Krios I-Titan Krios I at Diamond
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Pavol
Bardy
,
Conor I. W.
Macdonald
,
Paul C.
Kirchberger
,
Huw T.
Jenkins
,
Tibor
Botka
,
Lewis
Byrom
,
Nawshin T. B.
Alim
,
Daouda A. K.
Traore
,
Hannah C.
Koenig
,
Tristan R.
Nicholas
,
Maria
Chechik
,
Samuel J.
Hart
,
Johan P.
Turkenburg
,
James N.
Blaza
,
J. Thomas
Beatty
,
Paul C. M.
Fogg
,
Alfred A.
Antson
Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Microviruses are single-stranded DNA viruses infecting bacteria, characterized by T = 1 shells made of single jelly-roll capsid proteins. To understand how microviruses infect their host cells, we have isolated and studied an unusually large microvirus, Ebor. Ebor belongs to the proposed “Tainavirinae” subfamily of Microviridae and infects the model Alphaproteobacterium Rhodobacter capsulatus. Using cryogenic electron microscopy, we show that the enlarged capsid of Ebor is the result of an extended C-terminus of the major capsid protein. The extra packaging space accommodates genes encoding a lytic enzyme and putative methylase, both absent in microviruses with shorter genomes. The capsid is decorated with protrusions at its 3-fold axes, which we show to recognize lipopolysaccharides on the host surface. Cryogenic electron tomography shows that during infection, Ebor attaches to the host cell via five such protrusions. This attachment brings a single pentameric capsomer into close contact with the cell membrane, creating a special vertex through which the genome is ejected. Both subtomogram averaging and single particle analysis identified two intermediates of capsid opening, showing that the interacting penton opens from its center via the separation of individual capsomer subunits. Structural comparison with the model Bullavirinae phage phiX174 suggests that this genome delivery mechanism may be widely present across Microviridae.
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Mar 2025
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I03-Macromolecular Crystallography
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Shelby D.
Foor
,
Kalvis
Brangulis
,
Anil K.
Shakya
,
Vipin S.
Rana
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Sandhya
Bista
,
Chrysoula
Kitsou
,
Michael
Ronzetti
,
Adit B.
Alreja
,
Sara B.
Linden
,
Amanda S.
Altieri
,
Bolormaa
Baljinnyam
,
Inara
Akopjana
,
Daniel C.
Nelson
,
Anton
Simeonov
,
Osnat
Herzberg
,
Melissa J.
Caimano
,
Utpal
Pal
Diamond Proposal Number(s):
[35587]
Open Access
Abstract: Borrelia burgdorferi, the pathogen of Lyme disease, encodes many conserved proteins of unknown structure or function, including ones that serve essential roles in microbial infectivity. One such protein is BB0238, which folds into a two-domain protein, as we have determined by X-ray crystallography and AlphaFold analysis. The N-terminal domain begins with a helix-turn-helix motif (HTH), previously referred to as a tetratricopeptide repeat (TPR) motif, known to mediate protein-protein interactions. The fold of the C-terminal domain has been seen in proteins with a range of unrelated activities and thus does not infer function. In addition to its previously known binding partner BB0323, another essential borrelial virulence determinant, we show that BB0238 also binds a second protein, BB0108, a borrelial ortholog of the chaperone protein SurA and the peptidyl-prolyl cis/trans isomerase protein PrsA. An in vitro enzymatic assay confirmed the catalytic activity. We also determined the crystal structure of the catalytic domain of BB0108, which revealed the parvulin-type organization of the key catalytic residues. We show that BB0238 influences the proteolytic processing of BB0323, although the TPR/HTH motif is not involved in the process. Instead, we show that the motif stabilizes BB0238 in the host environment and facilitates tick-to-mouse pathogen transmission by aiding spirochete evasion of early host cellular immunity. Taken together, these studies highlight the biological significance of BB0238 and its interactions with multiple B. burgdorferi proteins essential for microbial infection.
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Oct 2023
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein (RBP), which facilitates host-cell attachment and plays a fundamental role in determining host range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently defined Narmovirus genus within the Paramyxoviridae family. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed β-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed β-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small-angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs.
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Sep 2023
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[21004, 22941, 22910]
Open Access
Abstract: Motile bacteria employ conserved chemotaxis networks to detect chemical gradients in their surroundings and effectively regulate their locomotion, enabling the location of essential nutrients and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSUs) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein, CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage φX174 E protein lysed Escherichia coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12-Å resolution. Using AlphaFold2, we further predict the atomic structures of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryo-electron tomography map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand-binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.
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Sep 2023
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Open Access
Abstract: Pathogenic spirochetes can alter their morphologies and behaviors to infect and survive within their hosts. Previous reports demonstrate that the formation of the so-called “round bodies” and biofilms, and chemotaxis are involved in spirochete pathogenesis. Here, we report a direct link between these cellular states that involve a new class of protein sensor with hitherto unclear function. Using cryo-electron microscopy, genetics, behavioral assays, and molecular modeling, we demonstrate that spirochetes regulate these behaviors in response to the small molecule S-adenosylmethionine (SAM) via a SAM sensor that is anchored to chemotaxis arrays. Furthermore, we establish an improved model for round body formation that now includes characterizations during log phase growth.
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Aug 2023
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I04-Macromolecular Crystallography
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Matthew
Singer
,
Tung
Dinh
,
Lev
Levintov
,
Arun S.
Annamalai
,
Juan S.
Rey
,
Lorenzo
Briganti
,
Nicola J.
Cook
,
Valerie E.
Pye
,
Ian A.
Taylor
,
Kyungjin
Kim
,
Alan N.
Engelman
,
Baek
Kim
,
Juan R.
Perilla
,
Mamuka
Kvaratskhelia
,
Peter
Cherepanov
Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target.
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Feb 2023
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment.
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Jan 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19242]
Open Access
Abstract: The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of ‘omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how “underground metabolism,” facilitated by enzyme substrate promiscuity, “rewires” Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism.
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Nov 2022
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I03-Macromolecular Crystallography
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Yao
Zhou
,
Kaizhou
Yan
,
Qijian
Qin
,
Olawale G.
Raimi
,
Chao
Du
,
Bin
Wang
,
Chukwuemeka Samson
Ahamefule
,
Bartosz
Kowalski
,
Cheng
Jin
,
Daniel M. F.
Van Aalten
,
Wenxia
Fang
Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals.
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Aug 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate.
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Jun 2022
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