Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[31371]
Open Access
Abstract: Magnetotactic bacteria, such as Magnetospirillum gryphiswaldense MSR-1, naturally produce magnetosomes—intracellular magnetic nanoparticles that enable navigation within geomagnetic fields. Magnetosomes hold significant potential for biomedical and biotechnological applications; however, key aspects of their biomineralization remain poorly understood. This study investigates how oxidative stress, induced by hydrogen peroxide and iron, influences magnetosome formation and bacterial physiology under aerobic and microaerobic conditions. Single-cell advanced microscopy and high-throughput techniques revealed that microaerobic conditions supported robust magnetosome production and larger magnetite crystals while maintaining low oxidative stress levels. In contrast, aerobic conditions suppressed magnetosome formation, reduced intracellular iron content, and increased reactive oxygen species (ROS) levels. High extracellular iron enhanced the formation of longer magnetosome chains in microaerobic cultures without causing toxicity but reduced cell viability under aerobic conditions. Hydrogen peroxide exposure caused mild damage and a 25% viability drop in magnetosome-producing cells but led to severe damage and an 80% viability drop in non-magnetosome-producing cells, along with chain fragmentation and smaller magnetite crystals. These results suggest that magnetosome-producing cells exhibit greater resilience to oxidative stress, potentially due to ROS scavenging properties of magnetosomes, and highlight the intricate interplay between oxidative stress, iron regulation, and magnetosome biomineralization. Single-cell analysis revealed heterogeneity in physiological responses, further demonstrating the complexity of these processes. These findings underscore the importance of monitoring physiological changes during production processes to enhance the efficiency and robustness of magnetosome synthesis. The insights gained provide a foundation for improving bioprocesses for large-scale production of high-quality magnetosomes, advancing their applications in biomedicine and biotechnology.
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Dec 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Paramyxoviral transmission between hosts may be, in part, attributed to the ability of the viral envelope-displayed receptor-binding protein (RBP) to bind to cell surface receptors of different host species. We sought to elucidate the architecture of the receptor-binding head region of the RBPs presented by jeilongviruses, a group of emerging and genetically unique paramyxoviruses belonging to the genus Jeilongvirus, family Paramyxoviridae. Structure determination of J and Beilong jeilongvirus RBPs reveals that the proteins exhibit a prototypical six-bladed β-propeller fold, present a binding site with residues associated with sialic acid recognition and hydrolysis, and bear a close structural relationship with sialic acid binding hemagglutinin-neuraminidase (HN)-type paramyxoviral RBPs. Additionally, unlike other paramyxoviruses, jeilongviruses encode an RBP with an unusually long C-terminal extension. In our dimeric Beilong virus RBP structure, we find that the C-terminal extension exchanges a hat-like domain with the central region of the β-propeller of the opposing protomer through domain-swapping. The hat-like domain occludes residues putatively associated with sialic acid binding and hydrolysis, providing a structural rationale for the absence of observed hemadsorption and neuraminidase activity. The insights gleaned from this analysis expand our appreciation of the structural palette available to the plastic paramyxoviral RBP and how their architectures may be adapted to regulate host-cell interactions at the cell surface.
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Nov 2025
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Krios I-Titan Krios I at Diamond
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Andreas
Schedlbauer
,
Xu
Han
,
Wouter
Van Bakel
,
Tatsuya
Kaminishi
,
Borja
Ochoa-Lizarralde
,
Idoia
Iturrioz
,
Retina
Çapuni
,
Ransford
Parry
,
Ronny
Zegarra
,
David
Gil-Carton
,
Jorge P.
López-Alonso
,
Kristina
Barragan Sanz
,
Letizia
Brandi
,
Claudio O.
Gualerzi
,
Paola
Fucini
,
Sean R.
Connell
Diamond Proposal Number(s):
[17171, 31586]
Open Access
Abstract: The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator fMet–tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B, and B1 with molecular masses of 643–658 Da). Previous biochemical and structural characterization of GE81112 indicates that the primary mechanism of action of this antibiotic is to (i) prevent the initiator fMet–tRNA from binding correctly to the P-site and (ii) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryo-EM, we have determined the binding site of GE81112 in initiation complexes (3.2–3.7 Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel but remote from the binding site of the initiation factors and initiator fMet–tRNA. This suggests that it acts allosterically to prevent the initiator fMet–tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.
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Oct 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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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
,
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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