B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31229]
Abstract: Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.
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Oct 2023
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin–antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3′-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2–FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2–FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.
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Oct 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.
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Sep 2023
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Sofiyah
Shaibullah
,
Nurshahirah
Shuhaimi
,
De-Sheng
Ker
,
Nurhikmah
Mohd-Sharif
,
Kok Lian
Ho
,
Aik-Hong
Teh
,
Jitka
Waterman
,
Thean-Hock
Tang
,
Rui-Rui
Wong
,
Sheila
Nathan
,
Rahmah
Mohamed
,
Min Jia
Ng
,
Shin-Yee
Fung
,
Mohd Anuar
Jonet
,
Mohd
Firdaus-Raih
,
Chyan Leong
Ng
Open Access
Abstract: Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.
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Sep 2023
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495]
Open Access
Abstract: Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.
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Sep 2023
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Abstract: Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.0 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.
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Sep 2023
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I02-Macromolecular Crystallography
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Sapir
Ofer
,
Fabian
Blombach
,
Amanda M.
Erkelens
,
Declan
Barker
,
Zoja
Soloviev
,
Samuel
Schwab
,
Katherine
Smollett
,
Dorota
Matelska
,
Thomas
Fouqueau
,
Nico
Van Der Vis
,
Nicholas A.
Kent
,
Konstantinos
Thalassinos
,
Remus T.
Dame
,
Finn
Werner
Open Access
Abstract: In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into ‘hypernucleosome’ particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.
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Sep 2023
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Open Access
Abstract: Fixed-target crystallography has become a widely used approach for serial crystallography at both synchrotron and X-ray free-electron laser (XFEL) sources. A plethora of fixed targets have been developed at different facilities and by various manufacturers, with different characteristics and dimensions and with little or no emphasis on standardization. These many fixed targets have good reasons for their design, shapes, fabrication materials and the presence or absence of apertures and fiducials, reflecting the diversity of serial experiments. Given this, it would be a Sisyphean task to design and manufacture a new standard fixed target that would satisfy all possible experimental configurations. Therefore, a simple standardized descriptor to fully describe fixed targets is proposed rather than a standardized device. This descriptor is a dictionary that could be read by fixed-target beamline software and straightforwardly allow data collection from fixed targets new to that beamline. The descriptor would therefore allow a much easier exchange of fixed targets between sources and facilitate the uptake of new fixed targets, benefiting beamlines, users and manufacturers. This descriptor was first presented at, and was developed following, a meeting of representatives from multiple synchrotron and XFEL sources in Hamburg in January 2023.
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Aug 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Olga
Moroz
,
Elena
Blagova
,
Andrey A.
Lebedev
,
Lars K.
Skov
,
Roland A.
Pache
,
Kirk M.
Schnorr
,
Lars
Kiemer
,
Esben P.
Friis
,
Søren
Nymand-Grarup
,
Li
Ming
,
Liu
Ye
,
Mikkel
Klausen
,
Marianne T.
Cohn
,
Esben G. W.
Schmidt
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[7864, 13587, 24948]
Open Access
Abstract: Muramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.
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Aug 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 24948]
Open Access
Abstract: Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.
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Aug 2023
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