I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen
effectors and initiate an immune response. Since their discovery, NLRs have been the focus of
protein engineering to improve disease resistance. However, this approach has proven challenging,
in part due to their narrow response specificity. Previously, we revealed the structural basis of
pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp
(Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile
of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an
effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for
AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik
variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in
complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results
provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors
where direct interaction between effectors and NLRs is established, particularly where this
interaction occurs via integrated domains.
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Sep 2019
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I03-Macromolecular Crystallography
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Timo
Vögtle
,
Sumana
Sharma
,
Jun
Mori
,
Zoltan
Nagy
,
Daniela
Semeniak
,
Cyril
Scandola
,
Mitchell J.
Geer
,
Christopher W.
Smith
,
Jordan
Lane
,
Scott
Pollack
,
Riitta
Lassila
,
Annukka
Jouppila
,
Alastair J.
Barr
,
Derek J.
Ogg
,
Tina D.
Howard
,
Helen J.
Mcmiken
,
Juli
Warwicker
,
Catherine
Geh
,
Rachel
Rowlinson
,
W. Mark
Abbott
,
Anita
Eckly
,
Harald
Schulze
,
Gavin J.
Wright
,
Alexandra
Mazharian
,
Klaus
Futterer
,
Sundaresan
Rajesh
,
Michael R.
Douglas
,
Yotis A.
Senis
Diamond Proposal Number(s):
[20026]
Open Access
Abstract: The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
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Aug 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.
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Jul 2019
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Krios II-Titan Krios II at Diamond
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Vasileios
Kargas
,
Pablo
Castro-hartmann
,
Norberto
Escudero-urquijo
,
Kyle
Dent
,
Christine
Hilcenko
,
Carolin
Sailer
,
Gertrude
Zisser
,
Maria J.
Marques-carvalho
,
Simone
Pellegrino
,
Leszek
Wawiórka
,
Stefan M. V.
Freund
,
Jane L.
Wagstaff
,
Antonina
Andreeva
,
Alexandre
Faille
,
Edwin
Chen
,
Florian
Stengel
,
Helmut
Bergler
,
Alan John
Warren
Diamond Proposal Number(s):
[14359, 15368, 16940, 17057, 18078, 18328]
Abstract: During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.
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May 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10088]
Open Access
Abstract: Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.
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May 2019
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[13848, 16052]
Open Access
Abstract: The quality of visual information that is available to an animal is limited by the size of its eyes. Differences in eye size can be observed even between closely related individuals, yet we understand little about how this affects vision. Insects are good models for exploring the effects of size on visual systems because many insect species exhibit size polymorphism. Previous work has been limited by difficulties in determining the 3D structure of eyes. We have developed a novel method based on x-ray microtomography to measure the 3D structure of insect eyes and to calculate predictions of their visual capabilities. We used our method to investigate visual allometry in the bumblebee Bombus terrestris and found that size affects specific aspects of vision, including binocular overlap, optical sensitivity, and dorsofrontal visual resolution. This reveals that differential scaling between eye areas provides flexibility that improves the visual capabilities of larger bumblebees.
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Feb 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar ‘kinked hairpin’ folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.
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Feb 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron preacinetobactin acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.
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Dec 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221]
Open Access
Abstract: The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily 2 helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5'-3' direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5'-3' translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesions might stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.
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Dec 2018
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I04-Macromolecular Crystallography
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Open Access
Abstract: Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo β-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.
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Dec 2018
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