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346 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae James M. Parkhurst , Adam D. Crawshaw , C. Alistair Siebert , Maud Dumoux , C. David Owen , Pedro Nunes , David Waterman , Thomas Glen , David I. Stuart , James H. Naismith , Gwyndaf Evans Iucrj 10 DOI: 10.1107/S2052252523001902 Open Access Show Abstract ▼ Abstract: Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.	May 2023
	Spatial- and Fourier-domain ptychography for high-throughput bio-imaging Shaowei Jiang , Pengming Song , Tianbo Wang , Liming Yang , Ruihai Wang , Chengfei Guo , Bin Feng , Andrew Maiden , Guoan Zheng Nature Protocols 5 DOI: 10.1038/s41596-023-00829-4 Show Abstract ▼ Abstract: First envisioned for determining crystalline structures, ptychography has become a useful imaging tool for microscopists. However, ptychography remains underused by biomedical researchers due to its limited resolution and throughput in the visible light regime. Recent developments of spatial- and Fourier-domain ptychography have successfully addressed these issues and now offer the potential for high-resolution, high-throughput optical imaging with minimal hardware modifications to existing microscopy setups, often providing an excellent trade-off between resolution and field of view inherent to conventional imaging systems, giving biomedical researchers the best of both worlds. Here, we provide extensive information to enable the implementation of ptychography by biomedical researchers in the visible light regime. We first discuss the intrinsic connections between spatial-domain coded ptychography and Fourier ptychography. A step-by-step guide then provides the user instructions for developing both systems with practical examples. In the spatial-domain implementation, we explain how a large-scale, high-performance blood-cell lens can be made at negligible expense. In the Fourier-domain implementation, we explain how adding a low-cost light source to a regular microscope can improve the resolution beyond the limit of the objective lens. The turnkey operation of these setups is suitable for use by professional research laboratories, as well as citizen scientists. Users with basic experience in optics and programming can build the setups within a week. The do-it-yourself nature of the setups also allows these procedures to be implemented in laboratory courses related to Fourier optics, biomedical instrumentation, digital image processing, robotics and capstone projects.	May 2023
	Fragment merging using a graph database samples different catalogue space than similarity search Stephanie Wills , Ruben Sanchez-Garcia , Tim Dudgeon , Stephen D. Roughley , Andy Merritt , Roderick E. Hubbard , James Davidson , Frank Von Delft , Charlotte M. Deane Journal Of Chemical Information And Modeling 9 DOI: 10.1021/acs.jcim.3c00276 Open Access Show Abstract ▼ Abstract: Fragment merging is a promising approach to progressing fragments directly to on-scale potency: each designed compound incorporates the structural motifs of overlapping fragments in a way that ensures compounds recapitulate multiple high-quality interactions. Searching commercial catalogues provides one useful way to quickly and cheaply identify such merges and circumvents the challenge of synthetic accessibility, provided they can be readily identified. Here, we demonstrate that the Fragment Network, a graph database that provides a novel way to explore the chemical space surrounding fragment hits, is well-suited to this challenge. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four crystallographic screening campaigns and contrast the results with a traditional fingerprint-based similarity search. The two approaches identify complementary sets of merges that recapitulate the observed fragment–protein interactions but lie in different regions of chemical space. We further show our methodology is an effective route to achieving on-scale potency by retrospective analyses for two different targets; in analyses of public COVID Moonshot and Mycobacterium tuberculosis EthR inhibitors, potential inhibitors with micromolar IC50 values were identified. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search.	May 2023
E02-JEM ARM 300CF	Cryogenic electron ptychographic single particle analysis with wide bandwidth information transfer Xudong Pei , Liqi Zhou , Chen Huang , Mark Boyce , Judy S. Kim , Emanuela Liberti , Yiming Hu , Takeo Sasaki , Peter D. Nellist , Peijun Zhang , David I. Stuart , Angus I. Kirkland , Peng Wang Nature Communications 14 DOI: 10.1038/s41467-023-38268-0 Diamond Proposal Number(s): [17918] Open Access Show Abstract ▼ Abstract: Advances in cryogenic transmission electron microscopy have revolutionised the determination of many macromolecular structures at atomic or near-atomic resolution. This method is based on conventional defocused phase contrast imaging. However, it has limitations of weaker contrast for small biological molecules embedded in vitreous ice, in comparison with cryo-ptychography, which shows increased contrast. Here we report a single-particle analysis based on the use of ptychographic reconstruction data, demonstrating that three dimensional reconstructions with a wide information transfer bandwidth can be recovered by Fourier domain synthesis. Our work suggests future applications in otherwise challenging single particle analyses, including small macromolecules and heterogeneous or flexible particles. In addition structure determination in situ within cells without the requirement for protein purification and expression may be possible.	May 2023
B21-High Throughput SAXS DL-SAXS-Offline SAXS and Sample Environment Development	Sample preparation of atherosclerotic plaque for SAXS/WAXS experimentation Rebecca R. Mackley , Steven Huband , Tara L. Schiller Acs Omega DOI: 10.1021/acsomega.3c00060 Diamond Proposal Number(s): [27756, 29720] Open Access Show Abstract ▼ Abstract: Atherosclerosis is often described as a single disease entity; however, the morphology of each plaque is unique to the individual. The field currently lacks a technique that can discriminate stable from unstable plaques, to identify those at risk of a thromboembolic event. Small- and wide-angle X-ray scattering (SAXS/WAXS) holds the potential to be able to identify key materials present in a plaque, such as cholesterol species, collagen, low-density lipoproteins (LDLs), and hydroxyapatite. Protocols have been established for the preparation of excised human atherosclerotic tissue that are investigated herein. This includes the fixing, sectioning, and substrate selection of the sample. Through several sample preparation methods, vast improvements have been made to sample-to-noise ratio and background subtraction.	Apr 2023
I13-2-Diamond Manchester Imaging	Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences Carles Bosch , Joerg Lindenau , Alexandra Pacureanu , Christopher J. Peddie , Marta Majkut , Andrew C. Douglas , Raffaella Carzaniga , Alexander Rack , Lucy Collinson , Andreas T. Schaefer , Heiko Stegmann Applied Physics Letters 122 DOI: 10.1063/5.0142405 Diamond Proposal Number(s): [20274] Open Access Show Abstract ▼ Abstract: Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.	Apr 2023
	A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography Swagatha Ghosh , Doris Zorić , Peter Dahl , Monika Bjelčić , Jonatan Johannesson , Emil Sandelin , Per Borjesson , Alexander Bjorling , Analia Banacore , Petra Edlund , Oskar Aurelius , Mirko Milas , Jie Nan , Anastasya Shilova , Ana Gonzalez , Uwe Mueller , Gisela Branden , Richard Neutze Journal Of Applied Crystallography 56, 449 - 460 DOI: 10.1107/S1600576723001036 Open Access Show Abstract ▼ Abstract: Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.	Apr 2023
I04-Macromolecular Crystallography	Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture Philipp Knyphausen , Mariana Rangel Pereira , Paul Brear , Marko Hyvonen , Lutz Jermutus , Florian Hollfelder Nature Communications 14 DOI: 10.1038/s41467-023-36099-7 Diamond Proposal Number(s): [18548, 25402] Open Access Show Abstract ▼ Abstract: Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.	Feb 2023
	Cryo-plasma FIB/SEM volume imaging of biological specimens 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 Elife 12 DOI: 10.7554/eLife.83623 Open Access Show Abstract ▼ 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.	Feb 2023
	Perspective: Structure determination of protein-ligand complexes at room temperature using X-ray diffraction approaches Michael A. Hough , Filippo Prischi , Jonathan A. R. Worrall Frontiers In Molecular Biosciences 10 DOI: 10.3389/fmolb.2023.1113762 Open Access Show Abstract ▼ Abstract: The interaction between macromolecular proteins and small molecule ligands is an essential component of cellular function. Such ligands may include enzyme substrates, molecules involved in cellular signalling or pharmaceutical drugs. Together with biophysical techniques used to assess the thermodynamic and kinetic properties of ligand binding to proteins, methodology to determine high-resolution structures that enable atomic level interactions between protein and ligand(s) to be directly visualised is required. Whilst such structural approaches are well established with high throughput X-ray crystallography routinely used in the pharmaceutical sector, they provide only a static view of the complex. Recent advances in X-ray structural biology methods offer several new possibilities that can examine protein-ligand complexes at ambient temperature rather than under cryogenic conditions, enable transient binding sites and interactions to be characterised using time-resolved approaches and combine spectroscopic measurements from the same crystal that the structures themselves are determined. This Perspective reviews several recent developments in these areas and discusses new possibilities for applications of these advanced methodologies to transform our understanding of protein-ligand interactions.	Jan 2023

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