Publications by authors named "Alan M Roseman"

15 Publications

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Theoretical and practical approaches to improve the performance of local correlation algorithms for volume data analysis and shape recognition.

Acta Crystallogr D Struct Biol 2021 Apr 30;77(Pt 4):447-456. Epub 2021 Mar 30.

Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PT, United Kingdom.

In this paper, several approaches to be used to accelerate algorithms for fitting an atomic structure into a given 3D density map determined by cryo-EM are discussed. Rotation and translation of the atomic structure to find similarity scores are used and implemented with discrete Fourier transforms. Several rotations can be combined into groups to accelerate processing. The finite resolution of experimental and simulated maps allows a reduction in the number of rotations and translations needed in order to estimate similarity-score values.
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http://dx.doi.org/10.1107/S2059798321001212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025886PMC
April 2021

The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure.

Nat Commun 2020 06 26;11(1):3252. Epub 2020 Jun 26.

Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester, LE1 7RH, UK.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function.
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http://dx.doi.org/10.1038/s41467-020-17078-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319964PMC
June 2020

The structural basis of translational control by eIF2 phosphorylation.

Nat Commun 2019 05 13;10(1):2136. Epub 2019 May 13.

Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, M13 9PT, UK.

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.
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http://dx.doi.org/10.1038/s41467-019-10167-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513899PMC
May 2019

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Nat Commun 2019 03 29;10(1):1456. Epub 2019 Mar 29.

Division of Structural Biology, University of Oxford, Wellcome Centre for Human Genetics, Oxford, OX3 7BN, UK.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.
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http://dx.doi.org/10.1038/s41467-019-09451-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441041PMC
March 2019

Imaging Three-Dimensional Elemental Inhomogeneity in Pt-Ni Nanoparticles Using Spectroscopic Single Particle Reconstruction.

Nano Lett 2019 02 25;19(2):732-738. Epub 2019 Jan 25.

School of Materials , University of Manchester , Oxford Road , Manchester M13 9PL , United Kingdom.

The properties of nanoparticles are known to critically depend on their local chemistry but characterizing three-dimensional (3D) elemental segregation at the nanometer scale is highly challenging. Scanning transmission electron microscope (STEM) tomographic imaging is one of the few techniques able to measure local chemistry for inorganic nanoparticles but conventional methodologies often fail due to the high electron dose imparted. Here, we demonstrate realization of a new spectroscopic single particle reconstruction approach built on a method developed by structural biologists. We apply this technique to the imaging of PtNi nanocatalysts and find new evidence of a complex inhomogeneous alloying with a Pt-rich core, a Ni-rich hollow octahedral intermediate shell and a Pt-rich rhombic dodecahedral skeleton framework with less Pt at ⟨100⟩ vertices. The ability to gain evidence of local surface enrichment that varies with the crystallographic orientation of facets and vertices is expected to provide significant insight toward the development of nanoparticles for sensing, medical imaging, and catalysis.
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http://dx.doi.org/10.1021/acs.nanolett.8b03768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378652PMC
February 2019

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils.

J Mol Biol 2018 10 16;430(21):4142-4155. Epub 2018 Aug 16.

Wellcome Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PT, UK; Division of Cell-Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PT, UK. Electronic address:

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.
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http://dx.doi.org/10.1016/j.jmb.2018.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193142PMC
October 2018

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

Acta Biomater 2017 04 10;52:21-32. Epub 2016 Dec 10.

Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, M13 9PT, UK. Electronic address:

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering.

Statement Of Significance: Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.
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http://dx.doi.org/10.1016/j.actbio.2016.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402720PMC
April 2017

Structural analysis of X-linked retinoschisis mutations reveals distinct classes which differentially effect retinoschisin function.

Hum Mol Genet 2016 12;25(24):5311-5320

Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for the construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ∼4.2Å resolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.
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http://dx.doi.org/10.1093/hmg/ddw345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418834PMC
December 2016

Collagen VI, conformation of A-domain arrays and microfibril architecture.

J Biol Chem 2011 Nov 9;286(46):40266-75. Epub 2011 Sep 9.

Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, United Kingdom.

Collagen VI is a ubiquitous extracellular matrix protein that assembles into beaded microfibrils that form networks linking cells to the matrix. Collagen VI microfibrils are typically formed from a heterotrimer of the α1, α2, and α3 chains. The α3 chain is distinct as it contains an extended N terminus with up to 10 consecutive von Willebrand factor type A-domains (VWA). Here, we use solution small angle x-ray scattering (SAXS) and single particle analysis EM to determine the nanostructure of nine of these contiguous A-domains. Both techniques reveal a tight C-shape conformation for the A-domains. Furthermore, using biophysical approaches, we demonstrate that the N-terminal region undergoes a conformational change and a proportion forms dimers in the presence of Zn(2+). This is the first indication that divalent cations interact with collagen VI A-domains. A three-dimensional reconstruction of tissue-purified collagen VI microfibrils was generated using EM and single particle image analysis. The reconstruction showed the intricate architecture of the collagen VI globular regions, in particular the highly structurally conserved C-terminal region and variations in the appearance of the N-terminal region. The N-terminal domains project out from the globular beaded region like angled radial spokes. These could potentially provide interactive surfaces for other cell matrix molecules.
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http://dx.doi.org/10.1074/jbc.M111.265595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220584PMC
November 2011

Structure of hepatitis B surface antigen from subviral tubes determined by electron cryomicroscopy.

J Mol Biol 2009 Jul 3;390(1):135-41. Epub 2009 May 3.

Medical Research Council, Cambridge, UK.

Hepatitis B virus consists of an icosahedral core containing the double-stranded DNA genome, enveloped by a membrane with embedded surface proteins. The crystal structure of the core protein has been solved but little information about the structure of the surface proteins has so far been available. There are three sizes of surface protein, small (S), medium (M) and large (L), which form disulfide-bonded homo- and heterodimers. The three proteins, expressed from different start sites in the coding sequence, share the common C-terminal S region; the M protein contains an additional preS2 sequence N-terminal to S, and the L protein a further preS1 sequence N-terminal to M. In infected individuals, the surface proteins are produced in huge excess over the amount needed for viral envelopment and are secreted as a heterogeneous mixture of isometric and tubular subviral particles. We have used electron cryomicroscopy to study tubular particles extracted from human serum. Helical Fourier-Bessel analysis was used to calculate a low-resolution map, although it showed that the tubes were quite disordered. From the symmetry derived from this analysis, we used single-particle methods to improve the resolution. We found that the tubes had a diameter of approximately 250 A, with spike-like features projecting from the membrane. In the plane of the membrane the proteins appear to be close packed. We propose a model for the packing arrangement of surface protein dimers in the tubes.
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http://dx.doi.org/10.1016/j.jmb.2009.04.059DOI Listing
July 2009

A structural model for maturation of the hepatitis B virus core.

Proc Natl Acad Sci U S A 2005 Nov 24;102(44):15821-6. Epub 2005 Oct 24.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

Hepatitis B virus, a widespread and serious human pathogen, replicates by reverse transcription of an RNA intermediate. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. Using electron cryomicroscopy, we compare the structures of the bacterially expressed RNA-containing core particle and the mature DNA-containing core particle extracted from virions. We show that the mature core contains 240 subunits in a T = 4 arrangement similar to that in expressed core (T is the triangulation number and the icosahedral shell contains 60 T subunits). During the infective cycle, the core assembles in an immature state around a complex of viral pregenomic RNA and polymerase. After reverse transcription with concomitant degradation of the RNA, the now mature core buds through a cellular membrane containing the surface proteins to become enveloped. Envelopment must not happen before reverse transcription is completed, so it has been hypothesized that a change in capsid structure may signal maturation. Our results show significant differences in structure between the RNA- and DNA-containing cores. One such difference is in a hydrophobic pocket, formed largely from residues that, on mutation, lead to abnormal secretion. We suggest that the changes we see are related to maturation and control of envelopment, and we propose a mechanism based on DNA synthesis for their triggering.
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http://dx.doi.org/10.1073/pnas.0504874102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276056PMC
November 2005

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.

Nat Struct Mol Biol 2004 Nov 10;11(11):1128-33. Epub 2004 Oct 10.

Electron Microscope Unit and Department of Chemistry, University of Cape Town, Rondebosch, South Africa.

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.
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http://dx.doi.org/10.1038/nsmb844DOI Listing
November 2004

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice.

Virology 2004 May;323(1):108-19

Institute of Virology, Charité School of Medicine, D-10098 Berlin, Germany.

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.
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http://dx.doi.org/10.1016/j.virol.2004.02.022DOI Listing
May 2004

Automatic particle selection: results of a comparative study.

J Struct Biol 2004 Jan-Feb;145(1-2):3-14

Center for Integrative Molecular Biosciences and Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.
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http://dx.doi.org/10.1016/j.jsb.2003.09.033DOI Listing
October 2004

Particle finding in electron micrographs using a fast local correlation algorithm.

Authors:
Alan M Roseman

Ultramicroscopy 2003 Apr;94(3-4):225-36

MRC-Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.

A versatile tool for selecting particles from electron micrographs, intended for single particle analysis and three-dimensional reconstruction, is presented. It is based on a local real-space correlation method. Real-space correlations calculated over a local area are suitable for finding small objects or patterns in a larger field. They provide a very sensitive measure-of-fit, partly due to local optimisation of the numerical scaling. It is equivalent to least squares with optimised scaling between the two objects being correlated. The only disadvantage of real-space methods is that they are slow to compute. A fast local correlation algorithm based on Fourier transforms has been developed, which is approximately two orders of magnitude faster than the explicit real-space formulation. The algorithm is demonstrated by application to the problem of locating images of macromolecules in transmission electron micrographs of unstained frozen hydrated specimens. This is a challenging computational problem because these images have low contrast and a low signal-to-noise ratio. Picking particles by hand is very time consuming and can be less accurate. The automated procedure gives a significant increase in speed, which is important if large numbers of particles have to be picked.
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http://dx.doi.org/10.1016/s0304-3991(02)00333-9DOI Listing
April 2003