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  1. Article ; Online: Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase

    Wenbo Li / Georgina C. Girt / Ashish Radadiya / James J. P. Stewart / Nigel G. J. Richards / James H. Naismith

    Open Biology, Vol 13, Iss

    2023  Volume 1

    Abstract: The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one ...

    Abstract The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.
    Keywords C-nucleoside ; X-ray crystallography ; formycin ; biosynthesis ; Biology (General) ; QH301-705.5
    Subject code 541
    Language English
    Publishing date 2023-01-01T00:00:00Z
    Publisher The Royal Society
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Plasma FIB milling for the determination of structures in situ

    Casper Berger / Maud Dumoux / Thomas Glen / Neville B.-y. Yee / John M. Mitchels / Zuzana Patáková / Michele C. Darrow / James H. Naismith / Michael Grange

    Nature Communications, Vol 14, Iss 1, Pp 1-

    2023  Volume 12

    Abstract: The authors harness plasma focused ion beams for pseudo-atomic structure determination, reporting increased throughput and automation in in situ structural biology to elucidate structure-function relationships inside cells and tissues. ...

    Abstract The authors harness plasma focused ion beams for pseudo-atomic structure determination, reporting increased throughput and automation in in situ structural biology to elucidate structure-function relationships inside cells and tissues.
    Keywords Science ; Q
    Language English
    Publishing date 2023-02-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Approaches to Using the Chameleon

    Talya S. Levitz / Miriam Weckener / Ivan Fong / James H. Naismith / Catherine L. Drennan / Edward J. Brignole / Daniel K. Clare / Michele C. Darrow

    Frontiers in Molecular Biosciences, Vol

    Robust, Automated, Fast-Plunge cryoEM Specimen Preparation

    2022  Volume 9

    Abstract: The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The ... ...

    Abstract The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.
    Keywords cryoEM specimen preparation ; vitrification ; automation ; self-wicking grids ; air-water interface issues ; preferred orientation ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2022-06-01T00:00:00Z
    Publisher Frontiers Media S.A.
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides

    Jinzhu Zhang / Minghai Tang / Yujie Chen / Dan Ke / Jie Zhou / Xinyu Xu / Wenxian Yang / Jianxiong He / Haohao Dong / Yuquan Wei / James H. Naismith / Yi Lin / Xiaofeng Zhu / Wei Cheng

    Nature Communications, Vol 12, Iss 1, Pp 1-

    2021  Volume 12

    Abstract: Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to ...

    Abstract Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb M
    Keywords Science ; Q
    Language English
    Publishing date 2021-12-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: The molecular basis of regulation of bacterial capsule assembly by Wzc

    Yun Yang / Jiwei Liu / Bradley R. Clarke / Laura Seidel / Jani R. Bolla / Philip N. Ward / Peijun Zhang / Carol V. Robinson / Chris Whitfield / James H. Naismith

    Nature Communications, Vol 12, Iss 1, Pp 1-

    2021  Volume 13

    Abstract: The Wzc–Wza complex forms part of the bacterial extracellular polysaccharides synthesis machinery, where cycling of the Wzc between phosphorylation states is crucial to both synthesis and export. Here the authors report the structure of the Wzc octamer ... ...

    Abstract The Wzc–Wza complex forms part of the bacterial extracellular polysaccharides synthesis machinery, where cycling of the Wzc between phosphorylation states is crucial to both synthesis and export. Here the authors report the structure of the Wzc octamer and provide insight into its regulation through phosphorylation.
    Keywords Science ; Q
    Language English
    Publishing date 2021-07-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: The use of nanobodies in a sensitive ELISA test for SARS-CoV-2 Spike 1 protein

    Georgina C. Girt / Abirami Lakshminarayanan / Jiandong Huo / Joshua Dormon / Chelsea Norman / Babak Afrough / Adam Harding / William James / Raymond J. Owens / James H. Naismith

    Royal Society Open Science, Vol 8, Iss

    2021  Volume 9

    Abstract: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in the fluid has important uses in biotechnology, and is integral to many point-of-care SARS-CoV-2 diagnostics. Sandwich enzyme-linked immunosorbent assays (ELISAs) are a ... ...

    Abstract Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in the fluid has important uses in biotechnology, and is integral to many point-of-care SARS-CoV-2 diagnostics. Sandwich enzyme-linked immunosorbent assays (ELISAs) are a sensitive, well-established method of measuring antigens in solutions. They use one ligand to capture and the other ligand to detect the target analyte. Detection is commonly achieved using colorimetric readout obtained upon the reaction of a substrate with HRP-conjugated secondary ligand. Nanobodies, the VHH domain of camelid antibodies, have expanded the repertoire of molecules used in antigen detection. Nanobodies' high affinity for target antigens, their compact structure, their high stability and ease of production has driven research into their use as diagnostic reagents. Guided by a structural understanding of epitopes on the receptor-binding domain of the SARS-CoV-2 Spike protein, we investigated various combinations of engineered nanobodies in a sandwich ELISA to detect the Spike protein of SARS-CoV-2. We have identified an optimal combination of nanobodies. These were selectively functionalized to further improve antigen capture, enabling the measurement of sub-picomolar amounts of SARS-CoV-2 Spike protein in solution. With this combination, the routine detection limit in samples inactivated by heat and detergent corresponded to less than seven focus-forming units of infectious SARS-CoV-2.
    Keywords ELISA ; nanobody ; SARS-CoV-2 ; Spike ; site-specific conjugation ; Science ; Q
    Subject code 500
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher The Royal Society
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Single Cell Cryo-Soft X-ray Tomography Shows That Each Chlamydia Trachomatis Inclusion Is a Unique Community of Bacteria

    Patrick Phillips / James M. Parkhurst / Ilias Kounatidis / Chidinma Okolo / Thomas M. Fish / James H. Naismith / Martin A. Walsh / Maria Harkiolaki / Maud Dumoux

    Life, Vol 11, Iss 842, p

    2021  Volume 842

    Abstract: Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It ... ...

    Abstract Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion.
    Keywords Chalmydia ; cryo-soft X ray tomography ; community ; single cell ; Science ; Q
    Language English
    Publishing date 2021-08-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  8. Article ; Online: Parakeet

    James M. Parkhurst / Maud Dumoux / Mark Basham / Daniel Clare / C. Alistair Siebert / Trond Varslot / Angus Kirkland / James H. Naismith / Gwyndaf Evans

    Open Biology, Vol 11, Iss

    a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

    2021  Volume 10

    Abstract: In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘ ... ...

    Abstract In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.
    Keywords digital twin ; electron microscopy ; tomography ; multislice simulation ; Biology (General) ; QH301-705.5
    Subject code 600
    Language English
    Publishing date 2021-10-01T00:00:00Z
    Publisher The Royal Society
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  9. Article ; Online: Cryo-plasma FIB/SEM volume imaging of biological specimens

    Maud Dumoux / Thomas Glen / Jake LR Smith / Elaine ML Ho / Luis MA Perdigão / Avery Pennington / Sven Klumpe / Neville BY Yee / David Andrew Farmer / Pui YA 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

    eLife, Vol

    2023  Volume 12

    Abstract: Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular 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 ...

    Abstract Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular 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. 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 20–50 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.
    Keywords HeLa cells ; Vero cells ; mouse tissue (brain) ; mouse tissue (cardiac) ; S. cerevisiae ; R. rubrum ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 500
    Language English
    Publishing date 2023-02-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  10. Article ; Online: Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

    Clarissa M. Czekster / Hannes Ludewig / Stephen A. McMahon / James H. Naismith

    Nature Communications, Vol 8, Iss 1, Pp 1-

    2017  Volume 10

    Abstract: Cyclic peptide macrocycles are promising anti-cancer and antimicrobial molecules. Here, the authors characterize the structure and catalytic mechanism of the prolyl oligopeptidase B from Basidiomycete fungi, showing that its dual macrocyclase-peptidase ... ...

    Abstract Cyclic peptide macrocycles are promising anti-cancer and antimicrobial molecules. Here, the authors characterize the structure and catalytic mechanism of the prolyl oligopeptidase B from Basidiomycete fungi, showing that its dual macrocyclase-peptidase activity is crucial for amatoxin macrocyclization.
    Keywords Science ; Q
    Language English
    Publishing date 2017-10-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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