I04-Macromolecular Crystallography
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Jogi
Madhuprakash
,
Amirali
Toghani
,
Hsuan
Pai
,
Madia
Harvey
,
Adam R.
Bentham
,
Benjamin A.
Seager
,
Enoch Lok Him
Yuen
,
Juan Carlos
De La Concepcion
,
David M.
Lawson
,
Clare E. M.
Stevenson
,
Angel
Vergara-Cruces
,
Lida
Derevnina
,
Tolga O.
Bozkurt
,
Mark J.
Banfield
,
Sophien
Kamoun
,
Mauricio P.
Contreras
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. We show that an effector from the late blight pathogen Phytophthora infestans interacts with the host protein NbTOL9a and a helper NLR to suppress immunity. We solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that, unlike other RXLR-LWY effectors, AVRcap1b has a previously unidentified L-shaped fold that defines a distinct structural family of effectors in the genus Phytophthora. We defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that do not bind NbTOL9a, impairing immune suppression. This suggests that ENTH binding is required for full virulence activity. Last, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. We propose a model in which the effector interconnects NbNRC2 with the NbTOL9a pathway. Our results illustrate a previously uncharacterized pathogen mechanism to hijack NLR pathways and suppress immunity.
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Jun 2026
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I04-Macromolecular Crystallography
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Edward S.
Hems
,
Joseph A.
Wright
,
Sergey A.
Nepogodiev
,
Rebecca
Devine
,
Corinne J.
Arnold
,
David L.
Hughes
,
Julia E. A.
Mundy
,
Sandra
Eltschkner
,
David M.
Lawson
,
Matthew I.
Hutchings
,
Barrie
Wilkinson
Diamond Proposal Number(s):
[32728, 39308]
Open Access
Abstract: Formicamycins and their biosynthetic precursors, the fasamycins, form part of the phenylnaphthacenoid family of polyketide natural products. A recent atroposelective total synthesis of formicamycin H brought into question our original stereochemical assignment of the axially chiral linkage between C-6 and C-7. To address this, we obtained an X-ray crystal structure for formicamycin H that unambiguously confirmed our original assignment as the Sa atropisomer. X-ray structures for multiple additional fasamycins and formicamycins confirmed that this is common to all congeners. However, these studies identified a compounded error made by us whereby several structures previously reported as para-methoxy were found to have ortho-methoxy groups on the hanging E-ring. To address this for congeners that did not crystallize or gave nondiffracting crystals, we turned to the surprisingly underutilized 1,n-ADEQUATE NMR experiment. In total, we generated X-ray structures for 15 phenylnaphthacenoid metabolites and by combining these results report the corrected structures for three formicamycins, six fasamycins, and three biosynthetic lactone intermediates, noting that several revised fasamycin structures now match previously reported naphthacemycins. Our results highlight the utility of 1,n-ADEQUATE experiments for regiochemical determination in polysubstituted aromatic molecules. Moreover, our investigations uncovered a potential deracemization step during biosynthesis of the formicamycin framework.
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Apr 2026
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[34108]
Open Access
Abstract: Understanding the molecular basis of regulated nitrogen (N2) fixation is essential for engineering N2-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.
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Sep 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Thomas C.
Mclean
,
Francisco
Balaguer-Pérez
,
Joshua
Chandanani
,
Christopher M.
Thomas
,
Clara
Aicart-Ramos
,
Sophia
Burick
,
Paul Dominic B.
Olinares
,
Giulia
Gobbato
,
Julia E. A.
Mundy
,
Brian T.
Chait
,
David M.
Lawson
,
Seth A.
Darst
,
Elizabeth A.
Campbell
,
Fernando
Moreno-Herrero
,
Tung
Le
Diamond Proposal Number(s):
[25108]
Open Access
Abstract: Examples of long-range gene regulation in bacteria are rare and generally thought to involve DNA looping. Here, using a combination of biophysical approaches including X-ray crystallography and single-molecule analysis for the KorB–KorA system in Escherichia coli, we show that long-range gene silencing on the plasmid RK2, a source of multi-drug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We show that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters up to 1.5 kb away. We resolved the tripartite crystal structure of a KorB–KorA–DNA co-complex, revealing that KorA latches KorB into a closed clamp state. DNA-bound KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Together, our findings explain the mechanistic basis for KorB role switching from a DNA-sliding clamp to a co-repressor and provide an alternative mechanism for long-range regulation of gene expression in bacteria.
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Jan 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108]
Open Access
Abstract: In this Letter, we present a small series of novel bacterial topoisomerase inhibitors (NTBIs) that exhibit both potent inhibition of Mycobacterium tuberculosis DNA gyrase and potent antimycobacterial activity. The disclosed crystal structure of M. tuberculosis DNA gyrase in complex with DNA and compound 5 from this NBTI series reveals the binding mode of an NBTI in the GyrA binding pocket and confirms the presence and importance of halogen bonding for the excellent on-target potency. In addition, we have shown that compound 5 is a promising M. tuberculosis DNA gyrase inhibitor, with an IC50 for M. tuberculosis gyrase of 0.096 μM, and it has potent activity against M. tuberculosis, with an IC50 of 0.165 μM.
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Nov 2024
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I04-Macromolecular Crystallography
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Nace
Zidar
,
Andrej
Emanuel Cotman
,
Wessel
Sinnige
,
Ondrej
Benek
,
Michaela
Barančokova
,
Anamarija
Zega
,
Lucija
Peterlin Mašič
,
Tihomir
Tomašič
,
Janez
Ilaš
,
Sara R.
Henderson
,
Julia E. A.
Mundy
,
Anthony
Maxwell
,
Clare E. M.
Stevenson
,
David M.
Lawson
,
Geert
Jan Sterk
,
Rodrigo
Tosso
,
Lucas
Gutierrez
,
Ricardo D.
Enriz
,
Danijel
Kikelj
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.
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Jun 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108]
Open Access
Abstract: In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05–Rpn10vWA complex at 2.17 Å resolution and of the SAP05–SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a “loop surface” with three protruding loops that form electrostatic interactions with ZnF, and a “sheet surface” featuring two β-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin–proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.
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Dec 2023
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Jon
Agirre
,
Mihaela
Atanasova
,
Haroldas
Bagdonas
,
Charles B.
Ballard
,
Arnaud
Basle
,
James
Beilsten-Edmands
,
Rafael J.
Borges
,
David G.
Brown
,
J. Javier
Burgos-Marmol
,
John M.
Berrisford
,
Paul S.
Bond
,
Iracema
Caballero
,
Lucrezia
Catapano
,
Grzegorz
Chojnowski
,
Atlanta G.
Cook
,
Kevin D.
Cowtan
,
Tristan I.
Croll
,
Judit É.
Debreczeni
,
Nicholas E.
Devenish
,
Eleanor J.
Dodson
,
Tarik R.
Drevon
,
Paul
Emsley
,
Gwyndaf
Evans
,
Phil R.
Evans
,
Maria
Fando
,
James
Foadi
,
Luis
Fuentes-Montero
,
Elspeth F.
Garman
,
Markus
Gerstel
,
Richard J.
Gildea
,
Kaushik
Hatti
,
Maarten L.
Hekkelman
,
Philipp
Heuser
,
Soon Wen
Hoh
,
Michael A.
Hough
,
Huw T.
Jenkins
,
Elisabet
Jiménez
,
Robbie P.
Joosten
,
Ronan M.
Keegan
,
Nicholas
Keep
,
Eugene B.
Krissinel
,
Petr
Kolenko
,
Oleg
Kovalevskiy
,
Victor S.
Lamzin
,
David M.
Lawson
,
Andrey
Lebedev
,
Andrew G. W.
Leslie
,
Bernhard
Lohkamp
,
Fei
Long
,
Martin
Maly
,
Airlie
Mccoy
,
Stuart J.
Mcnicholas
,
Ana
Medina
,
Claudia
Millán
,
James W.
Murray
,
Garib N.
Murshudov
,
Robert A.
Nicholls
,
Martin E. M.
Noble
,
Robert
Oeffner
,
Navraj S.
Pannu
,
James M.
Parkhurst
,
Nicholas
Pearce
,
Joana
Pereira
,
Anastassis
Perrakis
,
Harold R.
Powell
,
Randy J.
Read
,
Daniel J.
Rigden
,
William
Rochira
,
Massimo
Sammito
,
Filomeno
Sanchez Rodriguez
,
George M.
Sheldrick
,
Kathryn L.
Shelley
,
Felix
Simkovic
,
Adam J.
Simpkin
,
Pavol
Skubak
,
Egor
Sobolev
,
Roberto A.
Steiner
,
Kyle
Stevenson
,
Ivo
Tews
,
Jens M. H.
Thomas
,
Andrea
Thorn
,
Josep Triviño
Valls
,
Ville
Uski
,
Isabel
Uson
,
Alexei
Vagin
,
Sameer
Velankar
,
Melanie
Vollmar
,
Helen
Walden
,
David
Waterman
,
Keith S.
Wilson
,
Martyn
Winn
,
Graeme
Winter
,
Marcin
Wojdyr
,
Keitaro
Yamashita
Open Access
Abstract: The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
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Jun 2023
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I03-Macromolecular Crystallography
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Maša
Sterle
,
Martina
Durcik
,
Clare E. M.
Stevenson
,
Sara
Henderson
,
Petra Eva
Szili
,
Marton
Czikkely
,
David M.
Lawson
,
Anthony
Maxwell
,
Dominique
Cahard
,
Danijel
Kikelj
,
Nace
Zidar
,
Csaba
Pal
,
Lucija Peterlin
Mašič
,
Janez
Ilaš
,
Tihomir
Tomašič
,
Andrej Emanuel
Cotman
,
Anamarija
Zega
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 μg/mL for most Gram-positive strains and 4–16 μg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.
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Jun 2023
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I03-Macromolecular Crystallography
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Mauricio P.
Contreras
,
Hsuan
Pai
,
Muniyandi
Selvaraj
,
Amirali
Toghani
,
David M.
Lawson
,
Yasin
Tumtas
,
Cian
Duggan
,
Enoch Lok Him
Yuen
,
Clare E. M.
Stevenson
,
Adeline
Harant
,
Abbas
Maqbool
,
Chih-Hang
Wu
,
Tolga O.
Bozkurt
,
Sophien
Kamoun
,
Lida
Derevnina
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.
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May 2023
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