I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Most prokaryotes divide using filaments of the tubulin-like FtsZ protein, while some archaea employ instead ESCRT-III-like proteins and their filaments for cell division and cytokinesis. The alternative archaeal system comprises Cdv proteins and is thought to bear some resemblance to ESCRT-III-based membrane remodeling in other domains of life, including eukaryotes, especially during abscission. Here, we present biochemical, crystallographic, and cryo-EM studies of the Sulfolobus Cdv machinery. CdvA, an early non-ESCRT component, adopts a PRC‐domain/coiled-coil fold and polymerizes into long double-stranded helical filaments, mainly via hydrophobic interfaces. Monomeric CdvB adopts the canonical ESCRT-III fold in both a closed and a distinct “semiopen” conformation. Soluble CdvB2 filaments are composed of subunits in the closed state, appearing to transition to the open, active state only when polymerized on membranes. Short N-terminal amphipathic helices in all CdvB paralogues, B, B1, and B2, mediate membrane binding and are required for liposome recruitment in vitro. We provide a molecular overview of archaeal ESCRT-III-based cytokinesis machinery, the definitive demonstration that CdvB proteins are bona fide ESCRT-III homologues, and reveal the molecular basis for membrane engagement. Thus, we illuminate conserved principles of ESCRT-mediated membrane remodeling and extend them to an anciently diverged archaeal lineage.
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Jan 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26835]
Abstract: Pyruvate kinase (PK) is a crucial glycolytic protein involved in vital cellular processes ranging from cell proliferation to immune responses. The activity and functions of PK are tightly regulated by diverse mechanisms, including posttranslational Nϵ-lysine acetylation. Although previous studies have explored the impact of acetylation on selected lysine residues within the M2 isoform of PK (PKM2), a more comprehensive selection of acetylation sites and their respective effects on both PKM2 and the highly homologous PKM1 isoform is lacking. Here, we describe the structural, functional, and regulatory effects of site-specific acetylation on an expanded set of conserved lysines in PKM2 and selected lysines in PKM1. To study homogeneously acetylated proteins, we genetically encoded the incorporation of acetylated lysine into PKM variants expressed in bacteria and cultured mammalian cells. Our integrated biochemical, structural, and computational approach revealed K115 acetylation as an inhibitory modification in both PKM1 and PKM2 that stabilizes a closed active site conformation of the proteins. We also show that, in contrast to K115 acetylation, previously reported acetylation of K305 inhibits PKM2 but has no effect on the activity and oligomerization of PKM1. These findings propose the existence of both uniform and isoform-specific regulatory mechanisms of PKM, mediated by acetylation.
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Dec 2025
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I06-Nanoscience (XPEEM)
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Gregg
Wildenberg
,
Kevin M.
Boergens
,
Lola
Lambert
,
Ruiyu
Li
,
Allison
Craig
,
Michael K. L.
Man
,
Amin
Moradi
,
Janek
Rieger
,
Hengli
Duan
,
Sarnjeet S.
Dhesi
,
Gabriel
Karras
,
Francesco
Maccherozzi
,
Keshav
Dani
,
Rudolf
Tromp
,
Sense Jan
Van Der Molen
,
Sarah B.
King
,
Narayanan
Kasthuri
Diamond Proposal Number(s):
[40333]
Open Access
Abstract: Photoemission electron microscopy (PEEM) offers a potential third modality for large-volume connectomics alongside transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We image osmium stained, ultrathin brain sections on gold coated silicon at synaptic resolution using commercial PEEMs. At coarser resolution, we demonstrate that ultraviolet laser illumination enables gigavoxel-per-second acquisition rates without thermal damage. PEEM combines TEM-like parallel detection with SEM-compatible solid supports into a potentially scalable and cost-effective approach for large-volume connectomes.
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Nov 2025
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[28576]
Open Access
Abstract: Carboxysomes in cyanobacteria and certain proteobacteria enable efficient CO2 fixation by encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a semipermeable shell. Sequestered CA catalyze the rapid interconversion of CO2 and HCO3−, supplying elevated levels of CO2 to boost Rubisco carboxylation. Despite its essential role, the structure and encapsulation of CA within carboxysomes remain poorly understood. Here, we determined the molecular structure of α-carboxysomal CA from the model chemoautotrophic bacterium Halothiobacillus neapolitanus (HnCsoSCA). HnCsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that HnCsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. HnCsoSCA truncations suggest nonspecific interactions between HnCsoSCA and CsoS1A. We further show that HnCsoSCA bridges Rubisco and the shell facets. Our study offers insights into the assembly and encapsulation mechanisms of α-carboxysomes and provides the framework for reprogramming carboxysome structures for synthetic biology and biotechnological applications.
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Nov 2025
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I05-ARPES
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Cong
Li
,
Yang
Wang
,
Jianfeng
Zhang
,
Hongxiong
Liu
,
Wanyu
Chen
,
Guowei
Liu
,
Hanbin
Deng
,
Timur K.
Kim
,
Craig
Polley
,
Balasubramanian
Thiagarajan
,
Jiaxin
Yin
,
Youguo
Shi
,
Tao
Xiang
,
Oscar
Tjernberg
Diamond Proposal Number(s):
[34265, 39652]
Open Access
Abstract: For several decades, it was widely believed that a noninteracting disordered electronic system could only undergo an Anderson metal–insulator transition due to Anderson localization. However, numerous recent theoretical works have predicted the existence of a disorder-driven non-Anderson phase transition that differs from Anderson localization. The frustration lies in the fact that this non-Anderson disorder-driven transition has not yet been experimentally demonstrated in any system. Here, using angle-resolved photoemission spectroscopy, we present a case study of observing the non-Anderson disorder-driven transition by visualizing the electronic structure of the Weyl semimetal NdAlSi on surfaces with varying amounts of disorder. Our observations reveal that strong disorder can effectively suppress all surface states in the Weyl semimetal NdAlSi, including the topological surface Fermi arcs. This disappearance of surface Fermi arcs is associated with the vanishing of the topological invariant, indicating a quantum phase transition from a Weyl semimetal to a diffusive metal. These observations provide direct experimental evidence of the non-Anderson disorder-driven transition occurring in real quantum systems, a finding long anticipated by theoretical physicists.
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Oct 2025
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I04-Macromolecular Crystallography
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Anastasia
Polycarpou
,
Tara
Wagner-Gamble
,
Roseanna
Greenlaw
,
Lauren
O'Neill
,
Varsha
Kanabar
,
Alanoud
Alrehaili
,
Yusun
Jeon
,
Jonathan
Baker
,
Mona
Bafadhel
,
Hataf
Khan
,
Michael H.
Malim
,
Marco
Romano
,
Conrad A.
Farrar
,
Dorota
Smolarek
,
Rocio
Martinez-Nunez
,
Katie J.
Doores
,
Russell
Wallis
,
Linda S.
Klavinskis
,
Steven H.
Sacks
Open Access
Abstract: SARS-CoV-2 respiratory-tract infection affects both vaccinated and unvaccinated persons suggesting factors besides adaptive immunity are operative. We investigated the role of collectin-11 (CL-11), an epithelial-secreted carbohydrate-binding lectin that drives innate immunity and eliminates pathogens by complement activation. SARS-CoV-2, despite binding CL-11 to activate complement, was resistant to lysis. Remarkably, opsonization by CL-11 enhanced virus production by infected respiratory epithelial cells independently of complement. Furthermore, infected cells expressing SARS-CoV-2 spike protein displayed enhanced vulnerability to CL-11 binding and membrane attack by complement. The mechanism of enhanced infectivity was ablated in the presence of L-fucose, which occupied the extended carbohydrate-binding cleft of CL-11 in a crystallographic analysis of complexes between L-fucose and CL-11. Our study suggests pathogenicity of SARS-CoV-2 is related to complement-resistance together with enhanced infectivity and injury of respiratory epithelial cells mediated by locally released CL-11.
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Oct 2025
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labSAXS-Offline SAXS and Sample Environment Development
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Diamond Proposal Number(s):
[32442]
Open Access
Abstract: Spatial ordering of matter elicits exotic properties sometimes absent from a material’s constituents. A few highly mineralized natural materials achieve high toughness through delocalized damage, whereas synthetic particulate composites must trade toughness for mineral content. We test whether ordering the mineral phase in particulate composites through the formation of macroscopic colloidal crystals can trigger the same damage resistance found in natural materials. Our macroscopic silica rod-based anisotropic colloidal crystal composites are processed fully at room temperature and pressure, reach volume fractions of mineral higher than 80%, and aided by a ductile interface, unveil toughness up to two orders of magnitude higher than bulk silica through the collective movement of rods and damage delocalization over millimeter. These composites demonstrate key design rules to break free from conventionally accepted structural materials’ properties trade-off.
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Jun 2025
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B21-High Throughput SAXS
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Alexander
Leithner
,
Oskar
Staufer
,
Tanmay
Mitra
,
Falk
Liberta
,
Salvatore
Valvo
,
Mikhail
Kutuzov
,
Hannah
Dada
,
Jacob
Spaeth
,
Weijie
Zhou
,
Felix
Schiele
,
Sophia
Reindl
,
Herbert
Nar
,
Stefan
Hoerer
,
Maureen
Crames
,
Stephen
Comeau
,
David
Young
,
Sarah
Low
,
Edward
Jenkins
,
Simon J.
Davis
,
David
Klenerman
,
Andrew
Nixon
,
Noah
Pefaur
,
David
Wyatt
,
Omer
Dushek
,
Srinath
Kasturirangan
,
Michael L.
Dustin
Diamond Proposal Number(s):
[26256]
Open Access
Abstract: Bispecific T cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact (≤13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A–D) targeting CD3ε and Her2, designed to create varying intermembrane distances (A < B < C < D). Small-angle X-ray scattering (SAXS) and modeling of the conformational states of isolated TcEs and TcE–antigen complexes predicted close contacts (≤13 nm) for Formats A and B and far contacts (≥18 nm) for Formats C and D. In supported lipid bilayer (SLB) model interfaces, Formats A and B recruited, whereas Formats C and D repelled, CD2–CD58 interactions. Formats A and B also excluded bulky Quantum dots more effectively. SAXS also revealed that TcE–antigen complexes formed by Formats A and C were less flexible than complexes formed by Formats B and D. Functional data with Her2-expressing tumor cells showed cytotoxicity, surface marker expression, and cytokine release following the order A > B = C > D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A = B = C > D. Addition of close contact requiring CD58 costimulation revealed phospholipase C-γ activation matching cytotoxicity with A > B = C > D. Our findings suggest that when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close contact/high flexibility) and C (far contact/low flexibility).
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Jun 2025
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I04-Macromolecular Crystallography
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Jonathan J.
Ford
,
Javier
Santos-Aberturas
,
Edward S.
Hems
,
Joseph W.
Sallmen
,
Lena A. K.
Bögeholz
,
Guy
Polturak
,
Anne
Osbourn
,
Joseph A.
Wright
,
Marina V.
Rodnina
,
Danny
Vereecke
,
Isolde M.
Francis
,
Andrew W.
Truman
Diamond Proposal Number(s):
[32728]
Open Access
Abstract: The natural products actinonin and matlystatin feature an N-hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesized to produce HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make an HPS-like chemophore informed the structural recharacterization of lydiamycin via NMR and crystallography to show that it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing an uncommon self-immunity mechanism associated with the production of a PDF inhibitor. In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonization. This study highlights how a BGC can inform the structure, biochemical target, and ecological function of a natural product.
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May 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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