I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38313]
Open Access
Abstract: Malaria remains a major global health concern. The development of point-of-care diagnostic aptasensors could improve malaria detection, patient management, and eradication efforts. We selected and validated single-stranded DNA aptamers against Plasmodium falciparum lactate dehydrogenase (Pf-LDH) using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The five most abundant aptamers were validated with an enzyme-linked oligonucleotide assay (ELONA). The aptamer binding affinities to Pf-LDH varied between 0.35-3.08 nM. Truncation of aptamers enhanced binding affinities in most cases, with all truncated aptamers showing comparable or improved dissociation constants (Kd) relative to full-length aptamers, except Pf-LDH5t, which lost binding. Selectivity analysis against LDH from P. ovale, P. vivax, and P. malariae showed that Pf-LDH1 and Pf-LDH1t specifically recognized Pf-LDH (Kd = 2.74 ± 0.28 nM and 1.13 ± 0.26 nM, respectively), while Pf-LDH2, Pf-LDH3, Pf-LDH4, and their truncated forms, as well as Pf-LDH5, recognized both Pf-LDH and P. ovale LDH (Po-LDH). Crystallography of Pf-LDH4t revealed a binding site overlapping that of the previously characterized aptamer 2008 but involving distinct molecular interactions. These findings demonstrate the structural versatility of Plasmodium LDH specific aptamers and provide a novel Pf- and Po-LDH co-specific aptamer panel with strong potential for next-generation aptamer-based malaria diagnostics.
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Jun 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[31353]
Open Access
Abstract: Effective vaccines against malaria are urgently required. All components of the PfPCRCR complex are essential for erythrocyte invasion by Plasmodium falciparum and are potential vaccine immunogens against blood-stage malaria. Of these, PfRH5 has progressed furthest in clinical development, while PfCyRPA also induces parasite growth-inhibitory antibodies. Here, we used direct nanoparticle coupling and structure-guided design to generate improved PfCyRPA-based immunogens. PfCyRPA is a six-bladed β-propeller. Blades 1 and 2 are exposed in the PfPCRCR complex and contain the epitopes of the most potent known growth-inhibitory antibodies. We therefore performed structure-guided design to generate a correctly folded, thermostable epitope mimic, PfCyRPA-EM, containing blades 1 and 2. In a pre-clinical model, PfCyRPA-EM elicited antibodies that inhibited parasite growth at lower concentrations than those elicited by PfCyRPA. In addition, the higher thermostability of PfCyRPA-EM and its improved expression as an I53-50 nanoparticle fusion make it well-suited for clinical development, alone or with other immunogens.
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Mar 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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Among the biophysical techniques used in fragment-based drug discovery (FBDD) campaigns, crystallography is the most sensitive, allowing for the identification of low-affinity ligands and the characterization of protein–ligand complexes at atomic resolution. Although powerful, the proper application of this technique depends on obtaining crystals capable of diffracting X-rays at high resolution. Additionally, in crystallographic compound screening, the crystals must be resistant to multiple organic solvents used in chemical libraries, such as DMSO. In this protocol, we describe recombinant protein production suitable for crystallization and procedures for X-ray crystallographic screening of a library of 768 fragments. As a case study, we used the Schistosoma mansoni thioredoxin glutathione reductase (SmTGR), a redox enzyme with a key role in controlling oxidative stress in parasites of the Schistosoma genus, which causes schistosomiasis. As a validated pharmacological target, SmTGR is used in the development of new schistosomicidal drugs. The experimental pipeline includes SmTGR expression, purification, and crystallization, crystal soaking, diffraction data collection, and refinement. The 768 fragments from the Diamond-SGC Poised Library (DSPL) were individually soaked onto the crystals, and diffraction data were collected and processed at the I04-1 beamline of the Diamond Light Source synchrotron. Diffraction data were subsequently analyzed using PanDDA to identify fragment-binding events and to enable reliable detection of low-occupancy ligands within the protein crystal structures. In addition to the core experimental steps, this protocol incorporates systematic approaches to overcome limitations frequently encountered in crystallographic screening campaigns, including assessment of crystal solvent tolerance, acceleration of crystal mounting through the use of auxiliary devices, acoustic dispensing–based soaking of hundreds of fragments for low material consumption and high throughput, automated data collection, and efficient data analysis pipeline for the detection of weakly bound ligand. This protocol can be broadly applied to screen diverse compound sets against multiple targets amenable to crystallization.
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Mar 2026
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B21-High Throughput SAXS
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[24557, 39224]
Open Access
Abstract: Histone variants define distinct chromatin states by modulating the biophysical properties of nucleosomes. Variants play a particularly important role in the parasitic protist Trypanosoma brucei, which has unusual chromatin and lacks a canonical repressive heterochromatin system. Instead, T. brucei utilizes specialized divergent histone variants H3.V and H4.V. However, the biochemical basis of their repressive functions is unknown. Here, we determined the structure of the H3.V-H4.V nucleosome core particle and biochemically characterized variant-containing nucleosomes and nucleosome arrays, probing their unique properties. We discovered that surprisingly for repressive-state nucleosomes, H3.V promotes pronounced DNA splaying, largely via its N-terminal tail region, while retaining overall stability that is comparable to canonical nucleosomes. In contrast, H4.V exhibits near-identical binding to DNA but mediates a slight increase in histone octamer stability. The surface of the H3.V-H4.V nucleosome is altered and provides a differential platform for chromatin-binding proteins, linking the variants to parasite pathogenicity.
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Feb 2026
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I24-Microfocus Macromolecular Crystallography
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Guido C.
Paesen
,
Nathaniel S.
Chapman
,
Jonna B.
Westover
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Cynthia M.
Mcmillen
,
Natalia A.
Kuzmina
,
Emmett A.
Dews
,
Luke
Myers
,
Robert
Stass
,
Joel M.
Montgomery
,
Alexander
Bukreyev
,
Amy L.
Hartman
,
Brian B.
Gowen
,
James E.
Crowe
,
Thomas A.
Bowden
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Rift Valley fever virus (RVFV) poses a continued threat to human health and animal husbandry. Two neutralizing and protective human monoclonal antibodies (mAbs), RVFV-268 and RVFV-379, exhibit similar affinities and epitope footprints on the Gn glycoprotein component of the RVFV Gn-Gc capsomeric lattice. Here, we define fine details of the biophysical determinants of Gn recognition used by RVFV human monoclonal antibodies through studying an antibody encoded by a set of recombined genes not previously identified in RVFV antibodies. We find that RVFV-379 exhibits a larger footprint than that observed for RVFV-268 and other antibodies targeting the same region, which involves major contributions of both the light and heavy chains. RVFV-379 also uses an oblique angle of approach towards the virion surface that contrasts with the perpendicular angle of engagement observed for some other potently neutralizing human mAbs. Further, consistent with amino acid sequence variation within and proximal to the RVFV-379 epitope, in vitro neutralization screening reveals a limited degree of neutralization breadth across prevalent RVFV strains, suggesting that RVFV has fewer functional constraints at this region of the virus envelope. By dissecting the molecular determinants of mAb recognition of Gn, this integrated analysis refines strategies needed for the rational design of vaccines that can elicit a potent and species-wide protective antibody immune response to this important re-emerging pathogen.
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Feb 2026
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Ingra M.
Claro
,
Erika R.
Manuli
,
Camila A. M.
Da Silva
,
Thaís M.
Coletti
,
Philippe
Lemey
,
Ana Catharina
Nastri
,
Luciana Vilas Boas
Casadio
,
Amaro Nunes
Duarte-Neto
,
Joshua
Quick
,
Camila M.
Romano
,
Charles
Whittaker
,
Sarah C.
Hill
,
Carlos A.
Prete
,
Darlan S.
Candido
,
Filipe R. R.
Moreira
,
Mariana S.
Ramundo
,
Ian Nunes
Valença
,
Jaqueline G.
De Jesus
,
Flavia C. S.
Sales
,
Mariana S.
Cunha
,
Juliana M.
Guerra
,
Maria Cassia
Mendes-Correa
,
Tania R.
Tozetto-Mendoza
,
Marcilio Jorge
Fumagalli
,
Yeh-Li
Ho
,
Peter
Simmonds
,
Weng M.
Ng
,
Thomas A.
Bowden
,
William M.
De Souza
,
Oliver G.
Pybus
,
Anna S.
Levin
,
Nicholas
Loman
,
Ester C.
Sabino
,
Nuno R.
Faria
Open Access
Abstract: Between December 2019 and January 2020, two patients suspected of having severe yellow fever were admitted to a tertiary healthcare facility in São Paulo, Brazil, presenting with acute hemorrhagic syndrome and neurological alterations; both cases had fatal outcomes. Upon admission, both tested negative for yellow fever viral RNA, and Sabiá virus (SABV), a New World arenavirus, was identified as the causative pathogen. To date, only four humans naturally acquired SABV infections have been confirmed, all fatal and linked to rural settings. We applied next-generation sequencing to generate complete and near-complete genomes from two patients (SP17 and SP19). Existing molecular diagnostics failed to detect SABV; therefore, new molecular tests were developed. Genetic analyses of SP17 and SP19 genomes along with other arenaviruses, revealed that the new cases were genetically diverse, showing 93-98.2% amino acid identity at the NP level between SP17, SP19, and the 1990 reference strain (SPH114202). Time-scaled phylogenetic analyses confirmed that SP17 and SP19 were not epidemiologically linked and suggested that SABV has been circulating undetected in Brazil for over a century. Additionally, homology modeling and structure-based mapping provided insights into SABV receptor-binding sequence conservation, suggesting that SABV shares similar receptor binding structure compared to other clade B arenaviruses, despite some amino acid variation around receptor binding site. Our findings underscore the need for retrospective and prospective surveillance of undiagnosed hemorrhagic fever cases to assess the public health impact of SABV in Brazil.
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Feb 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Xiaomin
Ni
,
R. Blake
Richardson
,
Andre
Schutzer Godoy
,
Matteo P.
Ferla
,
Caroline
Kikawa
,
Jenke
Scheen
,
William W.
Hannon
,
Eda
Capkin
,
Noa
Lahav
,
Blake H.
Balcomb
,
Peter G.
Marples
,
Michael
Fairhead
,
Siyi
Wang
,
Eleanor P.
Williams
,
Charles W. E.
Tomlinson
,
Jasmin C.
Aschenbrenner
,
Ryan
Lithgo
,
Max
Winokan
,
Charline
Giroud
,
Isabela
Dolci
,
Rafaela Sachetto
Fernandes
,
Glaucius
Oliva
,
Anu V.
Chandran
,
Mary-Ann
Xavier
,
Martin A.
Walsh
,
Warren
Thompson
,
Jesse D.
Bloom
,
Nathaniel T.
Kenton
,
Alpha A.
Lee
,
Annette
Von Delft
,
Haim
Barr
,
Karla
Kirkegaard
,
Lizbe
Koekemoer
,
Daren
Fearon
,
Matthew J.
Evans
,
Frank
Von Delft
Diamond Proposal Number(s):
[32627]
Open Access
Abstract: The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 46 fragments with diverse scaffolds are identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we perform deep mutational scanning of the NS2B-NS3 protease. Merging fragment hits yields an extensive set of ‘mergers’, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points. In this work, we maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.
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Oct 2025
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Plasmodium falciparum is a eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and, like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, replicated by a dedicated replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol). This suggests that apPol can multitask and catalyse both replicative and lesion bypass synthesis. Replicative synthesis relies on a restrictive active site for high accuracy while lesion bypass typically requires an open active site. This raises the question: how does apPol combine the structural features of multiple DNA polymerases in a single protein? Using single-particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its undamaged DNA and nucleotide substrates in five pre-chemistry conformational states. We found that apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers-open state, we identified a nascent base pair checkpoint that preferentially selects Watson–Crick base pairs, an essential requirement for replicative synthesis. Taken together, these structural features might explain how apPol balances replicative and lesion bypass synthesis.
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Oct 2025
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I02-Macromolecular Crystallography
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Open Access
Abstract: The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.
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Sep 2025
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