I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Josephine H. R.
Maidment
,
Motoki
Shimizu
,
Adam R.
Bentham
,
Sham
Vera
,
Marina
Franceschetti
,
Apinya
Longya
,
Clare E. M.
Stevenson
,
Juan Carlos
De La Concepcion
,
Aleksandra
Bialas
,
Sophien
Kamoun
,
Ryohei
Terauchi
,
Mark J.
Banfield
Diamond Proposal Number(s):
[13467, 18565]
Open Access
Abstract: A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.
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May 2023
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I04-Macromolecular Crystallography
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Amit
Kumar
,
Oscar
Vadas
,
Nicolas
Dos Santos Pacheco
,
Xu
Zhang
,
Kin
Chao
,
Nicolas
Darvill
,
Helena Ø.
Rasmussen
,
Yingqi
Xu
,
Gloria Meng-Hsuan
Lin
,
Fisentzos A.
Stylianou
,
Jan Skov
Pedersen
,
Sarah L.
Rouse
,
Marc L.
Morgan
,
Dominique
Soldati-Favre
,
Stephen
Matthews
Diamond Proposal Number(s):
[23620]
Open Access
Abstract: The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.
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Apr 2023
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tamar
Skaist Mehlmam
,
Justin T.
Biel
,
Syeda Maryam
Azeem
,
Elliot R.
Nelson
,
Sakib
Hossain
,
Louise
Dunnett
,
Neil G.
Paterson
,
Alice
Douangamath
,
Romain
Talon
,
Danny
Axford
,
Helen
Orins
,
Frank
Von Delft
,
Daniel A.
Keedy
Diamond Proposal Number(s):
[15751, 18340, 23570]
Open Access
Abstract: Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.
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Mar 2023
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Maud
Dumoux
,
Thomas
Glen
,
Jake L. R.
Smith
,
Elaine M. L.
Ho
,
Luis Ma
Perdigão
,
Avery
Pennington
,
Sven
Klumpe
,
Neville B. Y.
Yee
,
David A.
Farmer
,
Pui Y. A.
Lai
,
William
Bowles
,
Ron
Kelley
,
Jürgen M
Plitzko
,
Liang
Wu
,
Mark
Basham
,
Daniel K.
Clare
,
C. Alistair
Siebert
,
Michele C.
Darrow
,
James H.
Naismith
,
Michael
Grange
Open Access
Abstract: Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.
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Feb 2023
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I04-Macromolecular Crystallography
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Julia
Takuno Hespanhol
,
Daniel Enrique
Sanchez-Limache
,
Gianlucca Gonçalves
Nicastro
,
Liam
Mead
,
Edgar Enrique
Llontop
,
Gustavo
Chagas-Santos
,
Chuck
Shaker Farah
,
Robson Francisco
De Souza
,
Rodrigo Da Silva
Galhardo
,
Andrew L.
Lovering
,
Ethel
Bayer-Santos
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: The type VI secretion system (T6SS) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here, we characterize the function of the SPI-22 T6SS of Salmonella bongori showing that it has antibacterial activity and identify a group of antibacterial T6SS effectors (TseV1–4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins with a DUF3396 domain (TsiV1–4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1–4 are evolutionarily related to enzymes involved in DNA repair. TseV3 recognizes specific DNA structures and preferentially cleave splayed arms, generating DNA double-strand breaks and inducing the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanisms regulating the activity of these toxins.
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Oct 2022
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Juliane
John
,
Oskar
Aurelius
,
Vivek
Srinivas
,
Patricia
Saura
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Philipp S.
Simon
,
Medhanjali
Dasgupta
,
Cindy
Pham
,
Sheraz
Gul
,
Kyle D.
Sutherlin
,
Pierre
Aller
,
Agata
Butryn
,
Allen M.
Orville
,
Mun Hon
Cheah
,
Shigeki
Owada
,
Kensuke
Tono
,
Franklin D
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Nicholas K.
Sauter
,
Vittal K
Yachandra
,
Junko
Yano
,
Ville R. I.
Kaila
,
Jan
Kern
,
Hugo
Lebrette
,
Martin
Högbom
Open Access
Abstract: Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
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Sep 2022
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B23-Circular Dichroism
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Lenka
Stejskal
,
Mphatso D.
Kalemera
,
Charlotte B.
Lewis
,
Machaela
Palor
,
Lucas
Walker
,
Tina
Daviter
,
William D.
Lees
,
David S.
Moss
,
Myrto
Kremyda-Vlachou
,
Zisis
Kozlakidis
,
Giulia
Gallo
,
Dalan
Bailey
,
William
Rosenberg
,
Christopher J. R.
Illingworth
,
Adrian J.
Shepherd
,
Joe
Grove
Diamond Proposal Number(s):
[22783]
Open Access
Abstract: E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.
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Jul 2022
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B21-High Throughput SAXS
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Open Access
Abstract: The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.
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Sep 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.
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Sep 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13467, 18565]
Open Access
Abstract: Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.
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Aug 2021
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