I13-1-Coherence
I13-2-Diamond Manchester Imaging
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Christoph
Rau
,
Darren J.
Batey
,
Shashidhara
Marathe
,
Leonard
Turpin
,
Kudakwashe
Jakata
,
Silvia
Cipiccia
,
Isabel
Anthony
,
Roberto
Volpe
,
Claus-Peter
Richter
,
Alessandra
Carriero
,
Maud
Dumoux
,
Jurgen E.
Schneider
,
Erica
Dall'Armellina
,
Marc W.
Holderied
,
Jan
Van Den Bulcke
Open Access
Abstract: We report about the experimental work related to hierarchical structures at the Diamond I13L beamlines. The I13-2 Imaging and I13-1 Coherence beamlines provide imaging with micro- and nano-resolution. The Diamond II upgrade for the synchrotron source and the OCTOPI upgrade for I13L provide new opportunities for expanding the existing scientific areas in multiscale and operando imaging. We describe the scientific research benefitting from the instrumental upgrade. Comparable recording times across all length scales will enable hierarchical operando imaging. With the implementation of automated high-throughput data acquisition and analysis, large numbers of samples will be analyzed.
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Nov 2024
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Open Access
Abstract: Background:
A key issue with the established method of cryo-electron tomography (Cryo- ET) often lies in the challenge of accurately locating complexes or proteins of interest within the crowded cellular environment of the generated tomograms. This project aims to use cryo-scanning transmission electron microscopy (STEM) to highlight regions of tomograms containing the protein of interest by tagging with a minimally-sized heavy atom nanoparticle for downstream processing. A fine balance must be achieved between resolution, signal-to-noise ratio (SNR), depth of focus, and damage to the lamella.
Methods:
A scan generator offers alternative (non-raster) methods of scanning the beam to offset damage and allow higher electron fluences to be used without compromising ice quality. Small raster and interleaved scans (< 200 nm) were compared in the same quantifoil hole of vitreous ice, using an equal overall electron fluence and flux.
Analysis of elastic scattering cross section theory yields an approximate minimal size of nanoparticles for detection in STEM of vitreous amorphous specimens. Single particle-like sample preparation was employed to freeze varying sizes of gold nanoparticles (0.8-4 nm) in different thicknesses of ice. These were subsequently exposed to STEM to assess a drop-off in SNR with increasing collection angles using an annular dark field detector.
Results:
Scanning in an alternative fashion using long dwell times demonstrated a significant reduction in mass loss. Raster scanning appeared to be marginally better than interleaved scanning when using shorter dwell times (20 μs), as evidenced by greater loss of intensity in the scanned areas, normalised to reference areas taken within the same hole of vitreous ice. However, using longer dwell times (250 μs or 500 μs) reversed this effect, showing raster scanning to be significantly more damaging than interleaved, melting the ice completely in thin samples for raster scanning, whilst maintaining the ice intact using an interleaved sequence.
Conclusions:
These findings provide valuable first steps toward optimizing cryo-STEM imaging for detecting nanoparticles and correlating these findings with in-situ Transmission Electron Microscopy (TEM).
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Oct 2024
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Open Access
Abstract: For cryo-electron tomography (cryo-ET) of beam-sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal-to-noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample-environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo-ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low-symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron-beam damage to the specimen is an issue and the first images acquired will transfer more high-resolution information than those acquired last. There is also an inherent trade-off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n-helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose-symmetric tilt schemes for cryo-ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose-symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition.
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Jun 2024
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Open Access
Abstract: Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.
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Nov 2023
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Krios I-Titan Krios I at Diamond
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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
Open Access
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.
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May 2023
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Electron cryo-tomography is an imaging technique for probing 3D structures with at the nanometer scale. This technique has been used extensively in the biomedical field to study the complex structures of proteins and other macromolecules. With the advancement in technology, microscopes are currently capable of producing images amounting to terabytes of data per day, posing great challenges for scientists as the speed of processing of the images cannot keep up with the ever-higher throughput of the microscopes. Therefore, automation is an essential and natural pathway on which image processing—from individual micrographs to full tomograms—is developing. In this paper, we present Ot2Rec, an open-source pipelining tool which aims to enable scientists to build their own processing workflows in a flexible and automatic manner. The basic building blocks of Ot2Rec are plugins which follow a unified application programming interface structure, making it simple for scientists to contribute to Ot2Rec by adding features which are not already available. In this paper, we also present three case studies of image processing using Ot2Rec, through which we demonstrate the speedup of using a semi-automatic workflow over a manual one, the possibility of writing and using custom (prototype) plugins, and the flexibility of Ot2Rec which enables the mix-and-match of plugins. We also demonstrate, in the Supplementary Material, a built-in reporting feature in Ot2Rec which aggregates the metadata from all process being run, and output them in the Jupyter Notebook and/or HTML formats for quick review of image processing quality. Ot2Rec can be found at https://github.com/rosalindfranklininstitute/ot2rec.
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Mar 2023
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Open Access
Abstract: An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.
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Mar 2023
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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
Open Access
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.
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Feb 2023
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I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Halina
Mikolajek
,
Miriam
Weckener
,
Z. Faidon
Brotzakis
,
Jiandong
Huo
,
Evmorfia V.
Dalietou
,
Audrey
Le Bas
,
Pietro
Sormanni
,
Peter J.
Harrison
,
Philip N.
Ward
,
Steven
Truong
,
Lucile
Moynie
,
Daniel K.
Clare
,
Maud
Dumoux
,
Joshua
Dormon
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Chelsea
Norman
,
Naveed
Hussain
,
Vinod
Vogirala
,
Raymond J.
Owens
,
Michele
Vendruscolo
,
James
Naismith
Diamond Proposal Number(s):
[27031, 27051, 29666]
Open Access
Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
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Jul 2022
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NONE-No attached Diamond beamline
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Charles J.
Buchanan
,
Ben
Gaunt
,
Peter J.
Harrison
,
Yun
Yang
,
Jiwei
Liu
,
Aziz
Khan
,
Andrew M.
Giltrap
,
Audrey
Le Bas
,
Philip N.
Ward
,
Kapil
Gupta
,
Maud
Dumoux
,
Tiong Kit
Tan
,
Lisa
Schimaski
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Sergio
Daga
,
Nicola
Picchiotti
,
Margherita
Baldassarri
,
Elisa
Benetti
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Chiara
Fallerini
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Francesca
Fava
,
Annarita
Giliberti
,
Panagiotis I.
Koukos
,
Matthew J.
Davy
,
Abirami
Lakshminarayanan
,
Xiaochao
Xue
,
Georgios
Papadakis
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Lachlan P.
Deimel
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Virgínia
Casablancas-Antràs
,
Timothy D. W.
Claridge
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Alexandre M. J. J.
Bonvin
,
Quentin J.
Sattentau
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Simone
Furini
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Marco
Gori
,
Jiandong
Huo
,
Raymond J.
Owens
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Christiane
Schaffitzel
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Imre
Berger
,
Alessandra
Renieri
,
James H.
Naismith
,
Andrew J.
Baldwin
,
Benjamin G.
Davis
Open Access
Abstract: Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.
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Jun 2022
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