Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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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.
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Dec 2024
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Yufei
Xiang
,
Jialu
Xu
,
Briana L.
Mcgovern
,
Anna
Ranzenigo
,
Wei
Huang
,
Zhe
Sang
,
Juan
Shen
,
Randy
Diaz-Tapia
,
Ngoc Dung
Pham
,
Abraham J. P.
Teunissen
,
M. Luis
Rodriguez
,
Jared
Benjamin
,
Derek J.
Taylor
,
Mandy M.t.
Van Leent
,
Kris M.
White
,
Adolfo
García-Sastre
,
Peijun
Zhang
,
Yi
Shi
Abstract: Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA’s modularity enables rapid, cost-effective production and adaptation to evolving pathogens.
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Oct 2024
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Open Access
Abstract: RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells. These methods evolve to be combined to achieve a wide resolution range from atomic to sub-nano resolutions, making correlative microscopy an emerging trend. The developments in microscopic methods provide multi-fold and spatial information, advancing our understanding of viral infections and providing critical tools for developing novel antiviral strategies and rapid responses to emerging viral threats.
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Sep 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20223, 21004]
Open Access
Abstract: Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3′ end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3′ oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.
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Jul 2024
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Talos-Talos Arctica at Diamond
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Paul B.
Klar
,
David G.
Waterman
,
Tim
Gruene
,
Debakshi
Mullick
,
Yun
Song
,
James B.
Gilchrist
,
C. David
Owen
,
Wen
Wen
,
Idan
Biran
,
Lothar
Houben
,
Neta
Regev-Rudzki
,
Ron
Dzikowski
,
Noa
Marom
,
Lukas
Palatinus
,
Peijun
Zhang
,
Leslie
Leiserowitz
,
Michael
Elbaum
Diamond Proposal Number(s):
[21004, 29812]
Open Access
Abstract: Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.
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Jul 2024
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Open Access
Abstract: The evolutionary pressures exerted by viral infections have led to the development of various cellular proteins with potent antiviral activities, some of which are known as antiviral restriction factors. TRIpartite Motif-containing protein 5 alpha (TRIM5α) is a well-studied restriction factor of retroviruses that exhibits virus- and host-species-specific functions in protecting against cross-primate transmission of specific lentiviruses. This specificity is achieved at the level of the host gene through positive selection predominantly within its C-terminal B30.2/PRYSPRY domain, which is responsible for the highly specific recognition of retroviral capsids. However, more recent work has challenged this paradigm, demonstrating TRIM5α as a restriction factor for retroelements as well as phylogenetically distinct viral families, acting similarly through the recognition of viral gene products via B30.2/PRYSPRY. This spectrum of antiviral activity raises questions regarding the genetic and structural plasticity of this protein as a mediator of the recognition of a potentially diverse array of viral molecular patterns. This review highlights the dynamic evolutionary footprint of the B30.2/PRYSPRY domain in response to retroviruses while exploring the guided ‘specificity’ conferred by the totality of TRIM5α’s additional domains that may account for its recently identified promiscuity.
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Jun 2024
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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.
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Dec 2023
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: The structure of chromatin plays pivotal roles in regulating gene transcription, DNA replication and repair, and chromosome segregation. This structure, however, remains elusive. Here, using cryo-FIB and cryo-ET, we delineate the 3D architecture of native chromatin fibres in intact interphase human T-lymphoblasts and determine the in situ structures of nucleosomes in different conformations. These chromatin fibres are not structured as uniform 30 nm one-start or two-start filaments but are composed of relaxed, variable zigzag organizations of nucleosomes connected by straight linker DNA. Nucleosomes with little H1 and linker DNA density are distributed randomly without any spatial preference. This work will inspire future high-resolution investigations on native chromatin structures in situ at both a single-nucleosome level and a population level under many different cellular conditions in health and disease.
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Oct 2023
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Anna T.
Gres
,
Karen A.
Kirby
,
William M.
Mcfadden
,
Haijuan
Du
,
Dandan
Liu
,
Chaoyi
Xu
,
Alexander J.
Bryer
,
Juan R.
Perilla
,
Jiong
Shi
,
Christopher
Aiken
,
Xiaofeng
Fu
,
Peijun
Zhang
,
Ashwanth C.
Francis
,
Gregory B.
Melikyan
,
Stefan G.
Sarafianos
Open Access
Abstract: HIV-1 capsid (CA) stability is important for viral replication. E45A and P38A mutations enhance and reduce core stability, thus impairing infectivity. Second-site mutations R132T and T216I rescue infectivity. Capsid lattice stability was studied by solving seven crystal structures (in native background), including P38A, P38A/T216I, E45A, E45A/R132T CA, using molecular dynamics simulations of lattices, cryo-electron microscopy of assemblies, time-resolved imaging of uncoating, biophysical and biochemical characterization of assembly and stability. We report pronounced and subtle, short- and long-range rearrangements: (1) A38 destabilized hexamers by loosening interactions between flanking CA protomers in P38A but not P38A/T216I structures. (2) Two E45A structures showed unexpected stabilizing CANTD-CANTD inter-hexamer interactions, variable R18-ring pore sizes, and flipped N-terminal β-hairpin. (3) Altered conformations of E45Aa α9-helices compared to WT, E45A/R132T, WTPF74, WTNup153, and WTCPSF6 decreased PF74, CPSF6, and Nup153 binding, and was reversed in E45A/R132T. (4) An environmentally sensitive electrostatic repulsion between E45 and D51 affected lattice stability, flexibility, ion and water permeabilities, electrostatics, and recognition of host factors.
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Sep 2023
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[21004, 22941, 22910]
Open Access
Abstract: Motile bacteria employ conserved chemotaxis networks to detect chemical gradients in their surroundings and effectively regulate their locomotion, enabling the location of essential nutrients and other important biological niches. The sensory apparatus of the chemotaxis pathway is an array of core-signaling units (CSUs) composed of transmembrane chemoreceptors, the histidine kinase CheA and an adaptor protein, CheW. Although chemotaxis pathways represent the best understood signaling systems, a detailed mechanistic understanding of signal transduction has been hindered by the lack of a complete structural picture of the CSU and extended array. In this study, we present the structure of the complete CSU from phage φX174 E protein lysed Escherichia coli cells, determined using cryo-electron tomography and sub-tomogram averaging to 12-Å resolution. Using AlphaFold2, we further predict the atomic structures of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly an all-atom CSU model, which we conformationally refine using our cryo-electron tomography map. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex, including novel interactions between neighboring receptor ligand-binding domains. Our results further elucidate previously unresolved interactions between individual CheA domains, including an anti-parallel P1 dimer and non-productive binding mode between P1 and P4, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation.
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Sep 2023
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