I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Tim
Nierhaus
,
Stephen H.
Mclaughlin
,
Frank
Bürmann
,
Danguole
Kureisaite-Ciziene
,
Sarah L.
Maslen
,
J. Mark
Skehel
,
Conny W. H.
Yu
,
Stefan M. V.
Freund
,
Louise F. H.
Funke
,
Jason W.
Chin
,
Jan
Lowe
Diamond Proposal Number(s):
[15916, 21426]
Abstract: During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA–FtsN interaction site in the IA–IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA–FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.
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Sep 2022
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I03-Macromolecular Crystallography
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Tomoyuki
Hatano
,
Saravanan
Palani
,
Dimitra
Papatziamou
,
Ralf
Salzer
,
Diorge P.
Souza
,
Daniel
Tamarit
,
Mehul
Makwana
,
Antonia
Potter
,
Alexandra
Haig
,
Wenjue
Xu
,
David
Townsend
,
David
Rochester
,
Dom
Bellini
,
Hamdi M. A.
Hussain
,
Thijs J. G.
Ettema
,
Jan
Lowe
,
Buzz
Baum
,
Nicholas P.
Robinson
,
Mohan
Balasubramanian
Diamond Proposal Number(s):
[21426]
Open Access
Abstract: The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events.
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Jun 2022
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I23-Long wavelength MX
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Matthew
Herdman
,
Andriko
Von Kugelgen
,
Danguole
Kureisaite-Ciziene
,
Ramona
Duman
,
Kamel
El Omari
,
Elspeth F.
Garman
,
Andreas
Kjaer
,
Dimitrios
Kolokouris
,
Jan
Lowe
,
Armin
Wagner
,
Phillip J.
Stansfeld
,
Tanmay A. M.
Bharat
Open Access
Abstract: Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.
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Nov 2021
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Krios I-Titan Krios I at Diamond
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Byung-Gil
Lee
,
Fabian
Merkel
,
Matteo
Allegretti
,
Markus
Hassler
,
Christopher
Cawood
,
Léa
Lecomte
,
Francis J.
O'Reilly
,
Ludwig R.
Sinn
,
Pilar
Gutierrez-Escribano
,
Marc
Kschonsak
,
Sol
Bravo
,
Takanori
Nakane
,
Juri
Rappsilber
,
Luis
Aragon
,
Martin
Beck
,
Jan
Lowe
,
Christian H.
Haering
Abstract: Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.
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Jul 2020
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.
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Jun 2020
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Krios III-Titan Krios III at Diamond
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Francesca
Coscia
,
Ajda
Taler-Verčič
,
Veronica T.
Chang
,
Ludwig
Sinn
,
Francis J.
O'Reilly
,
Thierry
Izore
,
Miha
Renko
,
Imre
Berger
,
Juri
Rappsilber
,
Dušan
Turk
,
Jan
Lowe
Diamond Proposal Number(s):
[17434]
Abstract: Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor–acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.
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Feb 2020
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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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.
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Sep 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Positioning of the division site in many bacterial species relies on the MinCDE system, which prevents the cytokinetic Z‐ring from assembling anywhere but the mid‐cell, through an oscillatory diffusion‐reaction mechanism. MinD dimers bind to membranes and, via their partner MinC, inhibit the polymerization of cell division protein FtsZ into the Z‐ring. MinC and MinD form polymeric assemblies in solution and on cell membranes. Here, we report the high‐resolution cryo‐EM structure of the copolymeric filaments of Pseudomonas aeruginosa MinCD. The filaments consist of three protofilaments made of alternating MinC and MinD dimers. The MinCD protofilaments are almost completely straight and assemble as single protofilaments on lipid membranes, which we also visualized by cryo‐EM.
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Jun 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15916]
Abstract: Structural maintenance of chromosomes (SMC)–kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC–kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head–kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.
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Mar 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Danguole
Kureisaite-Ciziene
,
Aravindan
Varadajan
,
Stephen H.
Mclaughlin
,
Marjolein
Glas
,
Alejandro
Montón Silva
,
Rosa
Luirink
,
Carolin
Mueller
,
Tanneke
Den Blaauwen
,
Tom N.
Grossmann
,
Joen
Luirink
,
Jan
Lowe
Open Access
Abstract: Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.
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Sep 2018
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