Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20287, 26703]
Open Access
Abstract: Bacterial mating, or conjugation, was discovered nearly 80 years ago as a process transferring genes from one bacterial cell (the donor) to another (the recipient). It requires three key multiprotein complexes in the donor cell: a DNA-processing machinery called the relaxosome, a double-membrane spanning type 4 secretion system (T4SS), and an extracellular appendage termed pilus. While the near-atomic resolution structures of the T4SS and pilus are already known, that of the relaxosome has not been reported to date. Here, we describe the cryo-EM structure of the fully assembled relaxosome encoded by the paradigm F plasmid in two different states corresponding to distinct functional steps along the DNA processing reaction. By varying the structures of model DNAs we delineate conformational changes required to initiate conjugation. Mutational studies of the various protein-protein and protein-DNA interaction hubs suggest a complex sensitive to trigger signals, that could arise from cell-to-cell contacts with recipient cells.
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May 2025
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Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Nattapong
Sanguankiattichai
,
Balakumaran
Chandrasekar
,
Yuewen
Sheng
,
Nathan
Hardenbrook
,
Werner W. A.
Tabak
,
Margit
Drapal
,
Farnusch
Kaschani
,
Clemens
Grünwald-Gruber
,
Daniel
Krahn
,
Pierre
Buscaill
,
Suzuka
Yamamoto
,
Atsushi
Kato
,
Robert
Nash
,
George
Fleet
,
Richard
Strasser
,
Paul D.
Fraser
,
Markus
Kaiser
,
Peijun
Zhang
,
Gail M.
Preston
,
Renier A. L.
Van Der Hoorn
Diamond Proposal Number(s):
[21004, 29812, 28713]
Abstract: The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo–electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
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Apr 2025
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[31586]
Abstract: Human H ferritin (HuHf) has excellent potential as a nanocarrier for the selective delivery of anticancer metal-based drugs to tumour cells. Here, we addressed the interaction of the gold monocarbene compound Au(NHC)Cl with HuHf by electrospray ionization-mass spectrometry (ESI-MS) measurements, which provide the metalation state of the protein subunits and demonstrate the involvement of protein cysteines in gold binding. The adduct between Au(NHC)Cl and HuHf was studied by cryo-EM measurements, resulting in a high-resolution 3D density map at 1.51 Å. The cryo-EM structure shows a novel tetranuclear gold(I) cluster, located in a surface pocket of each subunit where it is bound to Cys90 and Cys102. The short inter-metal distances are diagnostic of the occurrence of aurophilic interactions. The present work demonstrates the usefulness of cryo-EM to investigate the interactions between metal-based drugs and their protein targets/carriers also leveraging the strong signal of transition metal ions.
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Apr 2025
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Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[19865]
Open Access
Abstract: Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized σ factor, σ54. σ54 recruits RNAP to promoters of genes associated with stress responses and forms a stable closed complex that does not spontaneously isomerize to the open state where promoter DNA is melted out and competent for transcription. The σ54-mediated open complex formation requires specific AAA+ proteins (ATPases Associated with diverse cellular Activities) known as bacterial enhancer-binding proteins (bEBPs). We have now obtained structures of new intermediate states of bEBP-bound complexes during transcription initiation, which elucidate the mechanism of DNA melting driven by ATPase activity of bEBPs and suggest a mechanistic model that couples the Adenosine triphosphate (ATP) hydrolysis cycle within the bEBP hexamer with σ54 unfolding. Our data reveal that bEBP forms a nonplanar hexamer with the hydrolysis-ready subunit located at the furthest/highest point of the spiral hexamer relative to the RNAP. ATP hydrolysis induces conformational changes in bEBP that drives a vectoral transiting of the regulatory N terminus of σ54 into the bEBP hexamer central pore causing the partial unfolding of σ54, while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of the bEBP AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many other AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.
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Apr 2025
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Zhuoyao
Chen
,
Gamma
Chi
,
Timea
Balo
,
Xiangrong
Chen
,
Beatriz Ralsi
Montes
,
Steven C.
Clifford
,
Vincenzo
D'Angiolella
,
Timea
Szabo
,
Arpad
Kiss
,
Tibor
Novak
,
András
Herner
,
András
Kotschy
,
Alex N.
Bullock
Diamond Proposal Number(s):
[34631]
Open Access
Abstract: Neomorphic mutations and drugs can elicit unanticipated effects that require mechanistic understanding to inform clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. Here, we identify that these neomorphic interactions are mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 capture 2:1 and 2:2 KBTBD4-HDAC2 complexes, as well as a 2:1:1 KBTBD4-HDAC2-CoREST1 complex, at resolutions spanning 2.7 to 3.3 Å. The mutant and drug-induced complexes adopt similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 is identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshapes the same surface of KBTBD4 providing an example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4, while structure-activity relationship (SAR) analysis of UM171 reveals analog S234984 as a more potent molecular glue for future studies.
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Apr 2025
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[30316]
Open Access
Abstract: Giant viruses of protists are a diverse and likely ubiquitous group of organisms. Here, we describe Jyvaskylavirus, the first giant virus isolated from Finland. This clade B marseillevirus was found in Acanthamoeba castellanii from a composting soil sample in Jyväskylä, Central Finland. Its genome shares similarities with other marseilleviruses. Helium ion microscopy and electron microscopy of infected cells unraveled stages of the Jyvaskylavirus life cycle. We reconstructed the Jyvaskylavirus particle to 6.3 Å resolution using cryo-electron microscopy. The ~2500 Å diameter virion displays structural similarities to other Marseilleviridae giant viruses. The capsid comprises of 9240 copies of the major capsid protein, encoded by open reading frame (ORF) 184, which possesses a double jellyroll fold arranged in trimers forming pseudo-hexameric capsomers. Below the capsid shell, the internal membrane vesicle encloses the genome. Through cross-structural and -sequence comparisons with other Marseilleviridae using AI-based software in model building and prediction, we elucidated ORF142 as the penton protein, which plugs the 12 vertices of the capsid. Five additional ORFs were identified, with models predicted and fitted into densities that either cap the capsomers externally or stabilize them internally. The isolation of Jyvaskylavirus suggests that these viruses may be widespread in the boreal environment and provide structural insights extendable to other marseilleviruses.
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Mar 2025
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[33824]
Abstract: Chloroplasts are specialised organelles found in plant cells and some algae. Photosynthesis, the process by which light energy is converted into chemical energy, resulting in the production of oxygen and energy-rich organic compounds, takes place in chloroplasts. The number of chloroplasts per plant cell can vary widely, ranging from one in unicellular algae to up to 100 in plants like Arabidopsis and wheat. Chloroplasts have a unique transcription machinery that is more complex than their cyanobacterial ancestors. The plastid-encoded RNA polymerase (PEP) is a multi-subunit complex crucial for transcribing chloroplast genes, which are essential for photosynthesis and plant growth. Despite its importance, the roles of many PEP-associated proteins (PAPs) are poorly understood. Researchers from the John Innes Centre and Diamond Light Source aimed to study the structure of PEP to better understand its composition, assembly, and function. They used cryo-Electron Microscopy (cryo-EM) at the electron Bio Imaging Centre (eBIC) to achieve this goal, providing a detailed view of the PEP complex and its interactions with DNA and RNA. Their work was recently published in Cell.
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Jan 2025
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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Huanyu Z.
Li
,
Ashley C.
Pike
,
Yung-Ning
Chang
,
Dheeraj
Prakaash
,
Zuzana
Gelova
,
Josefina
Stanka
,
Christophe
Moreau
,
Hannah C.
Scott
,
Frank
Wunder
,
Gernot
Wolf
,
Andreea
Scacioc
,
Gavin
Mckinley
,
Helena
Batoulis
,
Shubhashish
Mukhopadhyay
,
Andrea
Garofoli
,
Adán
Pinto-Fernández
,
Benedikt M.
Kessler
,
Nicola A.
Burgess-Brown
,
Saša
Štefanić
,
Tabea
Wiedmer
,
Katharina L.
Duerr
,
Vera
Puetter
,
Alexander
Ehrmann
,
Syma
Khalid
,
Alvaro
Ingles-Prieto
,
Giulio
Superti-Furga
,
David B.
Sauer
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamics simulations to probe SPNS2’s substrate binding and transport. These results reveal the transporter’s binding mode to its native substrate S1P, the therapeutic FTY720-P, and the reported SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein’s mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein’s export activity and thereby lead to hearing loss.
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Jan 2025
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
Krios IV-Titan Krios IV at Diamond
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Anokhi
Shah
,
Xiaoli
Zhang
,
Matthew
Snee
,
Michael P.
Lockhart-Cairns
,
Colin W.
Levy
,
Thomas A.
Jowitt
,
Holly L.
Birchenough
,
Louisa
Dean
,
Richard
Collins
,
Rebecca J.
Dodd
,
Abigail R. E.
Roberts
,
Jan J.
Enghild
,
Alberto
Mantovani
,
Juan
Fontana
,
Clair
Baldock
,
Antonio
Inforzato
,
Ralf P.
Richter
,
Anthony J.
Day
Diamond Proposal Number(s):
[22724, 29338, 17773, 24447]
Open Access
Abstract: Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter-α-inhibitor (IαI) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC•HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric ‘Half-PTX3’ and a construct missing the 24 N-terminal residues (Δ1-24-PTX3). SAXS determined a length of ∼520 Å for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC•HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC•HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC•HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.
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Jan 2025
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Matthew C.
Gaines
,
Michail N.
Isupov
,
Mathew
Mclaren
,
Clara L.
Mollat
,
Risat U. I.
Haque
,
Jake K.
Stephenson
,
Shamphavi
Sivabalasarma
,
Cyril
Hanus
,
Daniel
Kattnig
,
Vicki A. M.
Gold
,
Sonja
Albers
,
Bertram
Daum
Diamond Proposal Number(s):
[25452, 32707]
Open Access
Abstract: Archaea produce various protein filaments with specialised functions. While some archaea produce only one type of filament, the archaeal model species Sulfolobus acidocaldarius generates four. These include rotary swimming propellers analogous to bacterial flagella (archaella), pili for twitching motility (Aap), adhesive fibres (threads), and filaments facilitating homologous recombination upon UV stress (UV pili). Here, we use cryo-electron microscopy to describe the structure of the S. acidocaldarius archaellum at 2.0 Å resolution, and update the structures of the thread and the Aap pilus at 2.7 Å and 2.6 Å resolution, respectively. We define features unique to archaella of the order Sulfolobales and compare their structure to those of Aap and threads in the context of the S-layer. We define distinct N-glycan patterns in the three filaments and identify a putative O-glycosylation site in the thread. Finally, we ascertain whether N-glycan truncation leads to structural changes in archaella and Aap.
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Dec 2024
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