I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11265]
Open Access
Abstract: Botulinum neurotoxins are the causative agents of botulism, a lethal paralytic disease, but are also one of the most commonly used therapeutics for the treatment of numerous neuromuscular conditions. These toxins recognise motor nerve terminals with high specificity and affinity by using a dual binding mechanism involving gangliosides and protein receptors. The initial recognition of gangliosides is crucial for the toxins’ potency. In this study, we employed a synaptosome-binding screening strategy to identify BoNT/A mutants with enhanced ganglioside-binding which translated into improved potency. X-ray crystallography and receptor-binding assays were used to elucidate the molecular mechanisms underlying the increased affinity or altered ganglioside selectivity of these mutants. Our findings provide a basis for the development of BoNT/A variants with enhanced therapeutic potential.
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Jun 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Muscle-type nicotinic acetylcholine receptor (AChR) is the key signaling molecule in neuromuscular junctions. Here, we present the structures of full-length human adult receptors in complex with Fab35 in α-bungarotoxin (αBuTx)-bound resting states and ACh-bound desensitized states. In addition to identifying the conformational changes during recovery from desensitization, we also used electrophysiology to probe the effects of eight previously unstudied AChR genetic variants found in patients with congenital myasthenic syndrome (CMS), revealing they cause either slow- or fast-channel CMS characterized by prolonged or abbreviated ion channel bursts. The combined kinetic and structural data offer a better understanding of both the AChR state transition and the pathogenic mechanisms of disease variants.
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May 2025
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B21-High Throughput SAXS
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Cristian Andres
Carmona-Carmona
,
Giovanni
Bisello
,
Rossella
Franchini
,
Gianluigi
Lunardi
,
Roberta
Galavotti
,
Massimiliano
Perduca
,
Rui P.
Ribeiro
,
Benny Danilo
Belviso
,
Alejandro
Giorgetti
,
Rocco
Caliandro
,
Patricia M.-J.
Lievens
,
Mariarita
Bertoldi
Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l-dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH-SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3-O-methyldopa) and a modified expression of key enzymes, such as dopamine beta-hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC-KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss-of-function and an altered profile of dopamine metabolites. By combining several structural approaches (X-ray crystallography, molecular dynamics, small angle X-ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss-of-function. Overall, the DDC-KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.
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May 2025
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I04-Macromolecular Crystallography
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Pekka
Kallunki
,
Florence
Sotty
,
Katarina
Willén
,
Michal
Lubas
,
Laurent
David
,
Malene
Ambjørn
,
Ann-Louise
Bergström
,
Louise
Buur
,
Ibrahim
Malik
,
Steffen
Nyegaard
,
Thomas Thiilmark
Eriksen
,
Berit O.
Krogh
,
Jeffrey B.
Stavenhagen
,
Kathrine J.
Andersen
,
Lars Ø.
Pedersen
,
Ersoy
Cholak
,
Edward N.
Van Den Brink
,
Rik
Rademaker
,
Tom
Vink
,
David
Satijn
,
Paul W. H. I.
Parren
,
Søren
Christensen
,
Line R.
Olsen
,
Josefine N.
Søderberg
,
Sandra
Vergo
,
Allan
Jensen
,
Jan
Egebjerg
,
Pernille Gry
Wulff-Larsen
,
Mikkel N.
Harndahl
,
Dina S. M.
Damlund
,
Kaare
Bjerregaard-Andersen
,
Karina
Fog
Open Access
Abstract: Amlenetug (Lu AF82422) is a human monoclonal antibody targeting α-synuclein in clinical development for multiple system atrophy. We describe a series of studies that characterize its functional properties and supported its selection as a viable clinical candidate. Amlenetug inhibits seeding induced in mouse primary neurons by various α-synuclein fibrillar assemblies and by aggregates isolated from MSA brain homogenate. In vivo, both co-injection of amlenetug with α-synuclein assemblies in mouse brain and peripheral administration inhibit α-synuclein seeding. Amlenetug inhibits uptake of α-synuclein seeds as well as accumulation of C-terminal truncated α-synuclein seeds and demonstrates binding to monomeric, aggregated, and truncated forms of human α-synuclein. The epitope of amlenetug was mapped to amino acids 112-117 and further characterized by crystallographic structure analysis. Based on our data, we hypothesize that targeting α-synuclein will potentially slow further disease progression by inhibiting further pathology development but be without impact on established pathology and symptoms.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[37045]
Open Access
Abstract: Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H2O2 and O2. This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 Å crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1’s mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structure’s unique conformations and active site interactions illuminate new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Moreover, we conducted a molecular docking analysis using SOD1 for potential radical scavengers and Abelson non-receptor tyrosine kinase (c-Abl, Abl1) inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions at the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.
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Apr 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[20287]
Open Access
Abstract: The Hsp70 chaperone system is capable of disassembling pathological aggregates such as amyloid fibres associated with serious degenerative diseases. Here we examine the role of the J-domain protein co-factor in amyloid disaggregation by the Hsc70 system. We used cryo-EM and tomography to compare the assemblies with wild-type DNAJB1 or inactive mutants. We show that DNAJB1 binds regularly along α-synuclein amyloid fibrils and acts in a 2-step recruitment of Hsc70, releasing DNAJB1 auto-inhibition before activating Hsc70 ATPase. The wild-type DNAJB1:Hsc70:Apg2 complex forms dense arrays of chaperones on the fibrils, with Hsc70 on the outer surface. When the auto-inhibition is removed by mutating DNAJB1 (ΔH5 DNAJB1), Hsc70 is recruited to the fibrils at a similar level, but the ΔH5 DNAJB1:Ηsc70:Apg2 complex is inactive, binds less regularly to the fibrils and lacks the ordered clusters. Therefore, we propose that 2-step activation of DNAJB1 regulates the ordered assembly of Hsc70 on the fibril. The localised, dense packing of chaperones could trigger a cascade of recruitment and activation to give coordinated, sequential binding and disaggregation from an exposed fibril end, as previously observed in AFM videos. This mechanism is likely to be important in maintaining a healthy cellular proteome into old age.
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Mar 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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Avinash V.
Dharmadhikari
,
Maria Alba
Abad
,
Sheraz
Khan
,
Reza
Maroofian
,
Tristan T.
Sands
,
Farid
Ullah
,
Itaru
Samejima
,
Yanwen
Shen
,
Martin A.
Wear
,
Kiara E.
Moore
,
Elena
Kondakova
,
Natalia
Mitina
,
Theres
Schaub
,
Grace K.
Lee
,
Christine H.
Umandap
,
Sara M.
Berger
,
Alejandro D.
Iglesias
,
Bernt
Popp
,
Rami
Abou Jamra
,
Heinz
Gabriel
,
Stefan
Rentas
,
Alyssa L.
Rippert
,
Christopher
Gray
,
Kosuke
Izumi
,
Laura K.
Conlin
,
Daniel C.
Koboldt
,
Theresa Mihalic
Mosher
,
Scott E.
Hickey
,
Dara V. F.
Albert
,
Haley
Norwood
,
Amy Feldman
Lewanda
,
Hongzheng
Dai
,
Pengfei
Liu
,
Tadahiro
Mitani
,
Dana
Marafi
,
Hatice Koçak
Eker
,
Davut
Pehlivan
,
Jennifer E.
Posey
,
Natalie C.
Lippa
,
Natalie
Vena
,
Erin L.
Heinzen
,
David B.
Goldstein
,
Cyril
Mignot
,
Jean-Madeleine
De Sainte Agathe
,
Nouriya Abbas
Al-Sannaa
,
Mina
Zamani
,
Saeid
Sadeghian
,
Reza
Azizimalamiri
,
Tahere
Seifia
,
Maha S.
Zaki
,
Ghada M. H.
Abdel-Salam
,
Mohamed S.
Abdel-Hamid
,
Lama
Alabdi
,
Fowzan Sami
Alkuraya
,
Heba
Dawoud
,
Aya
Lofty
,
Peter
Bauer
,
Giovanni
Zifarelli
,
Erum
Afzal
,
Faisal
Zafar
,
Stephanie
Efthymiou
,
Daniel
Gossett
,
Meghan C.
Towne
,
Raey
Yeneabat
,
Belen
Perez-Duenas
,
Ana
Cazurro-Gutierrez
,
Edgard
Verdura
,
Veronica
Cantarin-Extremera
,
Ana Do Vale
Marques
,
Aleksandra
Helwak
,
David
Tollervey
,
Sandeep N.
Wontakal
,
Vimla S.
Aggarwal
,
Jill A.
Rosenfeld
,
Victor
Tarabykin
,
Shinya
Ohta
,
James R.
Lupski
,
Henry
Houlden
,
William C.
Earnshaw
,
Erica E.
Davis
,
A. Arockia
Jeyaprakash
,
Jun
Liao
Open Access
Abstract: SPOUT1/CENP-32 encodes a putative SPOUT RNA methyltransferase previously identified as a mitotic chromosome associated protein. SPOUT1/CENP-32 depletion leads to centrosome detachment from the spindle poles and chromosome misalignment. Aided by gene matching platforms, here we identify 28 individuals with neurodevelopmental delays from 21 families with bi-allelic variants in SPOUT1/CENP-32 detected by exome/genome sequencing. Zebrafish spout1/cenp-32 mutants show reduction in larval head size with concomitant apoptosis likely associated with altered cell cycle progression. In vivo complementation assays in zebrafish indicate that SPOUT1/CENP-32 missense variants identified in humans are pathogenic. Crystal structure analysis of SPOUT1/CENP-32 reveals that most disease-associated missense variants are located within the catalytic domain. Additionally, SPOUT1/CENP-32 recurrent missense variants show reduced methyltransferase activity in vitro and compromised centrosome tethering to the spindle poles in human cells. Thus, SPOUT1/CENP-32 pathogenic variants cause an autosomal recessive neurodevelopmental disorder: SpADMiSS (SPOUT1 Associated Development delay Microcephaly Seizures Short stature) underpinned by mitotic spindle organization defects and consequent chromosome segregation errors.
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Feb 2025
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B21-High Throughput SAXS
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Open Access
Abstract: The pathological deposition of tau and amyloid-beta into insoluble amyloid fibrils are pathological hallmarks of Alzheimer’s disease. Molecular chaperones are important cellular factors contributing to the regulation of tau misfolding and aggregation. Here we reveal an Hsp90-independent mechanism by which the co-chaperone p23 as well as a molecular complex formed by two co-chaperones, p23 and FKBP51, modulates tau aggregation. Integrating NMR spectroscopy, SAXS, molecular docking, and site-directed mutagenesis we reveal the structural basis of the p23-FKBP51 complex. We show that p23 specifically recognizes the TPR domain of FKBP51 and interacts with tau through its C-terminal disordered tail. We further show that the p23-FKBP51 complex binds tau to form a dynamic p23-FKBP51-tau trimeric complex that delays tau aggregation and thus may counteract Hsp90-FKBP51 mediated toxicity. Taken together, our findings reveal a co-chaperone mediated Hsp90-independent chaperoning of tau protein.
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Jan 2025
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I24-Microfocus Macromolecular Crystallography
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Alex G.
Baldwin
,
David W.
Foley
,
Ross
Collins
,
Hyunah
Lee
,
D. Heulyn
Jones
,
Ben
Wahab
,
Loren
Waters
,
Josephine
Pedder
,
Marie
Paine
,
Gui Jie
Feng
,
Lucia
Privitera
,
Alexander
Ashall-Kelly
,
Carys
Thomas
,
Jason A.
Gillespie
,
Lauramariú
Schino
,
Delia
Belelli
,
Cecilia
Rocha
,
Gilles
Maussion
,
Andrea I.
Krahn
,
Thomas M.
Durcan
,
Jonathan M.
Elkins
,
Jeremy J.
Lambert
,
John R.
Atack
,
Simon E.
Ward
Open Access
Abstract: LIMKs are serine/threonine and tyrosine kinases responsible for controlling cytoskeletal dynamics as key regulators of actin stability, ensuring synaptic health through normal synaptic bouton structure and function. However, LIMK1 overactivation results in abnormal dendritic synaptic development that characterizes the pathogenesis of Fragile X Syndrome (FXS). As a result, the development of LIMK inhibitors represents an emerging disease-modifying therapeutic approach for FXS. We report the discovery of MDI-114215 (85), a novel, potent allosteric dual-LIMK1/2 inhibitor that demonstrates exquisite kinome selectivity. 85 reduces phospho-cofilin in mouse brain slices and rescues impaired hippocampal long-term potentiation in brain slices from FXS mice. We also show that LIMK inhibitors are effective in reducing phospho-cofilin levels in iPSC neurons derived from FXS patients, demonstrating 85 to be a potential therapeutic candidate for FXS that could have broad application to neurological disorders or cancers caused by LIMK1/2 overactivation and actin instability.
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Dec 2024
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