Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[19832]
Open Access
Abstract: CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the ‘crass fold’, that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.
|
May 2023
|
|
I04-Macromolecular Crystallography
|
Abstract: Phosphorylation of SARS-CoV-2 nucleoprotein recruits human cytosolic 14-3-3 proteins playing a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that 14-3-3 binding is triggered by phosphorylation of SARS-CoV-2 nucleoprotein at either of two pseudo-repeats centered at Ser197 and Thr205. According to fluorescence anisotropy, the pT205-motif, present in SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which collectively display an unforeseen pT205/pS197 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, inhibiting its dephosphorylation. This Ser/Arg-rich region is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting that the strength of 14-3-3/nucleoprotein interaction can be linked with the replicative fitness of the virus.
|
Nov 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Herman K. H.
Fung
,
Shelley
Grimes
,
Alexis
Huet
,
Robert L.
Duda
,
Maria
Chechik
,
Joseph
Gault
,
Carol V.
Robinson
,
Roger W.
Hendrix
,
Paul J.
Jardine
,
James F.
Conway
,
Christoph G..
Baumann
,
Alfred A.
Antson
Open Access
Abstract: Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.
|
Aug 2022
|
|
Krios II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[19832]
Open Access
Abstract: Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the “3-bases-in, 3-bases-out” conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123–139, which may serve as a valuable epitope for surveillance and diagnostics.
|
Jul 2021
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3– and AlF4–, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)−, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)− species, indicating the significance of this TSA for studies of biological motors.
|
Feb 2021
|
|
|
|
Yanchun
Peng
,
Alexander J.
Mentzer
,
Guihai
Liu
,
Xuan
Yao
,
Zixi
Yin
,
Danning
Dong
,
Wanwisa
Dejnirattisai
,
Timothy
Rostron
,
Piyada
Supasa
,
Chang
Liu
,
César
López-Camacho
,
Jose
Slon-Campos
,
Yuguang
Zhao
,
David I.
Stuart
,
Guido C.
Paesen
,
Jonathan M.
Grimes
,
Alfred A.
Antson
,
Oliver W.
Bayfield
,
Dorothy E. D. P.
Hawkins
,
De-Sheng
Ker
,
Beibei
Wang
,
Lance
Turtle
,
Krishanthi
Subramaniam
,
Paul
Thomson
,
Ping
Zhang
,
Christina
Dold
,
Jeremy
Ratcliff
,
Peter
Simmonds
,
Thushan
De Silva
,
Paul
Sopp
,
Dannielle
Wellington
,
Ushani
Rajapaksa
,
Yi-Ling
Chen
,
Mariolina
Salio
,
Giorgio
Napolitani
,
Wayne
Paes
,
Persephone
Borrow
,
Benedikt M.
Kessler
,
Jeremy W.
Fry
,
Nikolai F.
Schwabe
,
Malcolm G.
Semple
,
J. Kenneth
Baillie
,
Shona C.
Moore
,
Peter J. M.
Openshaw
,
M. Azim
Ansari
,
Susanna
Dunachie
,
Eleanor
Barnes
,
John
Frater
,
Georgina
Kerr
,
Oliver
Gould
,
Teresa
Lockett
,
Robert
Levin
,
Yonghong
Zhang
,
Ronghua
Jing
,
Ling-Pei
Ho
,
Richard J.
Cornall
,
Christopher P.
Conlon
,
Paul
Klenerman
,
Gavin R.
Screaton
,
Juthathip
Mongkolsapaya
,
Andrew
Mcmichael
,
Julian C.
Knight
,
Graham
Ogg
,
Tao
Dong
Open Access
Abstract: The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
|
Sep 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
Abstract: Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria. This is mediated by the steroidogenic acute regulatory protein (STARD1), containing a mitochondrial import sequence followed by a cholesterol‐binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia (LCAH), the mechanism of steroidogenesis regulation by STARD1 remains debatable. It has been hypothesized to involve a molten‐globule structural transition and interaction with 14‐3‐3 proteins. In this study, we aimed to address the structural basis for the 14‐3‐3‐STARD1 interaction. We show that, while the isolated START domain does not interact with 14‐3‐3, this interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity toward 14‐3‐3 and crystal structures of 14‐3‐3 complexes with Ser57 and Ser195 phosphopeptides suggest distinct roles of site‐specific phosphorylations in recruiting 14‐3‐3, to modulate STARD1 activity, processing and import to the mitochondria. Phosphorylation at Ser195 creates a unique conditional site that could only bind to 14‐3‐3 upon partial unfolding of the START domain. Overall, our findings on the interaction between 14‐3‐3 and STARD1 may have potential clinical implications for patients with LCAH.
|
Sep 2020
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Oliver W.
Bayfield
,
Evgeny
Klimuk
,
Dennis C.
Winkler
,
Emma L.
Hesketh
,
Maria
Chechik
,
Naiqian
Cheng
,
Eric C.
Dykeman
,
Leonid
Minakhin
,
Neil A.
Ranson
,
Konstantin
Severinov
,
Alasdair C.
Steven
,
Alfred A.
Antson
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Understanding molecular events during virus assembly and genome packaging is important for understanding viral life cycles, and the functioning of other protein–nucleic acid machines. The model system developed for the thermophilic bacteriophage P23-45 offers advantages over other systems. Cryo-EM reconstructions reveal modifications to a canonical capsid protein fold, resulting in capsids that are abnormally large for this virus class. Structural information on the portal protein, through which the genome is packaged, demonstrates that the capsid influences the portal’s conformation. This has implications for understanding how processes inside and outside the capsid can be coordinated.
|
Feb 2019
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.
|
Jan 2019
|
|
I24-Microfocus Macromolecular Crystallography
|
Benjamin
Cressiot
,
Sandra J.
Greive
,
Wei
Si
,
Tomas
Pascoa
,
Mehrnaz
Mojtabavi
,
Maria
Chechik
,
Huw T.
Jenkins
,
Xueguang
Lu
,
Ke
Zhang
,
Aleksei
Aksimentiev
,
Alfred A.
Antson
,
Meni
Wanunu
Diamond Proposal Number(s):
[13587]
Abstract: Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein’s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal’s DNA-ratchet function.
|
Nov 2017
|
|
