Faull, S.V., Lau, A.M., Martens, C., Ahdash, Z., Hansen, K., Yebenes, H., Schmidt, C., Beuron, F., Cronin, N.B., Morris, E.P., et al.
(2019). Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome. Nature communications,
Burgoyne, T., Heumann, J.M., Morris, E.P., Knupp, C., Liu, J., Reedy, M.K., Taylor, K.A., Wang, K. & Luther, P.K.
(2019). Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle. Proceedings of the national academy of sciences,
Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres..
Morris, E.P. & da Fonseca, P.C.
(2017). High-resolution cryo-EM proteasome structures in drug development. Acta crystallographica section d structural biology,
With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein–ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein–ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of thePlasmodium falciparum20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human andP. falciparum20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of theicr3dsoftware used for three-dimensional reconstruction..
Paul, D.M., Squire, J.M. & Morris, E.P.
(2017). Relaxed and active thin filament structures; a new structural basis for the regulatory mechanism. Journal of structural biology,
Squire, J.M., Paul, D.M. & Morris, E.P.
(2017). Myosin and Actin Filaments in Muscle: Structures and Interactions. ,
Bachmann, G., Richards, M.W., Winter, A., Beuron, F., Morris, E. & Bayliss, R.
(2016). A closed conformation of the
separase–securin complex. Open biology,
Mariotti, L., Templeton, C.M., Ranes, M., Paracuellos, P., Cronin, N., Beuron, F., Morris, E. & Guettler, S.
(2016). Tankyrase Requires SAM Domain-Dependent Polymerization to Support Wnt-β-Catenin Signaling. Molecular cell,
The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-β-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active β-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive β-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling..
Taylor, A.I., Beuron, F., Peak-Chew, S.-., Morris, E.P., Herdewijn, P. & Holliger, P.
(2016). Nanostructures from Synthetic Genetic Polymers. Chembiochem,
Burgoyne, T., Morris, E.P. & Luther, P.K.
(2015). Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle. Journal of molecular biology,
da Fonseca, P.C. & Morris, E.P.
(2015). Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core. Nature communications,
Coster, G., Frigola, J., Beuron, F., Morris, E.P. & Diffley, J.F.
(2014). Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol cell,
Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. .
On, K.F., Beuron, F., Frith, D., Snijders, A.P., Morris, E.P. & Diffley, J.F.
(2014). Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication. Embo j,
Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. .
Goodman, K.M., Kjær, S., Beuron, F., Knowles, P.P., Nawrotek, A., Burns, E.M., Purkiss, A.G., George, R., Santoro, M., Morris, E.P., et al.
(2014). RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association. Cell rep,
The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation..
Al-Khayat, H.A., Kensler, R.W., Squire, J.M., Marston, S.B. & Morris, E.P.
(2013). Atomic model of the human cardiac muscle myosin filament. Proc natl acad sci u s a,
Of all the myosin filaments in muscle, the most important in terms of human health, and so far the least studied, are those in the human heart. Here we report a 3D single-particle analysis of electron micrograph images of negatively stained myosin filaments isolated from human cardiac muscle in the normal (undiseased) relaxed state. The resulting 28-Å resolution 3D reconstruction shows axial and azimuthal (no radial) myosin head perturbations within the 429-Å axial repeat, with rotations between successive 132 Å-, 148 Å-, and 149 Å-spaced crowns of heads close to 60°, 35°, and 25° (all would be 40° in an unperturbed three-stranded helix). We have defined the myosin head atomic arrangements within the three crown levels and have modeled the organization of myosin subfragment 2 and the possible locations of the 39 Å-spaced domains of titin and the cardiac isoform of myosin-binding protein-C on the surface of the myosin filament backbone. Best fits were obtained with head conformations on all crowns close to the structure of the two-headed myosin molecule of vertebrate chicken smooth muscle in the dephosphorylated relaxed state. Individual crowns show differences in head-pair tilts and subfragment 2 orientations, which, together with the observed perturbations, result in different intercrown head interactions, including one not reported before. Analysis of the interactions between the myosin heads, the cardiac isoform of myosin-binding protein-C, and titin will aid in understanding of the structural effects of mutations in these proteins known to be associated with human cardiomyopathies..
Zhang, Z., Yang, J., Kong, E.H., Chao, W.C., Morris, E.P., da Fonseca, P.C. & Barford, D.
(2013). Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C). Biochem j,
Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C..
Lamber, E.P., Beuron, F., Morris, E.P., Svergun, D.I. & Mittnacht, S.
(2013). Structural insights into the mechanism of phosphoregulation of the retinoblastoma protein. Plos one,
The retinoblastoma susceptibility protein RB1 is a key regulator of cell proliferation and fate. RB1 operates through nucleating the formation of multi-component protein complexes involved in the regulation of gene transcription, chromatin structure and protein stability. Phosphorylation of RB1 by cyclin-dependent kinases leads to conformational alterations and inactivates the capability of RB1 to bind partner protein. Using small angle X-ray scattering in combination with single particle analysis of transmission electron microscope images of negative-stained material we present the first three-dimensional reconstruction of non-phosphorylated RB1 revealing an extended architecture and deduce the domain arrangement within the molecule. Phosphorylation results in an overt alteration of the molecular shape and dimensions, consistent with the transition to a compact globular architecture. The work presented provides what is to our knowledge the first description of the relative domain arrangement in active RB1 and predicts the molecular movement that leads to RB1 inactivation following protein phosphorylation..
Pinotsis, N., Chatziefthimiou, S.D., Berkemeier, F., Beuron, F., Mavridis, I.M., Konarev, P.V., Svergun, D.I., Morris, E., Rief, M. & Wilmanns, M., et al.
(2012). Superhelical Architecture of the Myosin Filament-Linking Protein Myomesin with Unusual Elastic Properties. Plos biology,
Spagnolo, L., Barbeau, J., Curtin, N.J., Morris, E.P. & Pearl, L.H.
(2012). Visualization of a DNA-PK/PARP1 complex. Nucleic acids res,
The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions..
He, J., Kulkarni, K., da Fonseca, P.C., Krutauz, D., Glickman, M.H., Barford, D. & Morris, E.P.
(2012). The structure of the 26S proteasome subunit Rpn2 reveals its PC repeat domain as a closed toroid of two concentric α-helical rings. Structure,
The 26S proteasome proteolyses ubiquitylated proteins and is assembled from a 20S proteolytic core and two 19S regulatory particles (19S-RP). The 19S-RP scaffolding subunits Rpn1 and Rpn2 function to engage ubiquitin receptors. Rpn1 and Rpn2 are characterized by eleven tandem copies of a 35-40 amino acid repeat motif termed the proteasome/cyclosome (PC) repeat. Here, we reveal that the eleven PC repeats of Rpn2 form a closed toroidal structure incorporating two concentric rings of α helices encircling two axial α helices. A rod-like N-terminal domain consisting of 17 stacked α helices and a globular C-terminal domain emerge from one face of the toroid. Rpn13, an ubiquitin receptor, binds to the C-terminal 20 residues of Rpn2. Rpn1 adopts a similar conformation to Rpn2 but differs in the orientation of its rod-like N-terminal domain. These findings have implications for understanding how 19S-RPs recognize, unfold, and deliver ubiquitylated substrates to the 20S core..
da Fonseca, P.C., He, J. & Morris, E.P.
(2012). Molecular model of the human 26S proteasome. Mol cell,
The 26S proteasome plays a fundamental role in eukaryotic homeostasis by undertaking the highly controlled degradation of a wide range of proteins, including key cellular regulators such as those controlling cell-cycle progression and apoptosis. Here we report the structure of the human 26S proteasome determined by cryo-electron microscopy and single-particle analysis, with secondary structure elements identified both in the 20S proteolytic core region and in the 19S regulatory particle. We have used this information together with crystal structures, homology models, and other biochemical information to construct a molecular model of the complete 26S proteasome. This model allows for a detailed description of the 20S core within the 26S proteasome and redefines the overall assignment of subunits within the 19S regulatory particle. The information presented here provides a strong basis for a mechanistic understanding of the 26S proteasome..
Bhanumathy, C., da Fonseca, P.C., Morris, E.P. & Joseph, S.K.
(2012). Identification of functionally critical residues in the channel domain of inositol trisphosphate receptors. J biol chem,
We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP(3)R) channel function. The residues selected were highly conserved in all three IP(3)R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pulldown assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP(3)R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating..
Enchev, R.I., Scott, D.C., da Fonseca, P.C., Schreiber, A., Monda, J.K., Schulman, B.A., Peter, M. & Morris, E.P.
(2012). Structural basis for a reciprocal regulation between SCF and CSN. Cell rep,
Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCF(Skp2/Cks1) and SCF(Fbw7) and found that CSN occludes both SCF functional sites-the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination..
Al-Khayat, H.A., Kensler, R.W., Squire, J.M., Marston, S.B. & Morris, E.P.
(2012). 3-Dimensional Structure of Human Cardiac Muscle Myosin Filaments by Electron Microscopy and Single Particle Analysis. Biophysical journal,
Morris, E.P., Rivera-Calzada, A., da Fonseca, P.C., Llorca, O., Pearl, L.H. & Spagnolo, L.
(2011). Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies. Nucleic acids res,
The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break..
da Fonseca, P.C., Kong, E.H., Zhang, Z., Schreiber, A., Williams, M.A., Morris, E.P. & Barford, D.
(2011). Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature,
The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ∼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C..
Schreiber, A., Stengel, F., Zhang, Z., Enchev, R.I., Kong, E.H., Morris, E.P., Robinson, C.V., da Fonseca, P.C. & Barford, D.
(2011). Structural basis for the subunit assembly of the anaphase-promoting complex. Nature,
The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates..
Paul, D.M., Squire, J.M. & Morris, E.P.
(2010). A novel approach to the structural analysis of partially decorated actin based filaments. J struct biol,
We describe a novel set of single particle based procedures for the structural analysis of electron microscope images of muscle thin filaments and other partially decorated actin based filaments. The thin filament comprises actin and the regulatory proteins tropomyosin and troponin in a 7:1:1M ratio. Prior to our work, structure analysis from electron microscope images of the thin filament has largely involved either helical averaging defined by the underlying actin helix or the use of single particle analysis but using a starting model as a reference structure. Our single particle based approach yields an accurate structure for the complete thin filament by avoiding the loss of information from troponin and tropomyosin associated with helical averaging and also removing the potential reference bias associated with the use of a starting model. The approach is more widely applicable to sub-stoichiometric complexes of F-actin and actin-binding proteins..
Anyatonwu, G., Khan, M.T., Schug, Z.T., da Fonseca, P.C., Morris, E.P. & Joseph, S.K.
(2010). Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors. J biol chem,
We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca2+-induced conformational changes of IP(3)Rs in their native membrane environment. We found that Ca2+ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca2+ induces a conformational change in the IP3R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca2+ were observed at similar to 0.5 mu M and was sensitive to inhibition by IP3. Ca2+ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1-225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca2+. However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca2+. We conclude the following: a) that large conformational changes induced by Ca2+ can be detected in IP(3)Rs in situ; b) these changes may be driven by Ca2+ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP3R may be relatively inaccessible to regulatory proteins unless Ca2+ is present..
Al-Khayat, H.A., Kensler, R.W., Morris, E.P. & Squire, J.M.
(2010). Three-Dimensional Structure of the M-region (Bare Zone) of Vertebrate Striated Muscle Myosin Filaments by Single-Particle Analysis. J mol biol,
The rods of anti-parallel myosin molecules overlap at the centre of bipolar myosin filaments to produce an M-region (bare zone) that is free of myosin heads Beyond the M-region edges, myosin molecules aggregate in a parallel fashion to yield the bridge regions of the myosin filaments Adjacent myosin filaments in striated muscle A-bands are cross-linked by the M-band Vertebrate striated muscle myosin filaments have a 3-fold rotational symmetry around their long axes In addition, at the centre of the M-region, there are three 2-fold axes perpendicular to the filament long axis, giving the whole filament dihedral 32-point group symmetry Here we describe the three-dimensional structure obtained by a single-particle analysis of the M-region of myosin filaments from goldfish skeletal muscle under relaxing conditions and as viewed in negative stain This is the first single-particle reconstruction of isolated M-regions The resulting three-dimensional reconstruction reveals details to about 55 angstrom resolution of the density distribution in the five main nonmyosin densities in the M-band (M6', M4', M1, M4 and M6) and in the myosin head crowns (P1, P2 and P3) at the M-region edges The outermost crowns in the reconstruction were identified specifically by their close similarity to the corresponding crown levels in our previously published bridge region reconstructions The packing of myosin molecules into the M-region structure is discussed, and some unidentified densities are highlighted (C) 2010 Elsevier Ltd All rights reserved.
Enchev, R.I., Schreiber, A., Beuron, F. & Morris, E.P.
(2010). Structural insights into the COP9 signalosome and its common architecture with the 26S proteasome lid and eIF3. Structure,
The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell expression and two-step purification of human CSN and demonstrate its functional assembly. We further obtain a three-dimensional structure of both native and recombinant CSN using electron microscopy and single particle analysis. Antibody labeling of CSN5 and segmentation of the structure suggest a likely subunit distribution and the architecture of its helical repeat subunits is revealed. We compare the structure of CSN with its homologous complexes, the 26S proteasome lid and eIF3, and propose a conserved architecture implying similar assembly pathways and/or conserved substrate interaction modes..
Wolfram, F., Morris, E. & Taylor, C.W.
(2010). Three-dimensional structure of recombinant type 1 inositol 1,4,5-trisphosphate receptor. Biochem j,
IP3Rs (inositol 1,4,5-trisphosphate receptors) are the intracellular channels that mediate release of Ca2+ from the endoplasmic reticulum in response to the many stimuli that evoke Ins(1,4,5)P3 formation. We characterized and purified type 1 IP3R heterologously expressed in Sf9 insect cells, and used the purified IP3R1 to determine its three-dimensional structure by electron microscopy and single-particle analysis. Recombinant IP3R1 has 4-fold symmetry with overall dimensions of approx. 19.5 nm x 19.5 nm x 17.5 nm. It comprises a small domain, which is likely to include the pore, linked by slender bridges to a large cytoplasmic domain with four petal-like regions. Our structures of recombinant IP3R1 and native cerebellar IP3R have similar appearances and dimensions. The only notable difference is the absence of a central stigma-like domain from the cytoplasmic region of recombinant IP3R1. The first structure of a recombinant IP3R is an important step towards developing three-dimensional structures of IP3R that better contribute to our understanding of the structural basis of IP3R activation..
Hodgkinson, J.L., Horsley, A., Stabat, D., Simon, M., Johnson, S., da Fonseca, P.C., Morris, E.P., Wall, J.S., Lea, S.M. & Blocker, A.J., et al.
(2009). Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat struct mol biol,
Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-angstrom resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component..
Al-Khayat, H.A., Morris, E.P. & Squire, J.M.
(2009). The 7-stranded structure of relaxed scallop muscle myosin filaments: Support for a common head configuration in myosin-regulated muscles. J struct biol,
Isolated relaxed myosin filaments from the myosin-regulated scallop striated adductor muscle have been reconstructed using electron microscopy and single particle analysis of negatively stained filaments. Three-dimensional reconstruction using 7-fold rotational symmetry but without imposed helical symmetry confirmed that the myosin head array is a 7-stranded, right-handed long-pitch 24/1 helix (or left-handed short-pitch 10/1 helix) with the whole structure having an axial repeat of 1440 K Reconstruction using the full helical symmetry revealed details of the myosin head density distribution within the head crowns in the relaxed scallop myosin filament. The resulting density distribution can best be explained by an arrangement in which the two heads from the same myosin molecule interact together within each crown in a compact parallel fashion along the filament axis. The configuration is consistent with the published configuration of the two heads within vertebrate smooth muscle myosin molecules observed in two-dimensional crystals of smooth muscle myosin and in the structure of tarantula myosin filaments. All these three muscle types are myosin-regulated, providing further support for a general motif of intramolecular interacting-heads structure in the relaxed state of myosin-regulated muscles as was proposed earlier by Woodhead et al. [Woodhead, J.L, Zhao, F.-Q., Craig, R., Egelman, E.H., Alamo, L, Padron, R.. 2005. Atomic model of a myosin filament in the relaxed state. Nature 436, 1195-1199]. However, the orientation of the Wendt structure is different from that found by Woodhead in that the outer head projects outwards and the inner head lies closer to the filament backbone, as in earlier work done on the insect flight muscle myosin filaments [AL-Khayat, H.A., Hudson, L, Reedy, M.K., Irving, T.C., Squire, J.M., 2003. Myosin head configuration in relaxed insect flight muscle: X-ray modelled resting crossbridges in a pre-power-stroke state are poised for actin binding. Biophys. J. 85, 1063-1079]. Possible species specific details that may differ between the scallop and the tarantula myosin filaments are also discussed. (C) 2009 Elsevier Inc. All rights reserved..
Remus, D., Beuron, F., Tolun, G., Griffith, J.D., Morris, E.P. & Diffley, J.F.
(2009). Concerted Loading of Mcm2-7 Double Hexamers around DNA during DNA Replication Origin Licensing. Cell,
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1 center dot Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication..
Altschuler, G.M., Dekker, C., McCormack, E.A., Morris, E.P., Klug, D.R. & Willison, K.R.
(2009). A single amino acid residue is responsible for species-specific incompatibility between CCT and alpha-actin. Febs lett,
Actin is dependent on the type-II chaperonin CCT (chaperonin containing TCP-1) to reach its native state. In vitro, yeast CCT folds yeast and also mammalian cytoplasmic (beta/gamma) actins but is now found to be incapable of folding mammalian skeletal muscle alpha-actin. Arrest of alpha-actin on yeast CCT at a folding cycle intermediate has been observed by electron microscopy. This discovery explains previous observations in vivo that yeast mutants expressing only the muscle actin gene are non-viable. Mutational analysis identified a single specific alpha-actin residue, Asn-297, that confers this species/isoform folding specificity. The implications of this incompatibility for chaperonin mechanism and actin-CCT co-evolution are discussed..
Paul, D.M., Morris, E.P., Kensler, R.W. & Squire, J.M.
(2009). Structure and orientation of troponin in the thin filament. J biol chem,
The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-A axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model..
Schug, Z.T., da Fonseca, P.C., Bhanumathy, C.D., Wagner, L., Zhang, X.C., Bailey, B., Morris, E.P., Yule, D.I. & Joseph, S.K.
(2008). Molecular characterization of the inositol 1,4,5-trisphosphate receptor pore-forming segment. J biol chem,
Specific residues in the putative pore helix, selectivity filter, and S6 transmembrane helix of the inositol 1,4,5-trisphosphate receptor were mutated in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel (C2533A, G2541A, G2545A, G2546A, and G2547A). Of the remaining three mutants, C2527A and R2543A were partially active and G2549A behaved like wild type receptor. Mutation of a putative glycine hinge residue in the S6 helix (G2586A) or a putative gating residue at the cytosolic end of S6 helix (F2592A) had minimal effects on function, although channel function was inactivated by G2586P and F2592D mutations. The mutagenesis data are interpreted in the context of a structural homology model of the inositol 1,4,5-trisphosphate receptor..
Al-Khayat, H.A., Morris, E.P., Kensler, R.W. & Squire, J.M.
(2008). Myosin filament 3D structure in mammalian cardiac muscle. J struct biol,
A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2 x 430 angstrom long, each of which was treated as an independent 'particle'. The resulting 40 angstrom resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 angstrom repeat, with successive crown rotations of approximately 60 degrees, 60 degrees and 0 degrees, rather than the regular 40 degrees for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac). (c) 2008 Elsevier Inc. All rights reserved..
da Fonseca, P.C. & Morris, E.P.
(2008). Structure of the human 26S proteasome: subunit radial displacements open the gate into the proteolytic core. J biol chem,
The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits..
Al-Khayat, H.A., Morris, E.P., Kensler, R.W. & Squire, J.M.
(2006). 3D structure of relaxed fish muscle myosin filaments by single particle analysis. J struct biol,
To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) single particle analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head 'crowns' in the 429 A myosin filament repeat. The analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60 degrees , 60 degrees and 0 degrees , rather than the regular 40 degrees characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated..
Morosinotto, T., Bassi, R., Frigerio, S., Finazzi, G., Morris, E. & Barber, J.
(2006). Biochemical and structural analyses of a higher plant photosystem II supercomplex of a photosystem I-less mutant of barley - Consequences of a chronic over-reduction of the plastoquinone pool. Febs j,
Photosystem II of higher plants is a multisubunit transmembrane complex composed of a core moiety and an extensive peripheral antenna system. The number of antenna polypeptides per core complex is modulated following environmental conditions in order to optimize photosynthetic performance. In this study, we used a barley (Hordeum vulgare) mutant, viridis zb63, which lacks photosystem I, to mimic extreme and chronic overexcitation of photosystem II. The mutation was shown to reduce the photosystem II antenna to a minimal size of about 100 chlorophylls per photosystem II reaction centre, which was not further reducible. The minimal photosystem II unit was analysed by biochemical methods and by electron microscopy, and found to consist of a dimeric photosystem II reaction centre core surrounded by monomeric Lhcb4 (chlorophyll protein 29), Lhcb5 (chlorophyll protein 26) and trimeric light-harvesting complex II antenna proteins. This minimal photosystem II unit forms arrays in vivo, possibly to increase the efficiency of energy distribution and provide photoprotection. In wild-type plants, an additional antenna protein, chlorophyll protein 24 (Lhcb6), which is not expressed in viridis zb63, is proposed to associate to this minimal unit and stabilize larger antenna systems when needed. The analysis of the mutant also revealed the presence of two distinct signalling pathways activated by excess light absorbed by photosystem II: one, dependent on the redox state of the electron transport chain, is involved in the regulation of antenna size, and the second, more directly linked to the level of photoinhibitory stress perceived by the cell, participates in regulating carotenoid biosynthesis..
Taylor, C.W., da Fonseca, P.C. & Morris, E.P.
(2004). IP3 receptors: the search for structure. Trends biochem sci,
Inositol (1,4,5)-trisphosphate receptors (IP3P) are intracellular Ca2+ channels that are regulated by Ca2+ and IP3, and are modulated by many additional signals. They thereby allow both receptors that stimulate IP3 formation and Ca2+ to control release of Ca2+ from intracellular stores. IP(3)Rs share many features with their close relatives, ryanodine receptors; each provides insight into the structure and function of the other. The structural basis of IP3R behaviour is beginning to emerge from intermediate-resolution structures of the complete IPA a 2.2-Angstrom structure of the IP3-binding core and comparisons with the pore structures of other tetrameric cation channels. The binding of IP3 to a site towards the N-terminal of each IP3R subunit promotes binding of Ca2+. This destabilizes an inhibitory interaction between N-terminal residues and a C-terminal 'gatekeeper' sequence, enabling the pore to open..
Paul, D., Patwardhan, A., Squire, J.M. & Morris, E.P.
(2004). Single particle analysis of filamentous and highly elongated macromolecular assemblies. J struct biol,
The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of approximately 20 degrees was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of approximately 25 A. If no out-of-plane views are available, an alternative approach is to use a prior 3D model as a reference for the initial angle assignment. Tests with the thin filament model indicated that reasonably accurate angular assignment can be made using a reference containing actin, but lacking the regulatory proteins tropomyosin and troponin. We also found that an adaptation of the exact filtered back projection method is required to allow the correct weighting of projection images in which the particle has a very large axial ratio. This adaptation resulted in significant improvements in the reconstruction..
Patwardhan, A., Paul, D., Al-Khayat, H.A. & Morris, E.P.
(2004). A measure for the angle between projections based on the extent of correlation between corresponding central sections. J mol biol,
A pre-condition for the ab initio assignment of Euler angles to a set of projections from an asymmetric object is that at least three of the available projections correspond to rotations about different axes. For symmetric objects this condition may be relaxed. There are some applications of single-particle electron microscopy, such as the reconstruction of filamentous macromolecular assemblies, where all available projections more-or-less correspond to rotations about a common rotation axis making it difficult to satisfy this condition. Here, a method has been developed to overcome this problem, based on the fact that the correlation between two central sections of the Fourier transform of a compact object will not be limited to an infinitesimal central line but will have a finite extent, which is related to the angle between the corresponding projections. Projections from model filaments, with different degrees of rotational symmetry about the long axis, have been used to test the methodology. The results show that angle determination is robust down to signal-to-noise ratios as low as 2 and that, in general, the error decreases as the degree of symmetry increases. The method has been used to assign angles to a set of negatively stained muscle thick filament projections to obtain an initial 3D reconstruction. The main features of the projections are seen to be faithfully reproduced in the reprojections from the reconstruction. A real-space adaptation of this method is also discussed..
Al-Khayat, H.A., Morris, E.P. & Squire, J.M.
(2004). Single particle analysis: a new approach to solving the 3D structure of myosin filaments. J muscle res cell motil,
Knowledge of the structure of muscle myosin filaments is essential for a proper understanding of sarcomere structure and how myosin heads interact with the actin filaments to produce force and movement. Two principal methods have been used to define the myosin head arrays in filaments in the relaxed state, namely modelling from low-angle X-ray diffraction data and image processing of electron micrographs of isolated filaments. Analysis of filament images by 3D helical reconstruction, which imposes total helical symmetry on the structure, is very effective in some cases, but it relies on the existence of very highly ordered preparations of straight filaments. Resolutions achieved to date are about 70 angstroms. Modelling of X-ray diffraction data recorded from whole relaxed fish or insect muscles has also been used as an independent method. Although the resolution of the diffraction data is often also about 70 angstroms, the effective resolution of the modelling is very much higher than this because additional very high resolution data (e.g. from protein crystallography) is included in the analysis. However, the X-ray diffraction method has to date provided only limited data on non-myosin thick filament proteins such as C-protein and titin and it cannot provide the polarity of the myosin head arrangement. Both the helical reconstruction and X-ray diffraction techniques have advantages and disadvantages, but their disadvantages are avoided in the new approach of single particle analysis of electron micrograph data. Even using the same micrographs as for helical reconstruction, the resolution can be extended by this method to about 50 angstroms or better. In addition, it is not necessary to assume that the myosin filaments are helical; a significant advantage in the case of vertebrate myosin filaments where there is a known crossbridge perturbation. Here we describe the principles of all these approaches, but particularly that of single particle analysis. We outline the application of single particle analysis to myosin filaments from vertebrate skeletal and insect flight (IFM) muscle myosin filaments..
da Fonseca, P.C., Morris, S.A., Nerou, E.P., Taylor, C.W. & Morris, E.P.
(2003). Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis. Proc natl acad sci u s a,
The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a tetrameric intracellular Ca(2+) channel, which mediates the release of Ca(2+) from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP(3)R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP(3)R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP(3)R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K(+) channel..
Luther, P.K. & Morris, E.P.
(2003). Cryoelectron microscopy of refrozen cryosections. J struct biol,
Cryoelectron microscopy makes it possible to record high-resolution detail from large and complex structures. However, its application to understanding cellular structure is limited by the requirement that samples should be no thicker than approximately 0.5-1 microm. Therefore it is important to develop the ability to section biological material so that it can be imaged in its native frozen state. Here we have adapted standard methods of preparing cryosections so that they can be imaged by cryoelectron microscopy. As used for immunolabeling, cryosections of chemically fixed, cryoprotected frozen rat cardiac muscle were thawed, applied to carbon-coated grids, and rinsed on a drop of buffer. The special step here is that the cryosections were then refrozen by being plunged into liquid ethane and imaged at approximately -180 degrees C in a 200-kV field-emission gun electron microscope. The unstained cryosections have good contrast, allowing the identification of optimum regions of the sample. Considerable fine detail is observed within the substructure of the sarcomere A-band and I-band. Fourier transform analysis of the micrographs shows that this method preserves high structural order, hence these sections are well-suited to 3D reconstruction. We conclude that this approach has considerable potential for obtaining intermediate- and high-resolution structural detail from bulk tissue..
Barber, J., Morris, E.P. & da Fonseca, P.C.
(2003). Interaction of the allophycocyanin core complex with photosystem II. Photochem photobiol sci,
Allophycocyanin core complexes were purified from the thermophilic cyanobacterium Thermosynechococcus elongatus and analysed by negative-stain electron microscopy and single-particle averaging. The purified complex was found to consist of three allophycocyanin cylinders. The single-particle analysis of end-on views of the complex revealed a mirror axis, indicative of two-fold symmetry. This observation allowed the assignment of the allophycocyanin base cylinders and the identification of their potential interaction sites with the thylakoid membrane and with the photosystem II reaction centre in particular. The T. elongatus allophycocyanin core projection map, together with published information on the structure of photosystem II for the same organism, was used to construct a model for the allophycocyanin core-photosystem II dimer supercomplex, from which docking sites between both complexes are suggested. The implications of this association for energy transfer from allophycocyanin to photosystem II are discussed..
Daniell, S.J., Kocsis, E., Morris, E., Knutton, S., Booy, F.P. & Frankel, G.
(2003). 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol microbiol,
The type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gram-negative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needle-like structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 A resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 A enclosing a central channel of 25 A diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one-start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 A per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS..
Nield, J., Morris, E.P., Bibby, T.S. & Barber, J.
(2003). Structural analysis of the photosystem I supercomplex of cyanobacteria induced by iron deficiency. Biochemistry,
Here we describe the three-dimensional structure of the newly discovered CP43'-photosystem I (PSI) supercomplex of cyanobacteria calculated by single-particle analysis of images obtained by electron cryomicroscopy (cryo-EM). This large membrane protein complex has a molecular mass of approximately 2 MDa and is found in cyanobacteria when grown in iron deficient media. It is composed of a reaction center trimer surrounded by 18 subunits of the chlorophyll a binding CP43'protein, encoded by the isiA gene, which increases the light harvesting capacity of PSI by approximately 70%. By modeling higher-resolution structural data obtained from X-ray crystallography into the three-dimensional (3D) cryo-EM map, we have been able to gain a better understanding of the structure and functional properties of this supermolecular complex. We have identified three separate clusters of chlorophyll molecules at the periphery of the PSI core which may aid energy transfer from the CP43' antenna ring to the reaction center. Moreover, it is shown that despite the replacement of ferredoxin with flavodoxin as an electron acceptor under iron stress conditions, the 3D map has density to accommodate the extrinsic proteins, PsaC, PsaD, and PsaE. The presence of these three proteins was also confirmed by immunoblotting..
Eakins, F., AL-Khayat, H.A., Kensler, R.W., Morris, E.P. & Squire, J.M.
(2002). 3D Structure of fish muscle myosin filaments. J struct biol,
Myosin filaments isolated from goldfish (Carassius auratus) muscle under relaxing conditions and viewed in negative stain by electron microscopy have been subjected to 3D helical reconstruction to provide details of the myosin head arrangement in relaxed muscle. Previous X-ray diffraction studies of fish muscle (plaice) myosin filaments have suggested that the heads project a long way from the filament surface rather than lying down flat and that heads in a single myosin molecule tend to interact with each other rather than with heads from adjacent molecules. Evidence has also been presented that the head tilt is away from the M-band. Here we seek to confirm these conclusions using a totally independent method. By using 3D helical reconstruction of isolated myosin filaments the known perturbation of the head array in vertebrate muscles was inevitably averaged out. The 3D reconstruction was therefore compared with the X-ray model after it too had been helically averaged. The resulting images showed the same characteristic features: heads projecting out from the filament backbone to high radius and the motor domains at higher radius and further away from the M-band than the light-chain-binding neck domains (lever arms) of the heads..
Squire, J.M., Eakins, F., Kensler, R.W., Morris, E.P. & Al-Khayat, H.A.
(2002). 3D reconstruction of isolated myosin filaments in fish muscle. Biophys j,
da Fonseca, P., Morris, E.P., Hankamer, B. & Barber, J.
(2002). Electron crystallographic study of photosystem II of the cyanobacterium Synechococcus elongatus. Biochemistry,
The determination of the structure of PSII at high resolution is required in order to fully understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by electron cryo-microscopy, and their analysis revealed they belong to the two-sided plane group p22(1)2(1), with unit cell parameters a = 121 A, b = 333 A, and alpha = 90 degrees. From these crystals, a projection map was calculated to a resolution of approximately 16 A. The reliability of this projection map is confirmed by its close agreement with the recently presented three-dimensional model of the same complex obtained by X-ray crystallography. Comparison of the projection map of the Synechococcus elongatus PSII complex with data obtained by electron crystallography of the spinach PSII core dimer reveals a similar organization of the main transmembrane subunits. However, some differences in density distribution between the cyanobacterial and higher plant PSII complexes exist, especially in the outer region of the complex between CP43 and cytochrome b(559) and adjacent to the B-helix of the D1 protein. These differences are discussed in terms of the number and organization of some of the PSII low molecular weight subunits..
Hankamer, B., Morris, E., Nield, J., Gerle, C. & Barber, J.
(2001). Three-dimensional structure of the photosystem II core dimer of higher plants determined by electron microscopy. J struct biol,
Here we report the first three-dimensional structure of a higher plant photosystem II core dimer determined by electron crystallography at a resolution sufficient to assign the organization of its transmembrane helices. The locations of 34 transmembrane helices in each half of the dimer have been deduced, 22 of which are assigned to the major subunits D1 (5), D2 (5), CP47 (6), and CP43 (6). CP47 and CP43, located on opposite sides of the D1/D2 heterodimer, are structurally similar to each other, consisting of 3 pairs of transmembrane helices arranged in a ring. Both CP47 and CP43 have densities protruding from the lumenal surface, which are assigned to the loops joining helices 5 and 6 of each protein. The remaining 12 helices within each half of the dimer are attributed to low-molecular-weight proteins having single transmembrane helices. Comparison of the subunit organization of the higher plant photosystem II core dimer reported here with that of its thermophilic cyanobacterial counterpart recently determined by X-ray crystallography shows significant similarities, indicative of a common evolutionary origin. Some differences are, however, observed, and these may relate to variations between the two classes of organisms in antenna linkage or thermostability..
Büchel, C., Morris, E., Orlova, E. & Barber, J.
(2001). Localisation of the PsbH subunit in photosystem II: a new approach using labelling of His-tags with a Ni(2+)-NTA gold cluster and single particle analysis. J mol biol,
Photosystem II core dimers were isolated from the green alga Chlamydomonas reinhardtii by Ni(2+)-affinity chromatography exploiting a 6 x His tag located at the N terminus of the PsbH protein. This protein is predicted to have a single transmembrane helix. In order to identify the location of PsbH within the photosystem II complex, the His-tagged core dimers were labelled using a Ni(2+)-NTA gold cluster and subjected to electron microscopy and image analysis. This new method enabled us to identify the location of the labelled His tag by statistical analysis of electron micrographs of the gold-labelled photosystem II complex. Comparison of these data with electron and X-ray crystallographic analysis of photosystem II indicates that the N terminus of PsbH is close to the two transmembrane helices of cytochrome b(559). Our analysis suggests that this approach is a powerful method to locate specific proteins within multisubunit complexes like photosystem II when crystallographic analysis is of insufficient resolution to directly identify amino acid side-chains. Moreover, it can be combined with cross-linking studies, and here we demonstrate that PsbH is a near neighbour of PsbX, which is consistent with the latter subunit being located close to the alpha and beta-subunits of cytochrome b(559). However, cross-linking between PsbH and PsbW was not detected despite the fact that the latter cross-linked with the alpha-subunit of cytochrome b(559)..
Morris, E.P. & Török, K.
(2001). Oligomeric structure of alpha-calmodulin-dependent protein kinase II. J mol biol,
The subunit stoichiometry and symmetry of the neuronal alpha-calmodulin-dependent protein kinase II (alphaCaMKII) is investigated in this report to understand the structural basis of its regulation and mechanism at the molecular level. Two preparations are studied, alphaCaMKII obtained by overexpression in baculovirus-transfected insect cells and CaMKII isolated from rat forebrain. The structures, are studied by electron microscopy and image analysis. Single-particle analysis of individual molecular images reveals a molecule with a circular outline and pronounced 6-fold rotational symmetry of the central part. The central part has an outer radius of approximately 6 nm and is composed of six lobes grouped around a hollow centre. The outer ring extends to approximately 15 nm and consists of 12 apparent domains. These data are interpreted in terms of a three-dimensional model of the alphaCaMKII complex consisting of 12 subunits, each corresponding to a single alphaCaMKII polypeptide chain. The inner ring corresponding to approximately one-third of the molecular mass of the complex is made up of the C-terminal association domains. The 12 association domains are arranged in two concentric hexagonal rings at different axial levels and in rotational register. The outer ring corresponding to the remaining molecular mass of the complex is made up of the 12 N-terminal catalytic domains located at an axial level halfway between the two levels of the association domains. The 6-fold symmetry of stacked association domains may derive from subunit arrangements corresponding to either the C6 or the D6 point group symmetries. The symmetry and the resulting subunit arrangement define the pattern and extent of regulatory autophosphorylation within the alphaCaMKII complex..
Nield, J., Orlova, E.V., Morris, E.P., Gowen, B., van Heel, M. & Barber, J.
(2000). 3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis. Nat struct biol,
Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet..
Morris, E.P., Hankamer, B.D. & Barber, J.
(2000). Three-dimensional structure of photosystem II determined by electron crystallography. Biophys j,
Kruse, O., Hankamer, B., Konczak, C., Gerle, C., Morris, E., Radunz, A., Schmid, G.H. & Barber, J.
(2000). Phosphatidylglycerol is involved in the dimerization of photosystem II. J biol chem,
Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PshTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1 Delta 3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1 Delta 3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two dimensional crystals of the native dimer..
Büchel, C., Morris, E. & Barber, J.
(2000). Crystallisation of CP43, a chlorophyll binding protein of photosystem II: an electron microscopy analysis of molecular packing. J struct biol,
Microcrystals of the chlorophyll binding protein, CP43, isolated from spinach thylakoid membranes have been studied by electron microscopy both in negative stain and in vitreous ice. Image analyses of three characteristic views show that the crystals are built of five different layers perpendicular to the c-axis. Each layer consists of different orientations of the CP43 protein. The unit cell derived from the end-on view (looking down the c-axis) shows an angle of 120 degrees, suggesting a threefold rotational symmetry. Both negative staining and cryo data are consistent with a hexagonal crystal lattice. Interpretation of the arrangement of the CP43 protein within this crystal lattice can be made based on 8- and 9-A electron crystallographic structures previously published that provide a model for the organisation of the transmembrane helices of CP43. Overall the analysis presented is consistent with X-ray diffraction data obtained from larger CP43 crystals and forms a framework on which to base further structural studies of this chlorophyll binding protein..
Barber, J., Morris, E. & Büchel, C.
(2000). Revealing the structure of the photosystem II chlorophyll binding proteins, CP43 and CP47. Biochim biophys acta,
A review of the structural properties of the photosystem II chlorophyll binding proteins, CP47 and CP43, is given and a model of the transmembrane helical domains of CP47 has been constructed. The model is based on (i) the amino acid sequence of the spinach protein, (ii) an 8 A three-dimensional electron density map derived from electron crystallography and (iii) the structural homology which the membrane spanning region of CP47 shares with the six N-terminal transmembrane helices of the PsaA/PsaB proteins of photosystem I. Particular emphasis has been placed on the position of chlorophyll molecules assigned in the 8 A three-dimensional map of CP47 (K.-H. Rhee, E.P. Morris, J. Barber, W. Kühlbrandt, Nature 396 (1998) 283-286) relative to histidine residues located in the transmembrane regions of this protein which are likely to form axial ligands for chlorophyll binding. Of the 14 densities assigned to chlorophyll, the model predicted that five have their magnesium ions within 4 A of the imidazole nitrogens of histidine residues. For the remaining seven histidine residues the densities attributed to chlorophylls were within 4-8 A of the imidazole nitrogens and thus too far apart for direct ligation with the magnesium ion within the tetrapyrrole head group. Improved structural resolution and reconsiderations of the orientation of the porphyrin rings will allow further refinement of the model..
Barber, J., Nield, J., Morris, E.P. & Hankamer, B.
(1999). Subunit positioning in photosystem II revisited. Trends biochem sci,
Hankamer, B., Morris, E.P. & Barber, J.
(1999). Revealing the structure of the oxygen-evolving core dimer of photosystem II by cryoelectron crystallography. Nat struct biol,
Here we present cryoelectron crystallographic analysis of an isolated dimeric oxygen-evolving complex of photosystem II (at a resolution of approximately 0.9 nm), revealing that the D1-D2 reaction center (RC) proteins are centrally located between the chlorophyll-binding proteins, CP43 and CP47. This conclusion supports the hypothesis that photosystems I and II have similar structural features and share a common evolutionary origin. Additional density connecting the two halves of the dimer, which was not observed in a recently described CP47-RC complex that did not include CP43, may be attributed to the small subunits that are involved in regulating secondary electron transfer, such as PsbH. These subunits are possibly also required for stabilization of the dimeric photosystem II complex. This complex, containing at least 29 transmembrane helices in its asymmetric unit, represents one of the largest membrane protein complexes studied at this resolution..
Knupp, C., Luther, P.K., Morris, E.P., Knight, D.P. & Squire, J.M.
(1999). Partially systematic molecular packing in the hexagonal columnar phase of dogfish egg case collagen. J struct biol,
The collagen that forms the egg case of the dogfish Scyliorhinus canicula is stored in bulk in the female nidamental glands. Here the collagen molecules are thought to undergo a series of distinct pH-dependent liquid crystalline aggregation phase changes before assembling into the final arrangement encountered in the mature egg case. One liquid crystalline phase is hexagonal with the centres of two adjacent hexagons about 36 nm apart. We have collected tilt series of the hexagonal phase from plastic sections of the nidamental gland and have produced a three-dimensional reconstruction of the collagen arrangement of this phase. The reconstruction features axial columns of protein density lying regularly on the vertices of hexagonal cells of edge length 21 nm. Each column is connected to three nearest neighbours by irregular sheets of protein, but there appear to be preferred molecular directions at about 40 degrees to 50 degrees to the columns. The reconstruction has been interpreted in terms of known interactions of this collagen in other assemblies. (C) 1999 Academic Press..
Barber, J., Rhee, K.H., Morris, E., Hankamer, B., Nield, J., Boekema, E. & Kuhlbrandt, W.
(1998). Towards the elucidation of the structure of photosystem II at high resolution. Biophys j,
Squire, J.M. & Morris, E.P.
(1998). A new look at thin filament regulation in vertebrate skeletal muscle. Faseb j,
It is 30 years since Ebashi and colleagues showed that Ca2+ ions directly affect regulation of the myosin-actin interaction in muscle through the action of tropomyosin and troponin on muscle thin filaments. It is more than 20 years since the idea was put forward that tropomyosin might act, at least in part, by changing its position on actin, thus uncovering or modifying the myosin binding site on actin when troponin molecules take up Ca2+. Since that time, a great deal of evidence for and against this steric blocking mechanism has been published: a structure for actin filaments at close to atomic resolution has been proposed, and the whole regulation story has become both more complicated and more subtle. Here we review structural and biochemical aspects of regulation in vertebrate skeletal muscle. We show that some basic ideas of the steric blocking mechanism remain valid. We also show that additional factors, such as troponin movements and structural changes within the actin monomers themselves, may be crucial. A number of the resulting regulation scenarios need to be distinguished..
Harford, J., Cantino, M., Chew, M., Denny, R., Hudson, L., Luther, P., Mendelson, R., Morris, E. & Squire, J.
(1998). Myosin crossbridge configurations in equilibrium states of vertebrate skeletal muscle Heads swing axially or turn upside-down between resting and rigor. Adv exp med biol,
The positions and orientations of the myosin heads in relaxed, active, rigor and S1-labelled fish muscle are being determined by analysis both of electron micrographs and of low-angle X-ray diffraction patterns. The X-ray analysis of resting muscle makes use of the head shape defined from the study of S1 crystals, with variable head configurational parameters being used on each of the three different 3-fold symmetric 14.3 nm-spaced 'crowns' of myosin heads within the 42.9 nm axial repeat of the myosin filaments. Diffraction patterns were stripped using CCP13 fibre diffraction software. Searches and optimisation were carried out using simulated annealing and local refinement procedures to give a 'best fit' relaxed structure with a crystallographic R-factor of about 4%. It had heads oriented all the same way up (i.e. with similar rotations around their own long axes) on the myosin filament, but with a small range of axial tilts. Head configuration in rigor fish muscle is being determined by X-ray diffraction and electron microscopy of normal rigor muscle and of skinned muscle soaked with extrinsic myosin S1. Computed 3-D reconstructions of acto-S1 using X-ray amplitudes and phases from electron microscopy are informative and help to analyse the X-ray diffraction data that extend axially to about 1 nm resolution. An ambiguity is the axial direction of the observed resting myosin head array relative to the known polarity of the actin filaments. One polarity would give little axial displacement (2-3 nm) between opposite ends of the resting and rigor heads, and in this case the heads would need to rotate around their own long axes by about 115 degrees to make a rigor attachment. The other (preferred) filament polarity would provide considerable axial swinging (14-15 nm) between the two states. We are attempting to define the absolute polarity of the resting muscle myosin head array using electron microscopy and image processing either of cryo-sections or of replicas from shadowed, freeze-fractured, rapidly frozen fish muscle fibres..
Rhee, K.H., Morris, E.P., Zheleva, D., Hankamer, B., Kuhlbrandt, W. & Barber, J.
(1997). Two-dimensional structure of plant photosystem II at 8-angstrom resolution. Nature,
The photosystem II complex, which is the most abundant membrane protein in chloroplasts, comprises the light-harvesting complex II and a reaction-centre core. The reaction centre uses the solar energy collected by the light-harvesting complex II to withdraw electrons from water, releasing oxygen into the atmosphere, It thus generates an electrochemical potential, providing the energy for carbon dioxide fixation and the synthesis of organic molecules, which make up the hulk of the biosphere(1). The structure of the light-harvesting complex II has been determined at 3.4-Angstrom resolution by electron crystallography(2), but the high-resolution structure of the photosystem II reaction centre and other core components remained unknown. We have grown well-ordered two-dimensional crystals of a sub-core complex containing the reaction centre from spinach thylakoid membranes and used electron crystallography to obtain a projection map of its structure at 8-Angstrom resolution. The features reveal the likely location of the key components that are active in electron transport, and suggest a structural homology and evolutionary links, not only with the purple bacterial reaction centre but also with the reaction centre of photosystem I..
Mendelson, R. & Morris, E.P.
(1997). The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography. P natl acad sci usa,
Surmises of how myosin subfragment I (S1) interacts with actin filaments in muscle contraction rest upon knowing the relative arrangement of the two proteins, Although there exist crystallographic structures for both S1 and actin, as well as electron microscopy data for the acto-S1 complex (AS1), modeling of this arrangement has so far only been done ''by eye.'' Here we report fitted AS1 structures obtained using a quantitative method that is both more objective and makes more complete use of the data, Using undistorted crystallographic results, the best-fit AS1 structure shows significant differences from that obtained by visual fitting, The best fit is produced using the F-actin model of Holmes ef al, [Holmes, K, C,, Popp, D,, Gebhard, W. & Kabsch, W, (1990) Nature (London) 347, 44-49], S1 residues at the ASI interface are now found at a higher radius as well as being translated axially and rotated azimuthally. Fits using S1 plus loops missing from the crystal structure were achieved using a homology search method to predict loop structures, These improved fits favor an arrangement in which the Loop at the 50- to 20-kDa domain junction of S1 is located near the N terminus of actin, Rigid-body movements of the lower 50-kDa domain, which further improve the fit, produce closure of the large 50-kDa domain cleft and bring conserved residues in the lower 50-kDa domain into an apparently appropriate orientation for close interaction with actin, This finding supports the idea that binding of ATP to AS1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50-kDa cleft of S1..
Barber, J., Nield, J., Morris, E.P., Zheleva, D. & Hankamer, B.
(1997). The structure, function and dynamics of photosystem two. Physiol plantarum,
One of the greatest challenges in modern photosynthesis research is to elucidate fully the structural and functional properties of photosystem two (PSII). This water-plastoquinone oxidoreductase is located in a membrane complex composed of more than 25 subunits. The primary and secondary structures of all known subunits which constitute the central core of PSII are reviewed. How these subunits interact with each other to produce the tertiary and quaternary structure of PSII in vivo is not fully understood. However, electron microscopy is helping to fill this gap in our knowledge both by single particle analysis and electron crystallography. These studies suggest that active PSII is dimeric, although the functional significance of this oligomeric state is not yet understood. Moreover, the elucidation of the structure of photosystem one (PSI) by X-ray crystallography has revealed features which are likely to be relevant to PSII structure. It seems highly likely that the D1 protein with CP43 and D2 protein with CP47 (summing 11 transmembrane helices in each case) will have structural similarities to the organisation of PsaA and PsaB. It is likely that the turnover of the D1 protein is aided by the relatively easy removal of CP43 from this arrangement of the PSII core..
Morris, E.P., Hankamer, B., Zheleva, D., Friso, G. & Barber, J.
(1997). The three-dimensional structure of a photosystem II core complex determined by electron crystallography. Structure,
BACKGROUND: Photosystem II (PSII) is a multisubunit protein complex which is embedded in the photosynthetic membranes of plants. It uses light energy to split water into molecular oxygen and reducing equivalents. PSII can be isolated with varying degrees of complexity in terms of its subunit composition and activity. To date, no three-dimensional (3-D) structure of the PSII complex has been determined which allows location of the proteins within the PSII complex and their orientation in relation to the thylakoid membrane. RESULTS: Two-dimensional (2-D) PSII core complex crystals composed of the two reaction centre proteins, D1 and D2, two chlorophyll-binding proteins, CP47 and CP43, cytb559 and associated low molecular weight proteins were formed after reconstituting the isolated complex into purified thylakoid lipids. Electron micrographs of negatively stained crystals were used for 2-D and 3-D image analyses. In the resulting maps, the PSII complex is composed of two halves related by twofold rotational symmetry, thus, confirming the dimeric nature of the complex; each monomer appears to contain five domains. Comparison of the 3-D images with platinum shadowed images of the crystals allowed the likely lumenal and stromal surfaces of the complex to be identified and regions contained within the membrane to be inferred. The projection structure of 2-D crystals of a smaller CP47-D1-D2-cytb559 complex was used to identify the domains apparently associated with CP43. CONCLUSION: The results indicate that PSII probably exists as a dimer in vivo. The extensive proteinaceous protrusions from the lumenal surface have been tentatively assigned to hydrophilic loops of CP47 and CP43; the positioning of these loops possibly implies their involvement in the water-splitting process..
Torok, K. & Morris, E.P.
(1996). Calmodulin dependent protein kinase II structure and interactions with TA-calmodulin. Biophys j,
Knupp, C., Chew, M., Morris, E. & Squire, J.
(1996). Three-Dimensional Reconstruction of a Collagen IV Analogue in the Dogfish Egg Case Wall. J struct biol,
The wall of the egg case of the dogfish (Scyliorhinus canicula) contains an analogue of collagen Types IV and VIII organised into a regular 3-dimensional network. It presumably provides both a protective and a filtering role for the eggs contained within it. Electron micrographs of longitudinal and transverse sections, including systematically tilted sections, have been used both to define, for the first time, the space group symmetry of the lattice and to carry out 3-D reconstruction of the unit cell contents. This cell was found to be tetragonal, space group I422, with a = b = 11 ± 1 nm, and c = 74 ± 4 nm. Consistent with this, projection symmetries were c2mm for the [1,0,0] view, p2mm for the [1,1,0] view, and p4mm for the projection down the c axis (the [0,0,1] view), and all observed reflections in the computed Fourier transform obeyed the rule h + k + l = 2n (n integer) for body-centred lattices. The 3-D reconstruction, the first electron micrograph 3-D reconstruction of a collagen-containing material, is interpreted in terms of variations of previous molecular models for this structure. Type IV collagen is a constituent of the basal lamina, where it forms a network with both structural and filtering properties. The dogfish egg case structure may throw light on the (less regular) collagen IV structure of the basal lamina..
SERPELL, L.C., SUNDE, M., FRASER, P.E., LUTHER, P.K., MORRIS, E.P., SANGREN, O., LUNDGREN, E. & BLAKE, C.C.
(1995). EXAMINATION OF THE STRUCTURE OF THE TRANSTHYRETIN AMYLOID FIBRIL BY IMAGE-RECONSTRUCTION FROM ELECTRON-MICROGRAPHS. J mol biol,
Familial amyloidotic polyneuropathies are autosomal-dominant, inherited disorders that are characterised by the aggregation of variant proteins in a fibrillar form and by the extracellular deposition of amyloid fibrils. In familial amyloidotic polyneuropathy type I the protein constituent is a variant transthyretin molecule that has a Val to Met substitution at residue 30. Patients with this form of the disease present with sensory and motor disturbances, widespread autonomic dysfunction and in some cases, vitreous opacities. We have used amyloid material from the vitreous humours of patients homozygous for this mutation and analysed the structure of the fibrils by thin section electron microscopy and image reconstruction. Cross-sectional images of 200 different fibrils were collected and aligned, manually at first and then with an automated process that uses iterative cross-correlation. The averaged cross-section calculated produced a detailed view of the fibril substructure. The diameter of the fibrils is about 130 Angstrom. In cross-section they exhibit 4-fold symmetry with four proto-filaments, each measuring 40 to 50 Angstrom across, arranged around a central hollow core. (C) 1995 Academic Press Limited.
Morris, E.P., Katayama, E. & Squire, J.M.
(1994). Evaluation of high-resolution shadowing applied to freeze-fractured, deep-etched particles: 3-D helical reconstruction of shadowed actin filaments. J struct biol,
Images of shadowed F-actin filaments on mica surfaces obtained using a quick-freeze, freeze-fracture, deep-etch technique were subjected to conventional 3-D helical reconstruction methods. Although the shadowing must vary systematically from subunit to subunit, the computed transforms of isolated filaments were characteristic of the helical actin transform. Helical reconstruction was therefore judged to be valid. The theoretical basis for such reconstruction is outlined. The reconstructions showed an average thin (about 3 nm) layer of shadow on the filament surface and both the outer and the inner surfaces of the shadow layer could be visualized. By comparison with the F-actin structure postulated by Holmes et al. (1990) on the basis of the known structure of the actin monomer, it is shown that, at the resolution considered, the inner surface of the shadow provides a reasonably faithful outline of the molecular surface. This, in turn, confirms that the original 3-D structure of the protein molecules has been well preserved throughout the whole preparation procedure up to the final replica. The "shadowed" filaments can thus be correlated axially and azimuthally with known actin structures and, in principle, features such as myosin head location on decorated filaments can be determined. The result emphasizes the amount of detail present in good quality images of shadowed particles and, in this case, shows that detailed evaluation of molecules labeling actin can be made..
MENDELSON, R.A. & MORRIS, E.
(1994). THE STRUCTURE OF F-ACTIN - RESULTS OF GLOBAL SEARCHES USING DATA FROM ELECTRON-MICROSCOPY AND X-RAY CRYSTALLOGRAPHY. J mol biol,
SJOSTROM, M., SQUIRE, J.M., LUTHER, P., MORRIS, E. & EDMAN, A.C.
(1991). CRYOULTRAMICROTOMY OF MUSCLE - IMPROVED PRESERVATION AND RESOLUTION OF MUSCLE ULTRASTRUCTURE USING NEGATIVELY STAINED ULTRATHIN CRYOSECTIONS. J microsc-oxford,
Ultrathin sections of rapidly frozen, briefly pre-treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in-vitro order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X-ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M-band and the bridge region of the A-band in great detail. To enhance the detail in the original images, one-dimensional (1-D) and 2-D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one-third of its original value, demonstrated here for the first time is the fact that the characteristic A-band lattice planes are preserved in these sections in 3-D. This confirms the usefulness of cryosections not just for 1-D and 2-D image processing, but also for 3-D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level..
MORRIS, E.P., SQUIRE, J.M. & FULLER, G.W.
(1991). THE 4-STRANDED HELICAL ARRANGEMENT OF MYOSIN HEADS ON INSECT (LETHOCERUS) FLIGHT-MUSCLE THICK FILAMENTS. J struct biol,
(1990). MUSCLE REGULATION - ON THE OTHER HAND. Nature,
Morris, E.P., Nneji, G. & Squire, J.M.
(1990). The three-dimensional structure of the nemaline rod Z-band. J cell biol,
(6 Pt 2),
In nemaline myopathy and some cardiac muscles, the Z-band becomes greatly enlarged and contains multiple layers of a zigzag structure similar to that seen in normal muscle. Because of the additional periodicity in the direction of the filament axis, these structures are particularly favorable for three-dimensional analysis since it becomes possible to average the data in all three dimensions and thus improve the reliability of the reconstruction. Individual views of the structure corresponding to tilted longitudinal and transverse sections were combined by matching the phases of common reflections. Examination of the tilted views strongly suggested that to the available resolution, the structure possesses fourfold screw symmetry along the actin filament axes. This symmetry could be used both in establishing the correct alignment for the combination of individual tilted views and to generate additional views not readily accessible in a single tilt series. The reconstruction shows actin filaments from one sarcomere surrounded by an array of four actin filaments with opposite polarity from the adjacent sacormere. The actin filaments show a right-handed twist and are connected by a structure that links adjacent filaments with the same polarity at the same axial level, then runs parallel to the filaments, and finally forms a link between two actin filaments whose polarity is opposite to that of the first pair. The connecting structure is probably composed of alpha-actinin which is located in Z-bands and cross-links actin filaments. The connecting structure may consist of two alpha-actinin molecules linking actin filaments of opposite polarity..
Enchev, R.I., Scott, D.C., da Fonseca, P.C., Schreiber, A., Monda, J.K., Schulman, B.A., Peter, M. & Morris, E.P.
Structural Basis for a Reciprocal Regulation
between SCF and CSN. Cell reports,
Abascal-Palacios, G., Ramsay, E.P., Beuron, F., Morris, E. & Vannini, A.
Structural basis of RNA Polymerase III transcription initiation. Nature,
Knupp, , Morris, & Squire,
The Interacting Head Motif Structure Does Not Explain the X-Ray Diffraction Patterns in Relaxed Vertebrate (Bony Fish) Skeletal Muscle and Insect (Lethocerus) Flight Muscle. Biology,
Unlike electron microscopy, which can achieve very high resolution but to date can only be used to study static structures, time-resolved X-ray diffraction from contracting muscles can, in principle, be used to follow the molecular movements involved in force generation on a millisecond timescale, albeit at moderate resolution. However, previous X-ray diffraction studies of resting muscles have come up with structures for the head arrangements in resting myosin filaments that are different from the apparently ubiquitous interacting head motif (IHM) structures found by single particle analysis of electron micrographs of isolated myosin filaments from a variety of muscle types. This head organization is supposed to represent the super-relaxed state of the myosin filaments where adenosine triphosphate (ATP) usage is minimized. Here we have tested whether the interacting head motif structures will satisfactorily explain the observed low-angle X-ray diffraction patterns from resting vertebrate (bony fish) and invertebrate (insect flight) muscles. We find that the interacting head motif does not, in fact, explain what is observed. Previous X-ray models fit the observations much better. We conclude that the X-ray diffraction evidence has been well interpreted in the past and that there is more than one ordered myosin head state in resting muscle. There is, therefore, no reason to question some of the previous X-ray diffraction results on myosin filaments; time-resolved X-ray diffraction should be a reliable way to follow crossbridge action in active muscle and may be one of the few ways to visualise the molecular changes in myosin heads on a millisecond timescale as force is actually produced..