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Open Access
Abstract: Determination of protein structures typically entails building a model that satisfies the collected experimental observations and its deposition in the Protein Data Bank. Experimental limitations can lead to unavoidable uncertainties during the process of model building, which result in the introduction of errors into the deposited model. Many metrics are available for model validation, but most are limited to consideration of the physico-chemical aspects of the model or its match to the experimental data. The latest advances in the field of deep learning have enabled the increasingly accurate prediction of inter-residue distances, an advance which has played a pivotal role in the recent improvements observed in the field of protein ab initio modelling. Here, new validation methods are presented based on the use of these precise inter-residue distance predictions, which are compared with the distances observed in the protein model. Sequence-register errors are particularly clearly detected and the register shifts required for their correction can be reliably determined. The method is available in the ConKit package (https://www.conkit.org).
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Dec 2022
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I03-Macromolecular Crystallography
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Josefin
Ahlqvist
,
Javier A.
Linares-Pastén
,
Andrius
Jasilionis
,
Martin
Welin
,
Maria
Håkansson
,
L. Anders
Svensson
,
Lei
Wang
,
Hildegard
Watzlawick
,
Arnþór
Ævarsson
,
Ólafur H.
Friðjónsson
,
Guðmundur Ó.
Hreggviðsson
,
Bernd
Ketelsen Striberny
,
Eirin
Glomsaker
,
Olav
Lanes
,
Salam
Al-Karadaghi
,
Eva
Nordberg Karlsson
Diamond Proposal Number(s):
[20028, 23282]
Open Access
Abstract: This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3′–5′ exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5′–3′ exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3′–5′ exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.
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Nov 2022
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Jill
Trewhella
,
Patrice
Vachette
,
Jan
Bierma
,
Clement
Blanchet
,
Emre
Brookes
,
Srinivas
Chakravarthy
,
Leonie
Chatzimagas
,
Thomas E.
Cleveland
,
Nathan
Cowieson
,
Ben
Crossett
,
Anthony P.
Duff
,
Daniel
Franke
,
Frank
Gabel
,
Richard E.
Gillilan
,
Melissa
Graewert
,
Alexander
Grishaev
,
J. Mitchell
Guss
,
Michal
Hammel
,
Jesse
Hopkins
,
Qingqui
Huang
,
Jochen S.
Hub
,
Greg L.
Hura
,
Thomas C.
Irving
,
Cy Michael
Jeffries
,
Cheol
Jeong
,
Nigel
Kirby
,
Susan
Krueger
,
Anne
Martel
,
Tsutomu
Matsui
,
Na
Li
,
Javier
Pérez
,
Lionel
Porcar
,
Thierry
Prange
,
Ivan
Rajkovic
,
Mattia
Rocco
,
Daniel J.
Rosenberg
,
Timothy M.
Ryan
,
Soenke
Seifert
,
Hiroshi
Sekiguchi
,
Dmitri
Svergun
,
Susana
Teixeira
,
Aurelien
Thureau
,
Thomas M.
Weiss
,
Andrew E.
Whitten
,
Kathleen
Wood
,
Xiaobing
Zuo
Open Access
Abstract: Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0–1 Å−1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å−1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å−1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.
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Nov 2022
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I24-Microfocus Macromolecular Crystallography
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Gyorgy
Babnigg
,
Darren A.
Sherrell
,
Youngchang
Kim
,
Jessica L.
Johnson
,
Boguslaw
Nocek
,
Kemin
Tan
,
Danny
Axford
,
Hui
Li
,
Lance
Bigelow
,
Lukas
Welk
,
Michael
Endres
,
Robin L.
Owen
,
Andrzej
Joachimiak
Abstract: Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre-sized droplets allow the very efficient use of protein samples and can produce large quantities of high-quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X-ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample-transport and data-collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50–500 pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data-collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X-ray beam by depositing an emulsion of droplets onto a silicon fixed-target serial device are provided. After three months of storage at 4°C, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample-delivery and data-collection strategy allows crystal droplets to be shipped and set aside until beamtime is available.
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Aug 2022
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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
,
Danny
Axford
,
Sam
Horrell
,
Pierre
Aller
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Robin L.
Owen
,
Martin A.
Walsh
,
Graeme
Winter
Diamond Proposal Number(s):
[26986, 27088]
Open Access
Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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Jun 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Å using crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser–His–Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.
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May 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Low-nanomolar binding constants were recorded for a series of six 2′-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues with acetylcholine-binding protein (AChBP). The crystal structures of three complexes with AChBP reveal details of molecular recognition in the orthosteric binding site and imply how the other three ligands bind. Comparisons exploiting AChBP as a surrogate for α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the key interactions are conserved. The ligands interact with the same residues as the archetypal nAChR agonist nicotine yet display greater affinity, thereby rationalizing their in vivo activity as potent antagonists of nicotine-induced antinociception. An oxyanion-binding site is formed on the periphery of the AChBP orthosteric site by Lys42, Asp94, Glu170 and Glu210. These residues are highly conserved in the human α4, β2 and α7 nAChR sequences. However, specific sequence differences are discussed that could contribute to nAChR subtype selectivity and in addition may represent a point of allosteric modulation. The ability to engage with this peripheral site may explain, in part, the function of a subset of ligands to act as agonists of α7 nAChR.
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Mar 2022
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Open Access
Abstract: Recently, there has been a dramatic improvement in the quality and quantity of data derived using cryogenic electron microscopy (cryo-EM). This is also associated with a large increase in the number of atomic models built. Although the best resolutions that are achievable are improving, often the local resolution is variable, and a significant majority of data are still resolved at resolutions worse than 3 Å. Model building and refinement is often challenging at these resolutions, and hence atomic model validation becomes even more crucial to identify less reliable regions of the model. Here, a graphical user interface for atomic model validation, implemented in the CCP-EM software suite, is presented. It is aimed to develop this into a platform where users can access multiple complementary validation metrics that work across a range of resolutions and obtain a summary of evaluations. Based on the validation estimates from atomic models associated with cryo-EM structures from SARS-CoV-2, it was observed that models typically favor adopting the most common conformations over fitting the observations when compared with the model agreement with data. At low resolutions, the stereochemical quality may be favored over data fit, but care should be taken to ensure that the model agrees with the data in terms of resolvable features. It is demonstrated that further re-refinement can lead to improvement of the agreement with data without the loss of geometric quality. This also highlights the need for improved resolution-dependent weight optimization in model refinement and an effective test for overfitting that would help to guide the refinement process.
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Feb 2022
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I04-Macromolecular Crystallography
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Josefin
Ahlqvist
,
Javier A.
Linares-Pastén
,
Maria
Håkansson
,
Andrius
Jasilionis
,
Karolina
Kwiatkowska-Semrau
,
Ólafur H.
Friðjónsson
,
Anna-Karina
Kaczorowska
,
Slawomir
Dabrowski
,
Arnþór
Ævarsson
,
Guðmundur Ó.
Hreggviðsson
,
Salam
Al-Karadaghi
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
Diamond Proposal Number(s):
[20028]
Open Access
Abstract: This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA–Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E–SD–EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.
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Feb 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19152, 11386]
Open Access
Abstract: Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Å resolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.
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Jan 2022
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