I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35120]
Open Access
Abstract: N-myc is a transcription factor, a powerful driver of cellular growth and an important oncoprotein. N-myc interacts with many factors, including the RNA Polymerase III assembly factor, TFIIIC, a six-subunit complex that is essential for the transcription of small, structured RNA. TFIIIC and N-myc mutually restrict each other’s chromatin association, and their complex contributes to quality control in mRNA transcription. We previously demonstrated that the intrinsically disordered transactivation domain of N-myc interacts directly with a sub-complex of TFIIIC, τA. Structural studies by others show that DNA binding of τA is largely mediated by TFIIIC3, leaving open the role of the DNA-binding domain of TFIIIC5. Here, we demonstrate that this domain is a binding site for two regions in the transactivation domain of N-myc, through an integrated approach combining NMR spectroscopy, hydrogen–deuterium exchange mass spectrometry, and interaction assays (pull-downs, ITC, fluorescence polarization, and co-immunoprecipitation). AlphaFold modelling predicts with high-confidence a binding mode for the higher affinity N-myc motif that overlaps with the predicted intramolecular binding site of the C-terminal acidic plug of TFIIIC5, removal of which enhances the binding of N-myc. This model elucidates how the N-myc:TFIIIC5 interaction competes with DNA and other interactions, providing a basis for their mutual regulation.
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Mar 2026
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Open Access
Abstract: Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm—a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D–S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D–S260D–Y271pCMF) further destabilizes the domain’s structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid–liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus.
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Mar 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Emma
Tarrant
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Isabel G.
Cormack
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Charlotte E.
Hunter
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Olesia
Werbowy
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Sebastian
Dorawa
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Lei
Wang
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Ida Helene
Steen
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Ruth-Anne
Sandaa
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Elísabet Eik
Guðmundsdóttir
,
Bernd
Ketelsen-Striberny
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Anna-Karina
Kaczorowska
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Tadeusz
Kaczorowski
,
Ehmke
Pohl
,
Stefanie
Freitag-Pohl
Diamond Proposal Number(s):
[39189]
Open Access
Abstract: This study describes the identification and characterization of two new extremophilic phage recombinases, UvsXt and UvsXp, discovered through metagenomic analysis within the Virus-X project, and explores their potential applications in biotechnology. DNA recombinases are essential for maintaining genome integrity across all kingdoms of life by facilitating homologous recombination and repairing double-stranded DNA breaks. Their capacity to bind and stabilize single-stranded DNA (ssDNA) has led to wide-ranging applications in molecular biology. UvsXt and UvsXp show homology with known bacterial RecA and viral UvsX recombinases, including conservation of key catalytic residues and DNA-binding motifs. Biochemical assays reveal that both enzymes exhibit superior DNA strand-exchange activity compared to Escherichia coli RecA. High-resolution crystal structures of UvsXt (2.0 Å) and UvsXp (2.6 Å) confirm a conserved RecA-like core fold, with distinct structural variation at the N-terminus responsible for oligomerization. However, in spite of their similarities, we show that neither enzyme is capable to functionally replace RecA in E. coli. Their remarkable thermostability and functionality across diverse chemical environments highlights their robustness for biotechnological use. Notably, UvsXt enhances loop-mediated isothermal amplification of viral RNA by stabilizing ssDNA intermediates. These findings expand the repertoire of thermostable recombinases with potential utility in diagnostic applications.
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Feb 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18566]
Open Access
Abstract: Zinc finger antiviral protein (ZAP) is a cytoplasmic protein central to host innate immunity to viral infection. ZAP has no intrinsic catalytic activity but inhibits viral replication by binding to CpG dinucleotides in cytoplasmic viral RNA and recruiting other factors to inhibit protein synthesis and target the RNA for degradation. KHNYN is a ZAP-binding protein required for ZAP-restriction of CpG-rich viral genomes. It contains an extended diKH, PIN nuclease, and CUElike domain, each of which are required for ZAP restriction of viral replication. Here, we report a structural, enzymological, and virological study of KHNYN’s essential PIN nuclease domain. Our crystal structure reveals an extended PIN domain (ex-PIN) containing a conserved N-terminal arm region required for domain stability and an active site tetra-Asp motif, which are both required for antiviral activity. Unlike the weak activity recently reported for the PIN domain, we demonstrate that the KHNYN ex-PIN domain is a highly active Mn2+-dependent single-stranded RNA endonuclease that cleaves with a preference for ApC, ApA, and UpA dinucleotides. These observations extend our view of KHNYN antiviral activity and suggest an unforeseen role for activation by manganese ions in the ZAP–KHNYN antiviral response.
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Dec 2025
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I04-Macromolecular Crystallography
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Xue
Han
,
Izaak N.
Beck
,
Moise
Mansour
,
Tom J.
Arrowsmith
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Roland
Barriot
,
Paul
Chansigaud
,
Carine
Pagès
,
Hussein
Hamze
,
Hatice
Akarsu
,
Laurent
Falquet
,
Peter
Redder
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Xibing
Xu
,
Tim R.
Blower
,
Pierre
Genevaux
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Toxin–antitoxin (TA) systems are central to bacterial immunity, genome maintenance, and pathogenicity. Toxins of TA systems use diverse strategies to control bacterial growth and represent attractive therapeutic targets to fight pathogens. In this work, we have investigated the toxic mechanism of the three RelE toxins of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis in humans. Structural studies showed that RelBE1, RelBE2, and RelBE3 TA complexes share conserved structural motifs distinct from the RelBE complex of Escherichia coli. Although RelE homologs have previously been reported to perform ribosome-dependent messenger RNA (mRNA) cleavage, detection of cleavage products by nEMOTE demonstrated that only RelE3 targets mRNA. In contrast, in vitro and in vivo analyses using Mycobacterium smegmatis and M. tuberculosis revealed that RelE1 is a site-specific RNase, able to cleave 16S rRNA from free 30S and formed 70S ribosomes, to release the anti-Shine–Dalgarno region and prevent translation. This stunning mode of action, which is likely shared with RelE2, demonstrates that there is broader diversity for toxic mechanisms within the widespread RelE family.
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Nov 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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[35324]
Open Access
Abstract: Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate, an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosylmethionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialized CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product 5′-methylthioadenosine-AMP reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analysis revealed a phage-encoded SAM-AMP lyase that degrades SAM-AMP efficiently in vitro, consistent with an anti-CRISPR function.
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Jul 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38144]
Open Access
Abstract: Bacteria encode a panoply of defence systems to overcome phage infection. In recent years, over 100 defence systems have been identified, with the majority of these found co-localized in defence islands. Although there has been much progress in understanding the mechanisms of anti-phage defence employed by bacteria, far less is known about their regulation before and during phage infection. Here, we describe RptR (RMS-proximal transcriptional regulator), a small transcriptional regulator of a defence island in enteropathogenic Escherichia coli composed of a toxin–antitoxin system, DarTG2, embedded within a Type I restriction–modification system (RMS). We determined the molecular structure of a RptR homodimer and, using transcriptional reporter and in vitro DNA binding assays, show that RptR represses the promoter of the defence island by binding to a series of three direct repeats in the promoter. Furthermore, we demonstrate, using the structural models of RptR validated with electrophoretic mobility shift assays, that the minimal RptR binding site is a 6-bp palindrome, TAGCTA. Both RptR and its binding site are highly conserved across diverse bacterial genomes with a strong genetic association with Type I RMS, highlighting the role of RptR as a novel regulatory component of an important mechanism for anti-phage defence in bacteria.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[20161, 21035, 25270]
Open Access
Abstract: The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein’s dynamic self-assembly. We show that the N-protein’s interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that the IDL exists in a concentration-dependent equilibrium between monomeric, dimeric, and trimeric states. The CC motif facilitates parallel, head-to-head oligomerization of N-protein dimers, transitioning between compact (closed) and extended (open) configurations depending on the interaction network within the IDL. This linker-driven assembly modulates phase separation, impacting the size, stability, and dynamics of biomolecular condensates. Here, we present the most comprehensive conformational landscape analysis of the N-protein to date, providing a detailed model of its self-assembly and phase separation. Our findings highlight how the structural plasticity of the IDL and CC-mediated interactions are pivotal to its roles in the SARS-CoV-2 life cycle.
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Jun 2025
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B21-High Throughput SAXS
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Open Access
Abstract: Complexes of nuclear factors 45 and 90 (NF45–NF90) play a multitude of roles in co- and post-transcriptional RNA processing, including regulating adenosine-to-inosine editing, cassette exon and back splicing, and splicing fidelity. NF45–NF90 complexes recognize double-stranded RNA (dsRNA) and, in human cells, primarily interact with Alu inverted repeats (AluIRs) that are commonly inserted into introns and other non-coding RNA regions. Intronic AluIRs of ∼300 bp can regulate splicing outcomes, such as generation of circular RNAs. We examined domain reorganization of NF45–NF90 domains on dsRNAs exceeding 50 bp to gain insight into its RNA recognition properties on longer dsRNAs. Using a combination of phylogenetic analysis, solution methods (including small angle X-ray scattering and quantitative cross-linking mass spectrometry), machine learning, and negative stain electron microscopy, we generated a model of NF45–NF90 complex formation on dsRNA. Our data reveal that different interactions of NF45–NF90 complexes allow these proteins to coat long stretches of dsRNA. This property of the NF45–NF90 complex has important implications for how long, nuclear dsRNAs are recognized in the nucleus and how this might promote (co)-regulation of specific RNA splicing and editing events that shape the mammalian transcriptome.
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Apr 2025
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