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Christopher D. O.
Cooper
,
Evgeny
Tankhilevich
,
Kim
Remans
,
Ahmed
El Marjou
,
Aled M.
Edwards
,
Andrew
Leach
,
Andrew
Quigley
,
Anja
Schutz
,
Anne
Zemella
,
Ario
De Marco
,
Åsa
Sivertsson
,
Barbara
Borgonovo
,
Ben
Kemp
,
Bjørn
Voldborg
,
Carlo
Carolis
,
Carter
Mitchell
,
Cecilia
Wickstrand
,
Dafydd R.
Owen
,
David
Drechsel
,
David
Jones
,
Diego A.
Oyarzún
,
Dominic
Esposito
,
Eric
Geertsma
,
Ewa
Krupinska
,
Frank
Bernhard
,
Frederico
Ferreira-Da-Silva
,
Hanna
Tegel
,
Ian
Hunt
,
Imre
Berger
,
James
Love
,
Jan
Dohnalek
,
Jelena
Thies
,
Jerome
Basquin
,
Jesse
Coker
,
Johannes
Buyel
,
Joop
Van Den Heuvel
,
Jurgen
Haustraete
,
Katharina L.
Dürr
,
Kelvin
Lau
,
Kristina
Hedfalk
,
Kristof
Bozovicar
,
Lihua
Liu
,
Lisa
Bamber
,
Luigi Angelo
Scietti
,
Malin
Bäckström
,
Maren
Schubert
,
Mark
Elvin
,
Martin
Pelosse
,
Matthew
Todd
,
Matthieu
Schapira
,
Miroslava
Alblova
,
Nicholas S.
Berrow
,
Nikolay
Marinchev Dobrev
,
Oleg
Brodsky
,
Ondřej
Vaněk
,
Opher
Gileadi
,
Pam
Dossang
,
Paola
Storici
,
Patrick
Celie
,
Paul
Wan
,
Peter A.
Loppnau
,
Rachel
Harding
,
Ray
Owens
,
Renaud
Vincentelli
,
Richard
Altman
,
Rick
Davies
,
Rob
Meijers
,
Robbert
Kim
,
Ruth
Saxl
,
Sabine
Suppmann
,
Sameer
Velankar
,
Sebastiano
Pasqualato
,
Sergio
Martinez Cuesta
,
Stephane
Petres
,
Steven
Harborne
,
Susanne
Witt
,
Svend
Kjaer
,
Tamar
Unger
,
Timothy
Craig
,
Tsafi
Danieli
,
Ugis
Sarkans
,
Wolfgang
Knecht
,
Wolfgang
Kuttenlochner
,
Yoav
Peleg
,
Lovisa
Holmberg Schiavone
,
Nicola A.
Burgess-Brown
Open Access
Abstract: As machine learning (ML) becomes increasingly important in protein engineering, synthetic biology, and bioprocess optimisation, the lack of consistent, complete, and standardised experimental reporting remains a major barrier to building effective models. EXPERT (EXpression and Purification Experimental Reporting Template) defines a structured framework for capturing protein expression and purification data across the community, to increase reproducibility across laboratories and support downstream ML applications. We propose here the essential data that should be recorded during expression and purification workflows by both novice and experienced protein production scientists. Its recommendations are informed by expert contributions from across academia and industry. The reporting requirements were distilled to a focused set of parameters deemed sufficient for workflow reproducibility and constructing robust ML models aimed at improving protein production. By deploying these guidelines, we hope to enable the collection of larger data sets to support future model building.
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Sep 2026
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B21-High Throughput SAXS
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Antonin
Galien
,
Stephanie
Hutin
,
Sarah
Le Hir
,
Michel
Paul
,
Pascal
Albanese
,
Sylvie
Kieffer-Jaquinod
,
Audrey
Guillotin
,
Nozomu
Iwabuchi
,
Kensaku
Maejima
,
Max H.
Nanao
,
Mark D.
Tully
,
Veronique
Hugouvieux
,
Chloe
Zubieta
Open Access
Abstract: Phytoplasmas are obligate plant pathogens that include species causing severe developmental abnormalities in diverse plant families. Phytoplasma infections may result in dwarfism, witches’ broom, virescence and phyllody, the conversion of reproductive organs into leaf-like structures. Phyllody is due to the activity of phytoplasma effector proteins, called phyllogens, that interact with the host MADS transcription factor (MTF) network. This interaction was believed to be specific for members of the AP1/SEP/AGL6 superclade of tetrameric MTFs, key regulators of floral meristem identity and organogenesis. Here, we determined the molecular and atomic basis for the interaction between the phyllogen from Onions Yellow phytoplasma (PHYLOY) and the floral MTFs, APETALA1, SEPALLATA and AGAMOUS using biochemical, structural and genetic experiments. The DNA-binding patterns of homo and heteromeric MTFs with PHYLOY are shown using electrophoretic mobility shift assays, revealing broader interaction patterns between PHYLOY and MTFs that depend on highly conserved hydrophobic residues. Point mutations of these residues allowed gain or loss of interaction between MTFs and PHYLOY. Expressing PHYLOY under the control of the SEP3 and AP1 promoter in Arabidopsis further demonstrated whorl specific phenotypes, recapitulating the in vitro interaction and DNA-binding patterns. These findings reveal how PHYLOY targets specific MTFs through structural mimicry, resulting in impaired MTF tetramer formation and homeotic conversion of floral organs due to loss of active MTF tetramers.
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Aug 2026
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Chitoporins (ChiPs) are chitooligosaccharide-specific outer membrane porins found in various Gram-negative bacteria and are critical for marine species that use chitin as a carbon and nitrogen source. Vibrio spp. ChiPs, exemplified by VhChiP from V. harveyi, are trimeric OmpC-like 16-stranded β-barrels with a long pore-confining loop L3 that interacts exclusively with long chain chitooligosaccharides. Here, using crystallography and electrophysiology, we show that two atypical ChiPs, Escherichia coli ChiP (EcChiP) and Serratia marcescens ChiP (SmChiP), are monomeric 18-stranded β-barrels with shorter L3 loops, which display weaker and less selective substrate interactions compared with their Vibrio counterparts. Structure-based phylogenetic analysis of bacterial ChiPs resolves three clades: monomeric ChiPs; clade A, trimeric ChiPs with an N-plug; and clade B, trimeric ChiPs lacking the characterized helical N-plug. These architectural differences likely reflect environmental selection. Marine Vibrio spp., living in chitin-rich environments, possess trimeric ChiPs that enable rapid chitin uptake, whereas soil-borne S. marcescens and multi-habitat E. coli encode monomeric ChiPs that we speculate are more suited to variable substrate availability.
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Aug 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. The environmental bacterial pathogen Chromobacterium violaceum possesses an active type VI secretion system (T6SS), which relies mainly on VgrG3 for its activity and role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and can intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented by RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 Å resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays indicated that RhsF-CT ADP-ribosylates RNA in vitro and that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.
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Jun 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844, 33658]
Open Access
Abstract: TonB-dependent uptake systems of gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrients—primarily siderophore-bound iron—to cross the highly impermeable outer membrane. Remarkably, the ferredoxin uptake system (Fus) of certain soft rot plant pathogens imports an entire host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homolog, plays two roles in facilitating import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the outer membrane receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. Here, we describe structures of the FusB homodimer as well as the FusB-ferredoxin complex and, using biophysical, biochemical, and mutagenesis approaches, we determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. The C-terminal domain of FusB (FusB-CTD) exists in a monomer-dimer equilibrium in vitro, with the homodimer stabilized by an intermolecular R241-D322 salt bridge. The “FusB-box” of FusA interacts with monomeric FusB-CTD, and FusA D53 outcompetes FusB D322 to bind R241. Upon ferredoxin binding, FusB-CTD undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces FusA from FusB with R241 forming an intramolecular salt bridge with D322 to stabilize the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import where FusB R241 acts as a molecular switch.
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Jun 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39641]
Open Access
Abstract: The β-barrel assembly machinery (BAM) inserts outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. In well-studied organisms such as Escherichia coli, BAM consists of BamA and four accessory lipoproteins (BamB-E) that coordinate substrate recognition, membrane remodeling, and β-barrel insertion. In the Lyme disease spirochete Borrelia burgdorferi, however, the BAM system is simplified, comprising only BamA (BB0795), BamD (BB0324), and BamB (BB0028), while lacking BamC and BamE. In addition, B. burgdorferi encodes a TamB homolog (BB0794), suggesting a hybrid BAM-TAM organization.
Here, we define the structural architecture of the spirochetal BAM-TAM system. AlphaFold3 modeling predicts that BamD binds to BamA periplasmic domains POTRA4-5, while BamB interacts with POTRA1, POTRA3, POTRA4, and POTRA5, forming a closed periplasmic ring distinct from the enterobacterial arrangement. Microscale thermophoresis confirms micromolar-affinity binding of BamD to BamA POTRA3-5, no detectable interaction with POTRA1-3, and loss of POTRA3-5 interaction upon mutation of key interface residues. Crystal structures of BamA POTRA1-2 and POTRA2-3 further define the architecture and flexibility of the periplasmic region.
Strikingly, structural modeling of the full complex suggests that the C-terminal β-strands of TamB complete the BamA β-barrel through β-strand augmentation, forming a continuous hybrid barrel with a hydrophobic exterior and hydrophilic interior. This arrangement may provide a direct structural link between periplasmic protein handling and membrane insertion.
Together, these findings support a reorganized BAM architecture in B. burgdorferi and suggest that the hybrid BAM-TAM organization is conserved across Lyme disease-associated Borrelia species, providing a framework for understanding outer membrane assembly in spirochetes.
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Jun 2026
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[31850]
Open Access
Abstract: Chordin is a cysteine-rich protein which acts as a regulator of bone morphogenetic protein (BMP) signalling in the extracellular matrix. Acting in concert with twisted gastrulation (TWSG1), chordin works as an antagonist of BMP signalling by binding tightly to the growth factor and is a vital component of the network of interactions that establish developmental signalling gradients. Chordin is known to interact with BMP ligands via its four von-Willebrand factor type C domains, but the function of the large central four CHRD domains were previously unknown. Here we show that these domains interact strongly with sulphated glycosaminoglycans (GAGs) and provide evidence for the location of the binding site using X-ray crystallographic analysis combined with mutagenesis and biophysical techniques. Additionally, we report the first recombinant expression and purification of the complete functional chordin, TWSG1, BMP2, BMP7 complex which was used to demonstrate that the four CHRD domains are largely redundant with respect to the role of chordin as an inhibitor of BMP ligands. We therefore propose that the four CHRD domains of chordin have relevance in the diffusion and localisation of chordin-TWSG1-BMP complexes at the tissue and organismal level, mediated by their interaction with GAGs or proteoglycans.
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Jun 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035]
Open Access
Abstract: The protein kinase C-related kinase (PKN) family of serine/threonine kinases consists of PKN1, PKN2 and PKN3, all of which are Rho family GTPase effectors. PKNs have three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils, which in two cases interact with Rho family GTPases, activating the kinase. The PKNs are implicated in several important cellular processes, including cytoskeletal regulation, cell adhesion, gene expression and cell cycle progression, and are also implicated in cancer. Here we have investigated the roles of the HR1 domains in PKN oligomerisation. We show that PKN1 HR1a is a dimer and that the HR1c domain drives further oligomerization. We have mapped the interactions between the HR1 domains and used an integrative approach to model HR1-containing PKN1 dimers. Biophysical analysis shows that RhoA forms a 1:2 complex with HR1a, resulting in a rearrangement of the HR1a dimer, an outcome supported by SAXS models. In contrast, Rac1 binds to monomeric HR1a, suggesting that this GTPase activates PKN1 via a different mechanism. These data provide structural insight into interactions between HR1 domains and the Rho family proteins and their potential consequences for PKN1 activation.
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Apr 2026
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I03-Macromolecular Crystallography
|
Open Access
Abstract: The minichromosome maintenance (MCM) complex is the replicative helicase in eukaryotes and archaea, unwinding genomic DNA upstream of DNA polymerase. The eukaryotic MCM complex forms from six different subunits (Mcm2–7), whereas in archaea, the complex is homohexameric. Both types of MCM can assemble into functional helicases in vitro in the absence of cofactors. However, despite being simpler in composition, we know little about how a homohexameric archaeal MCM assembles, largely because the field has lacked a convenient system to interrogate. Historically, characterization of archaeal MCMs has focused on proteins from thermophilic organisms, which typically form robust oligomers in solution. We have identified an uncharacterized MCM from the mesophilic archaeon Mancarchaeum acidophilum (MacMCM) that shows strong DNA unwinding activity at room temperature. Unexpectedly, apo-MacMCM is monomeric in solution, providing a first opportunity to investigate the mechanisms of assembly of an active homohexameric MCM complex in vitro. We show that MacMCM requires both ATP and DNA to form an active homohexamer, and that the C-terminal winged-helix domain impedes oligomerization. We report the 3D structure of MacMCM, which reveals similar numbers of interactions at subunit–subunit interfaces as eukaryotic MCMs but fewer than MCMs from thermophilic archaea. Finally, we show that installing subunit–subunit salt bridges from Sulfolobus solfataricus MCM into MacMCM promotes oligomerization. Heterohexameric eukaryotic MCMs evolved from a homomeric ancestor. Our results identify structural and ligand-driven mechanisms of assembly that are conserved between homomeric and heteromeric MCMs.
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Apr 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The malarial parasite, Plasmodium falciparum (Pf), utilizes aminopeptidases in the breakdown of hemoglobin-derived oligopeptides to release amino acids for protein synthesis during growth and asexual reproduction of erythrocytic stages of the parasite. However, a N-terminal peptide bond that involves proline is difficult to hydrolyze. Aminopeptidase P (APP) is capable of cleaving peptide bonds with proline in the second position. Inhibition of PfAPP is therefore an attractive strategy for developing therapeutics for the treatment of malaria by limiting the supply of amino acids at the erythrocytic stage. We employed the structure-activity relationship of an existing APP inhibitor, apstatin, to design a more potent PfAPP inhibitor by introducing a hydroxamic acid metal-binding group in place of the amino-alcohol of apstatin, and an aromatic P4′ moiety. A hydroxamic tetrapeptide with phenylalanine at P4′ (6d) greatly increased the inhibitory potency (apstatin Ki, 16 μM; 6d, Ki 685 nM). Replacing the P3′ proline of 6d with a 2-substituted piperidine (6e) further improved the potency (Ki, 24 nM). Crystal structure analysis of PfAPP in complex with 6d and 6e showed binding at the active site with coordination of the hydroxamic acid metal binding group to the di-metal center, and several protein-inhibitor interactions involving domains II and III. A comparison of PfAPP-6e with human APP1 indicated that the P4′ phenylalanine drives inhibitor potency and selectivity towards PfAPP, by forming an interaction with Tyr617 of the adjacent monomer within the dimer. The details presented here should be useful for the future design of potent and selective PfAPP inhibitors.
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Mar 2026
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