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Instruments / Technical Area	Publication Details	Published
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 Krios V-Titan Krios V at Diamond	A service-based approach to cryoEM facility processing pipelines at eBIC Anna Horstmann , Stephen Riggs , Yuriy Chaban , Daniel K. Clare , Guilherme De Freitas , David Farmer , Andrew Howe , Kyle L. Morris , Daniel Hatton Acta Crystallographica Section D Structural Biology 80 DOI: 10.1107/S2059798324000986 Open Access Show Abstract ▼ Abstract: Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.	Mar 2024
Krios III-Titan Krios III at Diamond	Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography Pranav N. M. Shah , James B. Gilchrist , Björn O. Forsberg , Alister Burt , Andrew Howe , Shyamal Mosalaganti , William Wan , Julika Radecke , Yuriy Chaban , Geoff Sutton , David I. Stuart , Mark Boyce Cell Host & Microbe 190 DOI: 10.1016/j.chom.2023.03.004 Diamond Proposal Number(s): [21004] Open Access Show Abstract ▼ Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.	Mar 2023
B21-High Throughput SAXS	CD44 and CD221 directed magnetic cubosomes for the targeted delivery of helenalin to rhabdomyosarcoma cells Hakmin Mun , Yuriy Chaban , Tanveer A. Tabish , Nanasaheb Thorat , Nathan Cowieson , C. David Owen , Helen E. Townley Nano Research 12 DOI: 10.1007/s12274-022-5037-4 Diamond Proposal Number(s): [29646] Open Access Show Abstract ▼ Abstract: Confining chemotherapy to tumour sites by means of active targeting nanoparticles (NPs) may increase the treatment effectuality while reducing potential side effects. Cubosomes are one of the next-generation drug delivery nanocarriers by virtue of their biocompatibility and bioadhesion, sizeable payload encapsulation and high thermostability. Herein, an active tumour targeting system towards rhabdomyosarcoma (RMS) cells was evaluated. Cubosomes were loaded with helenalin (a secondary metabolite from Arnica plants), which we have previously shown to induce apoptosis in RMS cells. The functionalization of the cubosomes was accomplished to enable binding to membrane receptors and translocation under a magnetic field. RMS cells overexpress CD44 and CD221 on their membrane surface and, therefore, hyaluronic acid (HA, a ligand for CD44) and antibodies (Abs) against CD221 were coupled to cubosomes via electrostatic attraction and the thiol-Michael reaction, respectively. Magnetization of the cubic phase NPs was achieved by embedding superparamagnetic iron oxide NPs (SPIONPs) into the cubic matrix. Single-function and multi-function cubosomes had Im3m cubic phase structures with well-organized lattice patterns. Conjugation with 2% HA or anti-CD221 half Abs and/or 1% SPIONPs showed significantly higher uptake into RMS cells compared to unfunctionalized cubosomes. CD44 and CD221 directed magnetic (triple-function) cubosomes were capable of internalizing into RMS cells in an energy-independent mechanism. Helenalin-laden triple functionalized cubosomes showed limited impact on the viability of control fibroblast cells, while they induced a high degree cytotoxicity against RMS cells. Profound tumour cell death was observed in both two-dimensional (2D) culture and three-dimensional (3D) tumour spheroids.	Oct 2022
	IceBreaker: Software for high-resolution single-particle cryo-EM with non-uniform ice Mateusz Olek , Kevin Cowtan , Donovan Webb , Yuriy Chaban , Peijun Zhang Structure 43 DOI: 10.1016/j.str.2022.01.005 Open Access Show Abstract ▼ Abstract: Despite the abundance of available software tools, optimal particle selection is still a vital issue in single-particle cryoelectron microscopy (cryo-EM). Regardless of the method used, most pickers struggle when ice thickness varies on a micrograph. IceBreaker allows users to estimate the relative ice gradient and flatten it by equalizing the local contrast. It allows the differentiation of particles from the background and improves overall particle picking performance. Furthermore, we introduce an additional parameter corresponding to local ice thickness for each particle. Particles with a defined ice thickness can be grouped and filtered based on this parameter during processing. These functionalities are especially valuable for on-the-fly processing to automatically pick as many particles as possible from each micrograph and to select optimal regions for data collection. Finally, estimated ice gradient distributions can be stored separately and used to inspect the quality of prepared samples.	Feb 2022
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	Structure of native HIV-1 cores and their interactions with IP6 and CypA Tao Ni , Yanan Zhu , Zhengyi Yang , Chaoyi Xu , Yuriy Chaban , Tanya Nesterova , Jiying Ning , Till Böcking , Michael W. Parker , Christina Monnie , Jinwoo Ahn , Juan R. Perilla , Peijun Zhang Science Advances 7 DOI: 10.1126/sciadv.abj5715 Diamond Proposal Number(s): [21004, 20223] Open Access Show Abstract ▼ Abstract: The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo–electron microscopy image quality. Using cryo–electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.	Nov 2021
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond Krios IV-Titan Krios IV at Diamond	A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair Kaiying Cheng , Martin Wilkinson , Yuriy Chaban , Dale B. Wigley Nature Structural & Molecular Biology 27, 71 - 77 DOI: 10.1038/s41594-019-0355-2 Diamond Proposal Number(s): [19865] Open Access Show Abstract ▼ Abstract: The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.	Jan 2020
Krios II-Titan Krios II at Diamond Krios III-Titan Krios III at Diamond Krios IV-Titan Krios IV at Diamond	Structural insights into the activation of ATM kinase Jianxiong Xiao , Mengjie Liu , Yilun Qi , Yuriy Chaban , Chao Gao , Beiqing Pan , Yuan Tian , Zishuo Yu , Jie Li , Peijun Zhang , Yanhui Xu Cell Research 29, 683 - 685 DOI: 10.1038/s41422-019-0205-0 Diamond Proposal Number(s): [21004] Show Abstract ▼ Abstract: Requested microscope - using Krios I for now.	Jul 2019
Krios I-Titan Krios I at Diamond Krios II-Titan Krios II at Diamond	Structure and dynamics of the yeast SWR1-nucleosome complex Oliver Willhoft , Mohamed Ghoneim , Chia-Liang Lin , Eugene Y. D. Chua , Martin Wilkinson , Yuriy Chaban , Rafael Ayala , Elizabeth A. Mccormack , Lorraine Ocloo , David S. Rueda , Dale B. Wigley Science 362 DOI: 10.1126/science.aat7716 Diamond Proposal Number(s): [14769] Show Abstract ▼ Abstract: The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo–electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis.	Oct 2018
Krios I-Titan Krios I at Diamond	Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones Martin Wilkinson , Lucy Troman , Wan Ak Wan Nur Ismah , Yuriy Chaban , Matthew B. Avison , Mark S. Dillingham , Dale B. Wigley Elife 5 DOI: 10.7554/eLife.22963 Open Access Show Abstract ▼ Abstract: Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.	Dec 2016
Krios I-Titan Krios I at Diamond	Mechanism for nuclease regulation in RecBCD Martin Wilkinson , Yuriy Chaban , Dale B Wigley Elife 5 DOI: 10.7554/eLife.18227 Diamond Proposal Number(s): [13275] Open Access Show Abstract ▼ Abstract: In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.	Sep 2016

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