Krios I-Titan Krios I at Diamond
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Tarik
Husremović
,
Vanessa
Meier
,
Lucas
Piëch
,
Katharina M.
Siess
,
Sumire
Antonioli
,
Irina
Grishkovskaya
,
Nikoleta
Kircheva
,
Silvia E.
Angelova
,
Karoline
Wenzl
,
Andreas
Brandstätter
,
Jiri
Veis
,
Fran
Miočić-Stošić
,
Dorothea
Anrather
,
Markus
Hartl
,
Linda
Truebestein
,
Luis M.
Cerron-Alvan
,
Martin
Leeb
,
Bojan
Žagrović
,
Stephan
Hann
,
Christoph
Bock
,
Egon
Ogris
,
Todor
Dudev
,
Nicholas A. T.
Irwin
,
David
Haselbach
,
Thomas A.
Leonard
Diamond Proposal Number(s):
[25222]
Open Access
Abstract: The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. Pleckstrin homology domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly found that PHLPP2 has no catalytic activity in vitro. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1,000 Mya, but that lost activity at the base of the metazoan lineage. Surface conservation indicates that while PHLPP2 has lost catalytic activity, it may have retained substrate binding. Finally, using phylogenomics, we identify coevolving genes consistent with a scaffolding role for PHLPP2 on membranes. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a focus on the noncatalytic roles of PHLPP1 and PHLPP2.
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Apr 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[36082, 30015]
Abstract: The distribution of sugars is crucial for plant energy, signaling, and defense mechanisms. Sugar Transport Proteins (STPs) are Sugar Porters (SPs) that mediate proton-driven cellular uptake of glucose. Some STPs also transport fructose, while others remain highly selective for only glucose. What determines this selectivity, allowing STPs to distinguish between compounds with highly similar chemical composition, remains unknown. Here, we present the structure of Arabidopsis thaliana STP6 in an inward-occluded conformational state with glucose bound and demonstrate its role as both a glucose and fructose transporter. We perform a comparative analysis of STP6 with the glucose-selective STP10 using in vivo and in vitro systems, demonstrating how different experimental setups strongly influence kinetic transport properties. We analyze the properties of the monosaccharide binding site and show that the position of a single methyl group in the binding site is sufficient to shuffle glucose and fructose specificity, providing detailed insights into the fine-tuned dynamics of affinity-induced specificity for sugar uptake. Altogether, these findings enhance our understanding of sugar selectivity in STPs and more broadly SP proteins.
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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
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Naito
Ishimoto
,
Joshua L. C.
Wong
,
Shan
He
,
Sally
Shirran
,
Olivia
Wright-Paramio
,
Chloe
Seddon
,
Nanki
Singh
,
Carlos
Balsalobre
,
Ravi R.
Sonani
,
Abigail
Clements
,
Edward H.
Egelmane
,
Gad
Frankel
,
Konstantinos
Beis
Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor–recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF and describe its cryoelectron-microscopic (cryo-EM) structure at 2.2 Å resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stoichiometric ratio of 1:1. While there were previous reports that the T-pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N and C termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxyl residues. Mutagenesis experiments revealed that loss of cyclization abolished pilus biogenesis and efficient plasmid transfer. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination.
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Apr 2025
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[34473]
Open Access
Abstract: Molluscan brains are composed of morphologically consistent and functionally interrogable neurons, offering rich opportunities for understanding how neural circuits drive behavior. Nonetheless, detailed component-level CNS maps are often lacking, total neuron numbers are unknown, and organizational principles remain poorly defined, limiting a full and systematic characterization of circuit operation. Here, we establish an accessible, generalizable approach, harnessing synchrotron X-ray tomography, to rapidly determine the three-dimensional structure of the multimillimeter-scale CNS of Lymnaea. Focusing on the feeding ganglia, we generate a full neuron-level reconstruction, revealing key design principles and revising cell count estimates upward threefold. Our atlas uncovers the superficial but also nonsuperficial ganglionic architecture, reveals the cell organization in normally hidden regions—ganglionic “dark sides”—and details features of single-neuron morphology, together guiding targeted follow-up functional investigation based on intracellular recordings. Using this approach, we identify three pivotal neuron classes: a command-like food-signaling cell type, a feeding central pattern generator interneuron, and a unique behavior-specific motoneuron, together significantly advancing understanding of the function of this classical control circuit. Combining our morphological and electrophysiological data, we also establish a functional CNS atlas in Lymnaea as a shared and scalable resource for the research community. Our approach enables the rapid construction of cell atlases in large-scale nervous systems, with key relevance to functional circuit interrogation in a diverse range of model organisms.
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Mar 2025
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mélanie
Loiodice
,
Elodie
Drula
,
Zak
Mciver
,
Svetlana
Antonyuk
,
Arnaud
Basle
,
Marcelo
Lima
,
Edwin A.
Yates
,
Dominic P.
Byrne
,
Jamie
Coughlan
,
Andrew
Leech
,
Shahram
Mesdaghi
,
Daniel J.
Rigden
,
Sophie
Drouillard
,
William
Helbert
,
Bernard
Henrissat
,
Nicolas
Terrapon
,
Gareth S. A.
Wright
,
Marie
Couturier
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970, 32677, 28406]
Open Access
Abstract: Acidic glycans are essential for the biology of multicellular eukaryotes. To utilize them, microbial life including symbionts and pathogens has evolved polysaccharide lyases (PL) that cleave their 1,4 glycosidic linkages via a β-elimination mechanism. PL family 33 (PL33) enzymes have the unusual ability to target a diverse range of glycosaminoglycans (GAGs), as well as the bacterial polymer, gellan gum. In order to gain more detailed insight into PL33 activities we recombinantly expressed 10 PL33 members derived from all major environments and further elucidated the detailed biochemical and biophysical properties of five, showing that their substrate specificity is conferred by variations in tunnel length and topography. The key amino acids involved in catalysis and substrate interactions were identified, and employing a combination of complementary biochemical, structural, and modeling approaches, we show that the tunnel topography is induced by substrate binding to the glycan. Structural and bioinformatic analyses revealed that these features are conserved across several lyase families as well as in mammalian GAG epimerases.
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Feb 2025
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I24-Microfocus Macromolecular Crystallography
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Shangwen
Luo
,
Xin-Rong
Li
,
Xiao-Tong
Gong
,
Alexey
Kulikovsky
,
Feng
Qu
,
Konstantinos
Beis
,
Konstantin
Severinov
,
Svetlana
Dubiley
,
Xinxin
Feng
,
Shi-Hui
Dong
,
Satish K.
Nair
Abstract: Infections caused by gram-negative pathogens continue to be a major risk to human health because of the innate antibiotic resistance endowed by their unique cell membrane architecture. Nature has developed an elegant solution to target gram-negative strains, namely by conjugating toxic antibiotic warheads to a suitable carrier to facilitate the active import of the drug to a specific target organism. Microcin C7 (McC) is a Trojan horse peptide–conjugated antibiotic that specifically targets enterobacteria by exploiting active import through oligopeptide transport systems. Here, we characterize the molecular mechanism of McC recognition by YejA, the solute binding protein of the Escherichia coli oligopeptide transporter. Structure-guided mutational and functional analysis elucidates the determinants of substrate recognition. We demonstrate that the peptide carrier can serve as a passport for the entry of molecules that are otherwise not taken into E. coli cells. We show that peptide conjugation can remodel the antibiotic spectrum of clinically relevant parent compounds. Bioinformatics analysis reveals a broad distribution of YejA-like transporters in only the Proteobacteria, underscoring the potential for the development of Trojan horse antibiotics that are actively imported into such gram-negative bacteria.
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Jan 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23248]
Abstract: Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer.
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Dec 2024
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I23-Long wavelength MX
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Diamond Proposal Number(s):
[31800]
Open Access
Abstract: Metal ions have important roles in supporting the catalytic activity of DNA-regulating enzymes such as topoisomerases (topos). Bacterial type II topos, gyrases and topo IV, are primary drug targets for fluoroquinolones, a class of clinically relevant antibacterials requiring metal ions for efficient drug binding. While the presence of metal ions in topos has been elucidated in biochemical studies, accurate location and assignment of metal ions in structural studies have historically posed significant challenges. Recent advances in X-ray crystallography address these limitations by extending the experimental capabilities into the long-wavelength range, exploiting the anomalous contrast from light elements of biological relevance. This breakthrough enables us to confirm experimentally the locations of Mg2+ in the fluoroquinolone-stabilized Streptococcus pneumoniae topo IV complex. Moreover, we can unambiguously identify the presence of K+ and Cl- ions in the complex with one pair of K+ ions functioning as an additional intersubunit bridge. Overall, our data extend current knowledge on the functional and structural roles of metal ions in type II topos.
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Oct 2024
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Roi
Asor
,
Anna
Olerinyova
,
Sean A.
Burnap
,
Manish S.
Kushwah
,
Fabian
Soltermann
,
Lucas S. P.
Rudden
,
Mario
Hensen
,
Mario
Hensen
,
Snežana
Vasiljevic
,
Juliane
Brun
,
Michelle
Hill
,
Liu
Chang
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Juthathip
Mongkolsapaya
,
Daming
Zhou
,
David I.
Stuart
,
Gavin R.
Screaton
,
Matteo T.
Degiacomi
,
Nicole
Zitzmann
,
Justin L. P.
Benesch
,
Weston B.
Struwe
,
Philipp
Kukura
Open Access
Abstract: Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.
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Oct 2024
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