Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
|
Nattapong
Sanguankiattichai
,
Balakumaran
Chandrasekar
,
Yuewen
Sheng
,
Nathan
Hardenbrook
,
Werner W. A.
Tabak
,
Margit
Drapal
,
Farnusch
Kaschani
,
Clemens
Grünwald-Gruber
,
Daniel
Krahn
,
Pierre
Buscaill
,
Suzuka
Yamamoto
,
Atsushi
Kato
,
Robert
Nash
,
George
Fleet
,
Richard
Strasser
,
Paul D.
Fraser
,
Markus
Kaiser
,
Peijun
Zhang
,
Gail M.
Preston
,
Renier A. L.
Van Der Hoorn
Diamond Proposal Number(s):
[21004, 29812, 28713]
Abstract: The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo–electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
|
Apr 2025
|
|
Krios II-Titan Krios II at Diamond
|
Yaqi
Sun
,
Yuewen
Sheng
,
Tao
Ni
,
Xingwu
Ge
,
Joscelyn
Sarsby
,
Philip J.
Brownridge
,
Kang
Li
,
Nathan
Hardenbrook
,
Gregory F.
Dykes
,
Nichola
Rockliffe
,
Claire E.
Eyers
,
Peijun
Zhang
,
Lu-Ning
Liu
Diamond Proposal Number(s):
[21004]
Open Access
Abstract: Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer (QconCAT)-based quantitative mass spectrometry, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.
|
Dec 2024
|
|
Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
|
Jianbing
Ma
,
Gangshun
Yi
,
Mingda
Ye
,
Craig
Macgregor-Chatwin
,
Yuewen
Sheng
,
Ying
Lu
,
Ming
Li
,
Qingrong
Li
,
Dong
Wang
,
Robert J. C.
Gilbert
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812]
Open Access
Abstract: The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.
|
Dec 2024
|
|
Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
|
Tao
Ni
,
Luiza
Mendonca
,
Yanan
Zhu
,
Andrew
Howe
,
Julika
Radecke
,
Pranav M.
Shah
,
Yuewen
Sheng
,
Anna-Sophia
Krebs
,
Helen M. E.
Duyvesteyn
,
Elizabeth
Allen
,
Teresa
Lambe
,
Cameron
Bisset
,
Alexandra
Spencer
,
Susan
Morris
,
David I.
Stuart
,
Sarah
Gilbert
,
Peijun
Zhang
Diamond Proposal Number(s):
[26987]
Open Access
Abstract: Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.
|
Sep 2023
|
|
Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
|
Yanan
Zhu
,
Christopher W.
Koo
,
C. Keith
Cassidy
,
Matthew C.
Spink
,
Tao
Ni
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Marisa
Martin-Fernandez
,
Juan
Shen
,
Yuewen
Sheng
,
Yun
Song
,
Zhengyi
Yang
,
Amy C.
Rosenzweig
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 29812]
Open Access
Abstract: Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.
|
Sep 2022
|
|
Krios I-Titan Krios I at Diamond
|
Open Access
Abstract: Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.
|
Jul 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 II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[26464]
Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
|
Jan 2022
|
|
B24-Cryo Soft X-ray Tomography
Krios II-Titan Krios II at Diamond
|
Luiza
Mendonca
,
Andrew
Howe
,
James B.
Gilchrist
,
Yuewen
Sheng
,
Dapeng
Sun
,
Michael L.
Knight
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Anna-Sophia
Krebs
,
Long
Chen
,
Julika
Radecke
,
Vivian D.
Li
,
Tao
Ni
,
Ilias
Kounatidis
,
Mohamed A.
Koronfel
,
Marta
Szynkiewicz
,
Maria
Harkiolaki
,
Marisa
Martin-Fernandez
,
William
James
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 26987]
Open Access
Abstract: Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
|
Jul 2021
|
|
E02-JEM ARM 300CF
|
Abstract: We show how gadolinium (Gd)-based metallofullerene (Gd3N@C80) molecules can be used to create single adatoms and nanoclusters on a graphene surface. An in situ heating holder within an aberration-corrected scanning transmission electron microscope is used to track the adhesion of endohedral metallofullerenes (MFs) to the surface of graphene, followed by Gd metal ejection and diffusion across the surface. Heating to 900 °C is used to promote adatom migration and metal nanocluster formation, enabling direct imaging of the assembly of nanoclusters of Gd. We show that hydrogen can be used to reduce the temperature of MF fragmentation and metal ejection, enabling Gd nanocluster formation on graphene surfaces at temperatures as low as 300 °C. The process of MF fragmentation and metal ejection is captured in situ and reveals that after metal release, the C80 cage opens further and fuses with the surface monolayer carbon glass on graphene, creating a highly stable carbon layer for further Gd adatom adhesion. Small voids and defects (∼1 nm) in the surface carbon glass act as trapping sites for Gd atoms, leading to atomic self-assembly of 2D monolayer Gd clusters. These results show that MFs can adhere to graphene surfaces at temperatures well above their bulk sublimation point, indicating that the surface bound MFs have strong adhesion to dangling bonds on graphene surfaces. The ability to create dispersed single Gd adatoms and Gd nanoclusters on graphene may have impact in spintronics and magnetism.
|
Sep 2018
|
|
