Krios I-Titan Krios I at Diamond
|
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.
|
May 2023
|
|
Krios I-Titan Krios I at Diamond
|
David J. K.
Swainsbury
,
Frederick
Hawkings
,
Elizabeth C.
Martin
,
Sabina
Musial
,
Jack H.
Salisbury
,
Philip J.
Jackson
,
David A.
Farmer
,
Matthew P.
Johnson
,
C. Alistair
Siebert
,
Andrew
Hitchcock
,
C. Neil
Hunter
Diamond Proposal Number(s):
[29785]
Open Access
Abstract: Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron–sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobacter sphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene–maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.
|
Mar 2023
|
|
|
|
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.
|
Feb 2023
|
|
Krios I-Titan Krios I at Diamond
|
Matthew S
Proctor
,
Lorna A.
Malone
,
David A.
Farmer
,
David J. K.
Swainsbury
,
Frederick R.
Hawkings
,
Federica
Pastorelli
,
Thomas Z.
Emrich-Mills
,
C. Alistair
Siebert
,
C. Neil
Hunter
,
Matthew P.
Johnson
,
Andrew
Hitchcock
Diamond Proposal Number(s):
[21004, 21005]
Open Access
Abstract: In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ~10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn molecule that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.
|
Jun 2022
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[23297]
Open Access
Abstract: In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.
|
Apr 2022
|
|
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
|
Diamond Proposal Number(s):
[26464, 28151]
Open Access
Abstract: Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that by collecting cryo-ET STA data using the same conditions as SPA, with both Correlated Double Sampling (CDS) and super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising data size on disk and area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).
|
Apr 2022
|
|
Krios I-Titan Krios I at Diamond
|
Pu
Qian
,
Alastair T.
Gardiner
,
Ivana
Šímová
,
Katerina
Naydenova
,
Tristan I.
Croll
,
Philip J.
Jackson
,
Nupur
Nupur
,
Miroslav
Kloz
,
Petra
Čubáková
,
Marek
Kuzma
,
Yonghui
Zeng
,
Pablo
Castro-Hartmann
,
Bart
Van Knippenberg
,
Kenneth N.
Goldie
,
David
Kaftan
,
Pavel
Hrouzek
,
Jan
Hájek
,
Jon
Agirre
,
C. Alistair
Siebert
,
David
Bína
,
Kasim
Sader
,
Henning
Stahlberg
,
Roman
Sobotka
,
Christopher J.
Russo
,
Tomáš
Polívka
,
C. Neil
Hunter
,
Michal
Koblížek
Diamond Proposal Number(s):
[29785]
Open Access
Abstract: Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.
|
Feb 2022
|
|
|
|
Open Access
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.
|
Oct 2021
|
|
Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
Talos-Talos Arctica at Diamond
|
Diamond Proposal Number(s):
[23047]
Open Access
Abstract: Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.
|
May 2021
|
|
I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
|
Wanwisa
Dejnirattisai
,
Daming
Zhou
,
Helen M.
Ginn
,
Helen M. E.
Duyvesteyn
,
Piyada
Supasa
,
James Brett
Case
,
Yuguang
Zhao
,
Thomas
Walter
,
Alexander J.
Mentzer
,
Chang
Liu
,
Beibei
Wang
,
Guido C.
Paesen
,
Jose
Slon-Campos
,
César
López-Camacho
,
Natasha M.
Kafai
,
Adam L.
Bailey
,
Rita E.
Chen
,
Baoling
Ying
,
Craig
Thompson
,
Jai
Bolton
,
Alex
Fyfe
,
Sunetra
Gupta
,
Tiong Kit
Tan
,
Javier
Gilbert-Jaramillo
,
William
James
,
Michael
Knight
,
Miles W.
Carroll
,
Donal
Skelly
,
Christina
Dold
,
Yanchun
Peng
,
Robert
Levin
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Paul
Klenerman
,
Nigel
Temperton
,
David R.
Hall
,
Mark A.
Williams
,
Neil G.
Paterson
,
Felicity
Bertram
,
C. Alistair
Siebert
,
Daniel K.
Clare
,
Andrew
Howe
,
Julika
Radecke
,
Yun
Song
,
Alain R.
Townsend
,
Kuan-Ying A.
Huang
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Michael S.
Diamond
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009, 26983]
Open Access
Abstract: Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike, and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50<0.1μg/ml) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryo-electron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
|
Feb 2021
|
|
