Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Jose Enrique
Gonzalez-Prada
,
Sulin
Liu
,
Chuhan
Shang
,
Chloe S.
Chernoff
,
Damian P.
Bright
,
Martin
Mortensen
,
Charlotte F.
Jones
,
Stephanie
Nestorow
,
Vikram Babu
Kasaragod
,
Wan-Na
Chen
,
Saad
Hannan
,
Jianchong
Zhou
,
Alexander W. E.
Dunn
,
Asma
Soltani
,
Richard J.
Turner
,
Natasha M.
Duggan
,
Yin
Yuan
,
Ayla A.
Wahid
,
Steven W.
Hardwick
,
Suzanne
Scott
,
Dimitri Y.
Chirgadze
,
Els
Pardon
,
Jan
Steyaert
,
A. Radu
Aricescu
,
Ole
Paulsen
,
David
Belin
,
Trevor G.
Smart
,
Paul S.
Miller
Diamond Proposal Number(s):
[31589, 37678]
Open Access
Abstract: γ-Aminobutyric acid type-A (GABAA) receptors are the principal mediators of inhibitory neurotransmission in the human central nervous system. The α2- and α3-containing subtypes have tightly controlled spatial expression profiles, which influence anxiety, nociception, epilepsy, and autism. α2/α3-Selective small molecules compromise on strength of effect (efficacy) to avoid off-subtype modulation. To break this pharmacological deadlock, we study here a panel of nanobodies (NBs) raised against α2- and α3-containing GABAA receptors. We identify subtype selective silent binders, positive allosteric modulators (PAMs), and inhibitors. Cryo–electron microscopy structures explain the binding modes and molecular mechanisms of action of representative NBs. Modulators exhibit distinct synaptic and extrasynaptic functional profiles in brain slices and neuronal networks and can reduce anxiety in vivo. These selective and efficacious NBs (whether inhibitors or positive modulators) enable strong yet precise pharmacological control of α2/α3-containing subtypes to advance basic research and as potential therapeutic leads to treat neuropsychiatric disorders.
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Jul 2026
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Krios I-Titan Krios I at Diamond
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Carys
Williams
,
Laura M.
Nocka
,
George
Hedger
,
Pragya
Parashara
,
Els
Pardon
,
Naomi R.
Latorraca
,
Ganesh V.
Pusapati
,
Parijat
Sarkar
,
Dorothy
Lartey
,
Lei
Gao
,
Ljiljana
Milenkovic
,
Rod
Chalk
,
Jan
Steyaert
,
Susan
Marqusee
,
Loic
Carrique
,
J. Fernando
Bazan
,
Sarah L.
Rouse
,
Jennifer H.
Kong
,
Christian
Siebold
,
Rajat
Rohatgi
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated with biophysical and functional studies, we visualized a human E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex attenuates Hedgehog signaling by ubiquitylating Smoothened (SMO), a G-protein-coupled receptor (GPCR) that transduces morphogen signals. A long helix in the MMM complex engages SMO using an intramembrane degron and extends into the cytoplasm to suspend an activated and precisely oriented RING domain below the plasma membrane. This architecture enables ubiquitylation of the cytoplasmic surface of SMO, reducing SMO abundance at primary cilia. Our structure provides insights into MEGF8 mutations, which cause multi-organ birth defects, and defines a paradigm for how transmembrane E3 ligases control the cell surface abundance of GPCRs and other signaling receptors.
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Jun 2026
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I24-Microfocus Macromolecular Crystallography
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Katleen
Van Nerom
,
Andres
Ainelo
,
Kyo
Coppieters ‘t Wallant
,
Ariel
Talavera-Perez
,
Dannele
Echemendia-Blanco
,
Sarah
Peeters
,
Brahim
El Khalfaoui Oulali
,
Hedvig
Tamman
,
Tatsuaki
Kurata
,
Mohammad
Roghanian
,
Chloé
Martens
,
Els
Pardon
,
Jan
Steyaert
,
Vasili
Hauryliuk
,
Abel
Garcia-Pino
Open Access
Abstract: Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HDON) state, a relaxed catalytically inactive (HDOFF/SYNTHOFF) state, and an elongated synthesis-competent (SYNTHON) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIANtr, ACP, NirD and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by stabilizing Rel and RelA in defined conformational states. Nanobodies that sequester the TGS domain of RelA prevent activation by deacylated tRNA on starved ribosomes, strongly inhibiting (p)ppGpp synthesis and suppressing Escherichia coli virulence in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HDON/SYNTHOFF conformation. Structural and biochemical analyses show that nanobodies, like endogenous allosteric regulators, restrict the conformational landscape of long RSH enzymes, establishing them as powerful tools for dissecting RSH function and as frameworks for developing protein-based RSH modulators.
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May 2026
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Miguel
Berbeira-Santana
,
Claudia
Peregrina
,
Kosuke
Okuda
,
Jin Chuan
Zhou
,
Maria
Carrasquero-Ordaz
,
Amy V.
Roberts
,
Anne E.
Thomas
,
Evert
Haanappel
,
Matthieu
Chavent
,
Kamel
El Omari
,
Lindsay A.
Baker
,
Daniel T.
Pederick
,
Els
Pardon
,
Jan
Steyaert
,
U. Valentin
Nägerl
,
Daniel
Del Toro
,
Elena
Seiradake
Open Access
Abstract: Cortical migration is a complex process in which neurons migrate along radial glial cells (RGC) to form functional layers. Teneurins (Ten1-4) play a role by interacting with Latrophilins (Lphn/ADGRL1-3). Teneurins are also known as cell adhesion molecules, but how homophilic and heterophilic Teneurin interactions are integrated is unknown. Here, single-particle-cryo-EM data of Ten2 shows that canonical Latrophilin-binding is sterically incompatible with Ten2-dimerisation, making these interactions exclusive. We engineered surface mutations that specifically disrupt Ten2-Ten2 or Ten2-Latrophilin interactions. These are transferrable to Ten4, suggesting conserved binding mechanisms. Proteomics, in-vivo-gene-editing and super-resolution-microscopy show that Ten4 is expressed along RGC fibres and that migrating neurons switch from low-to-high Ten4-expression. Ten4 expression is highest in the cortical plate where Ten4-Ten4 interactions reduce RGC-attachment. In the intermediate zone, Ten4-Latrophilin interactions are required to promote neuron-RGC association. The results show how Ten4 orchestrates different stages of cortical migration by using a structural/functional switch between high-affinity Lphn interactions and low-affinity homophilic interactions, underpinning the integration of distinct migration programmes.
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Apr 2026
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NONE-No attached Diamond beamline
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Deniz
Bicer
,
Rei
Matsuoka
,
Aurélien F. A.
Moumbock
,
Preethi
Sukumar
,
Albert
Suades
,
Harish
Cheruvara
,
Andrew
Quigley
,
David
Drew
,
Els
Pardon
,
Jan
Steyaert
,
Peter J. F.
Henderson
,
Martin
Caffrey
,
Julia J.
Griese
,
Emmanuel
Nji
Open Access
Abstract: Under conditions of extreme acidity, the lysine-specific permease, LysP, not only mediates the import of L-lysine it also interacts with the transcriptional regulator, CadC, to activate expression of the cadAB operon. This operon encodes the lysine decarboxylase, CadA, which converts lysine to cadaverine while consuming a cytoplasmic proton, and the antiporter, CadB, which exports protonated cadaverine in exchange for extracellular lysine. Together, these processes contribute to cytoplasmic pH homeostasis and support bacterial acid resistance - a mechanism essential for the survival of pathogenic bacteria in acidic host environments. Here, we present the cryo-EM structure of LysP from Pseudomonas aeruginosa in an inward-occluded conformation (3.2–5.3 Å resolution), bound to L-lysine and a nanobody. L-Lysine is coordinated by hydrophobic contacts, cation–π interactions, and by hydrogen bonding mostly with polar uncharged residues. Reconstitution of LysP into proteoliposomes confirms specific L-lysine transport, which is competitively inhibited by L-4-thialysine. These findings provide a structural framework for understanding selective lysine recognition and inhibition, with implications for antibacterial drug design.
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Dec 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31589]
Open Access
Abstract: Uncoupling protein 1 (UCP1, SLC25A7) is responsible for the thermogenic properties of brown adipose tissue. Upon fatty acid activation, UCP1 facilitates proton leakage, dissipating the mitochondrial proton motive force to release energy as heat. Purine nucleotides are considered to be the only inhibitors of UCP1 activity, binding to its central cavity to lock UCP1 in a proton-impermeable conformation. Here we show that pyrimidine nucleotides can also bind and inhibit its proton-conducting activity. All nucleotides bound in a pH-dependent manner, with the highest binding affinity observed for ATP, followed by dTTP, UTP, GTP and CTP. We also determined the structural basis of UTP binding to UCP1, showing that binding of purine and pyrimidine nucleotides follows the same molecular principles. We find that the closely related mitochondrial dicarboxylate carrier (SLC25A10) and oxoglutarate carrier (SLC25A11) have many cavity residues in common, but do not bind nucleotides. Thus, while UCP1 has evolved from dicarboxylate carriers, no selection for nucleobase specificity has occurred, highlighting the importance of the pH-dependent nucleotide binding mechanism mediated via the phosphate moieties.
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Feb 2025
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I03-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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Audrey
Le Bas
,
Bradley R.
Clarke
,
Tanisha
Teelucksingh
,
Micah
Lee
,
Kamel
El Omari
,
Andrew M.
Giltrap
,
Stephen A.
Mcmahon
,
Hui
Liu
,
John H.
Beale
,
Vitaliy
Mykhaylyk
,
Ramona
Duman
,
Neil G.
Paterson
,
Philip N.
Ward
,
Peter J.
Harrison
,
Miriam
Weckener
,
Els
Pardon
,
Jan
Steyaert
,
Huanting
Liu
,
Andrew
Quigley
,
Benjamin G.
Davis
,
Armin
Wagner
,
Chris
Whitfield
,
James H.
Naismith
Diamond Proposal Number(s):
[33941]
Open Access
Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.
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Jan 2025
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I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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James
Hillier
,
Yuguang
Zhao
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Tao-Hsin
Chang
,
Gangshun
Yi
,
Helen M. E.
Duyvesteyn
,
Jing
Yu
,
Weixian
Lu
,
Els
Pardon
,
Jan
Steyaert
,
Yanan
Zhu
,
Tao
Ni
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[19946, 28713]
Open Access
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
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Aug 2024
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Baptiste
Fischer
,
Tomasz
Uchanski
,
Aidana
Sheryazdanova
,
Simon
Gonzalez
,
Alexander N.
Volkov
,
Elke
Brosens
,
Thomas
Zogg
,
Valentina
Kalichuk
,
Steven
Ballet
,
Wim
Versees
,
Anna A.
Sablina
,
Els
Pardon
,
Alexandre
Wohlkonig
,
Jan
Steyaert
Diamond Proposal Number(s):
[17150]
Open Access
Abstract: Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Karin E. J.
Roedstroem
,
Alexander
Cloake
,
Janina
Sörmann
,
Agnese
Baronina
,
Kathryn H. M.
Smith
,
Ashley C. W.
Pike
,
Jackie
Ang
,
Peter
Proks
,
Marcus
Schewe
,
Ingelise
Holland-Kaye
,
Simon R.
Bushell
,
Jenna
Elliott
,
Els
Pardon
,
Thomas
Baukrowitz
,
Raymond J.
Owens
,
Simon
Newstead
,
Jan
Steyaert
,
Elisabeth P.
Carpenter
,
Stephen J.
Tucker
Diamond Proposal Number(s):
[15433, 19301]
Open Access
Abstract: Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
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May 2024
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