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Abstract: The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.

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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.

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Abstract: Background incl. aims: Antibiotic resistance is a global health crisis with the ever growing need to develop novel antibiotics and strategies to treat resistant infections. Bacteriophage therapy is often highlighted as an alternative approach due to its high specificity to kill a certain bacterial strain. However, bacteriophage propagate through a replication cycle within the target bacterium, with the potential to generate mutations with detrimental consequences. Strains of Pseudomonas sp. produce phage tail-like bacteriocins (PTLBs) which have evolved from bacteriophage. Although sharing many similarities, they differ from bacteriophage lacking a capsid and therefore the ability to replicate. These unique features highlight the potential of PTLBs as an alternative therapy to treat bacterial infections as they can be tittered to a specific dose. However, for PTLBs to be implemented as a bactericidal treatment, further information is needed regarding their structure, mechanism of action and how they recognise their target strains. We have isolated a contracting PTLB from an environmental strain of P. veronii and determined its structure by cryo-EM. Methods: The PTLB was purified by ammonium sulphate precipitation and visualised by cryo-EM. The structure of the PTLB was determined using a combination approach of single particle and helical analysis. Results: The structure was determined of a new clade of contracting PTLBs in both its uncontracted and contracted states. We also identified the lack of a ‘ruler protein’ for the purified PTLBs, observing varying lengths in the collected micrographs. Conclusion: We solved the structure of a novel contracting PTLB and show that it shares structural similarities with the previously characterised contractile nanomachine from P. aeruginosa. We also observe that inconsistent lengths of PTLBs does not appear to affect the lethality of the PTLB to its target strain.

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Abstract: Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F–Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.

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Abstract: The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.