterodimers, conjoined by a random coil linker, assemble back-to-back to form a tripartite DLP chain with extreme flexibility. In vitro, tetramerisation triggers GTPase activity and induces lipid binding. Liposomes are readily tethered and form tubes at high tetramer concentration. Our results provide a direct mechanism for the long-range binding and bridging of opposing membranes by a bacterial DLP pair. They also provide broad mechanistic and structural insights that are relevant to other heterotypic DLP complexes.	Aug 2018
I02-Macromolecular Crystallography	LeoA, B and C from enterotoxigenic Escherichia coli (ETEC) are bacterial dynamins Kate Michie , Anders Boysen , Harry H. Low , Jakob Møller-Jensen , Jan Lowe , Michael Hensel Plos One 9 (9) DOI: 10.1371/journal.pone.0107211 PMID: 25203511 Diamond Proposal Number(s): [8547] Open Access Show Abstract ▼ Abstract: Escherichia coli (ETEC) strain H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP). Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.	Sep 2014

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