I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Fouzia
Bano
,
Suneale
Banerji
,
Tao
Ni
,
Dixy E.
Green
,
Kalila R.
Cook
,
Iain W.
Manfield
,
Paul L.
Deangelis
,
Emanuele
Paci
,
Martin
Lepšík
,
Robert J. C.
Gilbert
,
Ralf P.
Richter
,
David G.
Jackson
Open Access
Abstract: Immune surveillance involves the continual migration of antigen-scavenging immune cells from the tissues to downstream lymph nodes via lymphatic vessels. To enable such passage, cells first dock with the lymphatic entry receptor LYVE-1 on the outer surface of endothelium, using their endogenous hyaluronan glycocalyx, anchored by a second hyaluronan receptor, CD44. Why the process should require two different hyaluronan receptors and by which specific mechanism the LYVE-1•hyaluronan interaction enables lymphatic entry is however unknown. Here we describe the crystal structures and binding mechanics of murine and human LYVE-1•hyaluronan complexes. These reveal a highly unusual, sliding mode of ligand interaction, quite unlike the conventional sticking mode of CD44, in which the receptor grabs free hyaluronan chain-ends and winds them in through conformational re-arrangements in a deep binding cleft, lubricated by a layer of structured waters. Our findings explain the mode of action of a dedicated lymphatic entry receptor and define a distinct, low tack adhesive interaction that enables migrating immune cells to slide through endothelial junctions with minimal resistance, while clinging onto their hyaluronan glycocalyx for essential downstream functions.
|
Mar 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 II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[21004]
Open Access
Abstract: Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the central enzyme for converting atmospheric CO2 into organic molecules, playing a crucial role in the global carbon cycle. In cyanobacteria and some chemoautotrophs, Rubisco complexes, along with carbonic anhydrase, are enclosed within specific proteinaceous microcompartments, known as carboxysomes. The polyhedral carboxysome shell ensures a dense packaging of Rubisco and creates a high-CO2 internal environment to facilitate the fixation of CO2. Rubisco and carboxysomes have been popular targets for bioengineering, with the intent of enhancing plant photosynthesis, crop yields, and biofuel production. However, efficient generation of Form 1B Rubisco and cyanobacterial β-carboxysomes in heterologous systems remains challenging. Here, we developed genetic systems to efficiently engineer functional cyanobacterial Form 1B Rubisco in E. coli, by incorporating Rubisco assembly factor Raf1 and modulating the RbcL/S stoichiometry. We further accomplished effective reconstitution of catalytically active β-carboxysomes in E. coli with cognate Form 1B Rubisco by fine-tuning the expression levels of individual β-carboxysome components. In addition, we investigated the encapsulation mechanism of Rubisco into carboxysomes via constructing hybrid carboxysomes; this was achieved by creating a chimeric encapsulation peptide incorporating SSLDs that permits the encapsulation of Form 1B Rubisco into α-carboxysome shells. Our study provides insights into the assembly mechanisms of plant-like Form 1B Rubisco and its encapsulation principles in both β-carboxysomes and hybrid carboxysomes, and highlights the inherent modularity of carboxysome structures. The findings lay the framework for rational design and repurposing of CO2-fixing modules in bioengineering applications, e.g. crop engineering, biocatalyst production, and molecule delivery.
|
Dec 2024
|
|
I03-Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
|
James
Hillier
,
Yuguang
Zhao
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Tao-Hsin
Chang
,
Gangshun
Yi
,
Helen M. E.
Duyvesteyn
,
Jing
Yu
,
Weixian
Lu
,
Els
Pardon
,
Jan
Steyaert
,
Yanan
Zhu
,
Tao
Ni
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[19946, 28713]
Open Access
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
|
Aug 2024
|
|
|
|
Abstract: In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.
|
Dec 2023
|
|
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
|
Tao
Ni
,
Qiuyao
Jiang
,
Pei Cing
Ng
,
Juan
Shen
,
Hao
Dou
,
Yanan
Zhu
,
Julika
Radecke
,
Gregory F.
Dykes
,
Fang
Huang
,
Lu-Ning
Liu
,
Peijun
Zhang
Diamond Proposal Number(s):
[29812, 28713]
Open Access
Abstract: Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.
|
Sep 2023
|
|
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
|
Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.
|
Oct 2022
|
|
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 III-Titan Krios III at Diamond
|
Diamond Proposal Number(s):
[21004, 20223, 21005]
Open Access
Abstract: Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. They encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase catalyzing carbon fixation inside a proteinaceous shell. How Rubisco complexes pack within the carboxysomes is unknown. Using cryo-electron tomography, we determine the distinct 3D organization of Rubisco inside two distant α-carboxysomes from a marine α-cyanobacterium Cyanobium sp. PCC 7001 where Rubiscos are organized in three concentric layers, and from a chemoautotrophic bacterium Halothiobacillus neapolitanus where they form intertwining spirals. We further resolve the structures of native Rubisco as well as its higher-order assembly at near-atomic resolutions by subtomogram averaging. The structures surprisingly reveal that the authentic intrinsically disordered linker protein CsoS2 interacts with Rubiscos in native carboxysomes but functions distinctively in the two α-carboxysomes. In contrast to the uniform Rubisco-CsoS2 association in the Cyanobium α-carboxysome, CsoS2 binds only to the Rubiscos close to the shell in the Halo α-carboxysome. Our findings provide critical knowledge of the assembly principles of α-carboxysomes, which may aid in the rational design and repurposing of carboxysome structures for new functions.
|
Jul 2022
|
|
