I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Classical molecules encoded by the major histocompatibility complex (MHC) are central to immune responses. Compared to typical mammals, the chicken MHC is small and simple, determining life or death from economically important pathogens like avian influenza virus and Marek’s disease virus (MDV). Several genes within the tightly linked chicken MHC have been suggested to determine resistance and susceptibility to MDV, but it was a surprise to find that the dominantly expressed class II molecule from the resistant B2 haplotype employed a novel peptide-binding mode with a decamer core sequence compared to the susceptible B19 haplotype with a typical nonamer core. We examined the crystal structure of the dominantly expressed class II molecule from another resistant haplotype, B21, which is extremely frequent in commercial chicken flocks, to find that it bound the same MDV peptide with both nonamer and decamer cores, revealing an unexpected plasticity of binding that potentially increases the immune response to this devastating pathogen.
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Apr 2026
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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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I04-Macromolecular Crystallography
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Ansgar
Zoch
,
Gabriela
Konieczny
,
Tania
Auchynnikava
,
Birgit
Stallmeyer
,
Nadja
Rotte
,
Madeleine
Heep
,
Rebecca V.
Berrens
,
Martina
Schito
,
Yuka
Kabayama
,
Theresa
Schöpp
,
Sabine
Kliesch
,
Brendan
Houston
,
Liina
Nagirnaja
,
Moira K.
O’bryan
,
Kenneth I.
Aston
,
Donald F.
Conrad
,
Juri
Rappsilber
,
Robin C.
Allshire
,
Atlanta G.
Cook
,
Frank
Tüttelmann
,
Dónal
O’carroll
Diamond Proposal Number(s):
[18515]
Open Access
Abstract: In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.
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Feb 2024
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Jon
Agirre
,
Mihaela
Atanasova
,
Haroldas
Bagdonas
,
Charles B.
Ballard
,
Arnaud
Basle
,
James
Beilsten-Edmands
,
Rafael J.
Borges
,
David G.
Brown
,
J. Javier
Burgos-Marmol
,
John M.
Berrisford
,
Paul S.
Bond
,
Iracema
Caballero
,
Lucrezia
Catapano
,
Grzegorz
Chojnowski
,
Atlanta G.
Cook
,
Kevin D.
Cowtan
,
Tristan I.
Croll
,
Judit É.
Debreczeni
,
Nicholas E.
Devenish
,
Eleanor J.
Dodson
,
Tarik R.
Drevon
,
Paul
Emsley
,
Gwyndaf
Evans
,
Phil R.
Evans
,
Maria
Fando
,
James
Foadi
,
Luis
Fuentes-Montero
,
Elspeth F.
Garman
,
Markus
Gerstel
,
Richard J.
Gildea
,
Kaushik
Hatti
,
Maarten L.
Hekkelman
,
Philipp
Heuser
,
Soon Wen
Hoh
,
Michael A.
Hough
,
Huw T.
Jenkins
,
Elisabet
Jiménez
,
Robbie P.
Joosten
,
Ronan M.
Keegan
,
Nicholas
Keep
,
Eugene B.
Krissinel
,
Petr
Kolenko
,
Oleg
Kovalevskiy
,
Victor S.
Lamzin
,
David M.
Lawson
,
Andrey
Lebedev
,
Andrew G. W.
Leslie
,
Bernhard
Lohkamp
,
Fei
Long
,
Martin
Maly
,
Airlie
Mccoy
,
Stuart J.
Mcnicholas
,
Ana
Medina
,
Claudia
Millán
,
James W.
Murray
,
Garib N.
Murshudov
,
Robert A.
Nicholls
,
Martin E. M.
Noble
,
Robert
Oeffner
,
Navraj S.
Pannu
,
James M.
Parkhurst
,
Nicholas
Pearce
,
Joana
Pereira
,
Anastassis
Perrakis
,
Harold R.
Powell
,
Randy J.
Read
,
Daniel J.
Rigden
,
William
Rochira
,
Massimo
Sammito
,
Filomeno
Sanchez Rodriguez
,
George M.
Sheldrick
,
Kathryn L.
Shelley
,
Felix
Simkovic
,
Adam J.
Simpkin
,
Pavol
Skubak
,
Egor
Sobolev
,
Roberto A.
Steiner
,
Kyle
Stevenson
,
Ivo
Tews
,
Jens M. H.
Thomas
,
Andrea
Thorn
,
Josep Triviño
Valls
,
Ville
Uski
,
Isabel
Uson
,
Alexei
Vagin
,
Sameer
Velankar
,
Melanie
Vollmar
,
Helen
Walden
,
David
Waterman
,
Keith S.
Wilson
,
Martyn
Winn
,
Graeme
Winter
,
Marcin
Wojdyr
,
Keitaro
Yamashita
Open Access
Abstract: The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
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Jun 2023
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Open Access
Abstract: Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.
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Jan 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18515]
Open Access
Abstract: Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5′ untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13550]
Open Access
Abstract: Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. The majority of RTT missense mutations disrupt the interaction of the MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the “NCoR/SMRT interaction domain” (NID) of MeCP2 directly contacts transducin-beta like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.
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Mar 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
Data acquisition
Diagnostics
Health Physics
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Diamond Proposal Number(s):
[135500]
Open Access
Abstract: Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 angstrom. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits.
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Jun 2016
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7613, 9487]
Open Access
Abstract: Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs.
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Dec 2015
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1225, 7613]
Open Access
Abstract: Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common ‘DZF’ domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins.
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Jul 2012
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