I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Julian M.
Ludäscher
,
Emma
Scaletti Hutchinson
,
Guillem
Vila-Julià
,
Ann-Sofie
Jemth
,
Saher
Shahid
,
Elisee
Wiita
,
Israel
Cabeza De Vaca
,
Szymon
Pach
,
Lukas
Gajdos
,
Swati
Aggarwal
,
Ellen
Walse
,
Oliver
Mortusewicz
,
Thomas
Helleday
,
Jens
Carlsson
,
Pal
Stenmark
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.
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Jun 2026
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I04-Macromolecular Crystallography
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Rashi
Goel
,
Kristina
Jevdokimenko
,
Ronja
Rehm
,
Jannik
Hentze
,
Paola
Agüi-Gonzalez
,
Momchil
Ninov
,
Erik
Maier
,
Yin
Wu
,
Felix
Lange
,
Agata
Witkowska
,
Svenja
Bolz
,
Francesca
Pennacchietti
,
Martina
Damenti
,
Natalie
Kaempf
,
Vladimir
Khayenko
,
Carles
Calatayud
,
Viveka Nand
Malviya
,
Natali L.
Chanaday
,
Emma
Scaletti Hutchinson
,
Hao
Liu
,
Kirsten
Weyand
,
Valentin
Schwarze
,
Daniela
Ivanova
,
Tristan P.
Wallis
,
Christopher
Small
,
Hans M.
Maric
,
Merja
Joensuu
,
Michael A.
Cousin
,
Frédéric A.
Meunier
,
Patrik
Verstreken
,
Ilaria
Testa
,
Ege T.
Kavalali
,
Volker
Haucke
,
Stefan
Jakobs
,
Henning
Urlaub
,
Nils
Brose
,
Benjamin H.
Cooper
,
Pal
Stenmark
,
Felipe
Opazo
,
Reinhard
Jahn
,
Silvio O.
Rizzoli
,
Eugenio F.
Fornasiero
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.
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May 2026
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I03-Macromolecular Crystallography
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Huazhang
Shu
,
Julian M.
Ludäscher
,
Sushma
Sharma
,
Seher
Alam
,
Lilian
Frank
,
Emma
Scaletti Hutchinson
,
Marianna
Tampere
,
Chloé
Lévêque
,
André B. P.
Van Kuilenburg
,
Nicholas C. K.
Valerie
,
Mikael
Altun
,
Andrei
Chabes
,
Pal
Stenmark
,
Sean G.
Rudd
,
Si Min
Zhang
Diamond Proposal Number(s):
[36026]
Open Access
Abstract: Molnupiravir is a nucleoside analogue antiviral drug against RNA viruses, including its clinical indication SARS-CoV-2. Whilst its mechanism-of-action is well defined, host factors that regulate its therapeutic responses have not been thoroughly deciphered and characterized. Here we show that uridine cytidine kinases (UCKs), key enzymes in pyrimidine salvage, effectively phosphorylate and thereby bioactivate N4-hydroxycytidine (NHC) – the active compound of molnupiravir, thus dictating its anti-SARS-CoV-2 efficacy and furthermore selectivity. In vitro, both isoforms of UCKs (UCK1 and UCK2) effectively phosphorylated NHC, where the structural basis of the catalysis was further deciphered via the first complete substrate bound co-crystal structure of UCK, i.e., UCK1-NHC-AMPPNP. In SARS-CoV-2-infected cells, UCK2 knockdown via siRNA hampered the intracellular accumulation of the tri-phosphorylated antiviral metabolite of NHC, resulting in a 10-fold reduction of the antiviral efficacy, and surprisingly, 2-fold reduction of its selectivity, which were critically recapitulated in a dose-dependent manner using a pan-UCK inhibitor. Altogether, this work underscores UCKs as pivotal players in upholding molnupiravir efficacy and therapeutic window of molnupiravir, and furthermore as pharmacologically tractable targets for tailoring the drug response.
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May 2026
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I24-Microfocus Macromolecular Crystallography
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Ying
Xu
,
Reed M.
Stubbendieck
,
Raghuvir
Viswanatha
,
Ajda
Krc
,
Lisa S.
Baik
,
Won Se
Suh
,
Yanhui
Hu
,
Huan
Wang
,
Linxiang
Yin
,
Enzo
Mameli
,
Anne
Van Der Meij
,
John R.
Carlson
,
Andrew C.
Doxey
,
Pal
Stenmark
,
Norbert
Perrimon
,
Cameron R.
Currie
,
Min
Dong
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR–Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.
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Apr 2026
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I04-Macromolecular Crystallography
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Pyung-Gang
Lee
,
Linxiang
Yin
,
Xin
Wei
,
Jingyuan
Shi
,
Geoffrey
Masuyer
,
Travis G.
Wentz
,
Pengsheng
Chen
,
Ying
Xu
,
Junjie
Liang
,
Haonan
Zhang
,
Sara
Persson Kosenina
,
Briallen
Lobb
,
Michael
Mansfield
,
Sarjeet S.
Gill
,
Sabine
Pellett
,
Pal
Stenmark
,
Andrew C.
Doxey
,
Min
Dong
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Insecticidal bacterial proteins play key roles in insect-bacteria interactions and have been used as biopesticides. Here, we identify two insecticidal proteins in Paeniclostridium ghonii, designated PG-toxin 1 (PG1) and PG-toxin 2 (PG2), which are homologs of botulinum neurotoxins (BoNTs). Unlike BoNTs, PG1 and PG2 contain two separate proteins: One is the protease light chain (LC), and the other is the heavy chain containing the translocation domain and the receptor binding domain. Crystal and cryo–electron microscopy structures show a conserved BoNT-like architecture but without an interchain disulfide bond. Functional characterizations establish that the LCs of PG1 and PG2 cleave insect synaptosomal–associated protein 25 (SNAP25), but not human or rat SNAP25, and microinjection of PG1 and PG2 caused paralysis and death in Drosophila and Aedes mosquitoes. These findings identified unique two-component BoNT-like insecticidal proteins, revealing insights into the evolution of the BoNT family of toxins, and broadening our understanding of bacteria that can be used for biopest controls.
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Nov 2025
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I03-Macromolecular Crystallography
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Emily C.
Hank
,
Nicholas D.
D'Arcy-Evans
,
Emma R.
Scaletti
,
Carlos
Benítez-Buelga
,
Olov
Wallner
,
Florian
Ortis
,
Kaixin
Zhou
,
Liuzhen
Meng
,
Alicia
Del Prado
,
Patricia
Calvo
,
Ingrid
Almlöf
,
Elisée
Wiita
,
Karen
Nierlin
,
Sara
Kosenina
,
Andreas
Krämer
,
Alice
Eddershaw
,
Mario
Kehler
,
Maeve
Long
,
Ann-Sofie
Jemth
,
Holly
Dawson
,
Josephine
Stewart
,
Adam
Dickey
,
Mikhael E.
Astorga
,
Marek
Varga
,
Evert J.
Homan
,
Martin
Scobie
,
Stefan
Knapp
,
Leandro
Sastre
,
Pal
Stenmark
,
Miguel
De Vega
,
Thomas
Helleday
,
Maurice
Michel
Diamond Proposal Number(s):
[15806, 21625]
Open Access
Abstract: Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.
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Oct 2025
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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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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Listeria monocytogenes is a ubiquitous, psychrotrophic human pathogen that can cause listeriosis, a serious illness for vulnerable populations. Some foods, such as Hispanic-style fresh cheeses like queso fresco, pose a specific risk because there are no widely accepted or available methods for L. monocytogenes mitigation that are both effective and able to maintain the properties of the products. Listeria-specific bacteriophages encode endolysins that are able to cleave the peptidoglycan layer of L. monocytogenes cells externally, showing promise as a potential solution to this problem. PlyP100, from the GRAS Listeria phage P100, is one such endolysin that can prevent the growth of L. monocytogenes in both lab culture conditions and a miniaturized queso fresco model. In this work, we aimed to understand the structural and functional properties of PlyP100. An AlphaFold prediction suggested the presence of three separate domains (D1, D2, and D3). By solving a crystal structure of D1 and assessing various domain truncations, we present evidence that D1 is responsible for catalytic activity, D3 is sufficient for cell wall binding, and D2 is necessary for full function of the enzyme against live cells. Additionally, we performed point mutations in D1 and compared PlyP100 to proteins with similar structures, including S. pneumoniae LytA and Listeria endolysin Ply511, in order to understand its specific enzymatic mechanism and target strain specificity. These insights into the structure and function of PlyP100 will aid future work aiming to engineer better endolysins as safe food antimicrobials.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
Abstract: NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two “NrdR boxes” upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR–ATP–dATP and EcoNrdR–ADP–dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR–ATP–dATP and novel filaments of EcoNrdR–ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR–ATP–dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.
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Mar 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Andreas
Luttens
,
Duc Duy
Vo
,
Emma R.
Scaletti
,
Elisée
Wiita
,
Ingrid
Almlöf
,
Olov
Wallner
,
Jonathan
Davies
,
Sara
Kosenina
,
Liuzhen
Meng
,
Maeve
Long
,
Oliver
Mortusewicz
,
Geoffrey
Masuyer
,
Flavio
Ballante
,
Maurice
Michel
,
Evert
Homan
,
Martin
Scobie
,
Christina
Kalderén
,
Ulrika
Warpman Berglund
,
Andrii V.
Tarnovskiy
,
Dmytro S.
Radchenko
,
Yurii S.
Moroz
,
Jan
Kihlberg
,
Pål
Stenmark
,
Thomas
Helleday
,
Jens
Carlsson
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging optimization process. To expand the explored chemical space, we employ structure-based docking to evaluate orders-of-magnitude larger libraries than those used in traditional fragment screening. We computationally dock a set of 14 million fragments to 8-oxoguanine DNA glycosylase (OGG1), a difficult drug target involved in cancer and inflammation, and evaluate 29 highly ranked compounds experimentally. Four of these bind to OGG1 and X-ray crystallography confirms the binding modes predicted by docking. Furthermore, we show how fragment elaboration using searches among billions of readily synthesizable compounds identifies submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cells. Comparisons of virtual screening strategies to explore a chemical space of 1022 compounds illustrate that fragment-based design enables enumeration of all molecules relevant for inhibitor discovery. Virtual fragment screening is hence a highly efficient strategy for navigating the rapidly growing combinatorial libraries and can serve as a powerful tool to accelerate drug discovery efforts for challenging therapeutic targets.
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Feb 2025
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