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Entry
Database: PDB / ID: 5tby
TitleHUMAN BETA CARDIAC HEAVY MEROMYOSIN INTERACTING-HEADS MOTIF OBTAINED BY HOMOLOGY MODELING (USING SWISS-MODEL) OF HUMAN SEQUENCE FROM APHONOPELMA HOMOLOGY MODEL (PDB-3JBH), RIGIDLY FITTED TO HUMAN BETA-CARDIAC NEGATIVELY STAINED THICK FILAMENT 3D-RECONSTRUCTION (EMD-2240)
Components
  • Myosin light chain 3
  • Myosin regulatory light chain 2, ventricular/cardiac muscle isoform
  • Myosin-7
KeywordsCONTRACTILE PROTEIN / CONTRACTILE PROTEIN Hypertrophic or dilated cardiomyopathy beta-cardiac myosin II Myosin interacting-heads motif
Function / homology
Function and homology information


myosin II heavy chain binding / regulation of slow-twitch skeletal muscle fiber contraction / muscle cell fate specification / regulation of the force of skeletal muscle contraction / muscle myosin complex / adult heart development / cardiac myofibril / regulation of the force of heart contraction / muscle fiber development / cardiac myofibril assembly ...myosin II heavy chain binding / regulation of slow-twitch skeletal muscle fiber contraction / muscle cell fate specification / regulation of the force of skeletal muscle contraction / muscle myosin complex / adult heart development / cardiac myofibril / regulation of the force of heart contraction / muscle fiber development / cardiac myofibril assembly / transition between fast and slow fiber / cardiac muscle hypertrophy in response to stress / positive regulation of the force of heart contraction / regulation of striated muscle contraction / actin-dependent ATPase activity / myosin filament / positive regulation of ATPase activity / A band / skeletal muscle contraction / microfilament motor activity / striated muscle contraction / muscle filament sliding / myosin complex / I band / structural constituent of muscle / myofibril / myosin heavy chain binding / heart contraction / ventricular cardiac muscle tissue morphogenesis / ATP metabolic process / actin monomer binding / cardiac muscle contraction / stress fiber / muscle contraction / sarcomere / regulation of heart rate / post-embryonic development / Z disc / negative regulation of cell growth / actin filament binding / heart development / calmodulin binding / cytoskeleton / ATPase activity / calcium ion binding / ATP binding / cytosol
EF-hand domain / EF-Hand 1, calcium-binding site / IQ motif, EF-hand binding site / Myosin head, motor domain / EF-hand domain / Myosin, N-terminal, SH3-like / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin tail / Myosin IQ motif-containing domain superfamily ...EF-hand domain / EF-Hand 1, calcium-binding site / IQ motif, EF-hand binding site / Myosin head, motor domain / EF-hand domain / Myosin, N-terminal, SH3-like / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin tail / Myosin IQ motif-containing domain superfamily / Myosin tail / Myosin N-terminal SH3-like domain / Kinesin motor domain superfamily / Myosin head (motor domain) / P-loop containing nucleoside triphosphate hydrolase
Myosin light chain 3 / Myosin regulatory light chain 2, ventricular/cardiac muscle isoform / Myosin-7
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 20 Å
AuthorsALAMO, L. / WARE, J.S. / PINTO, A. / GILLILAN, R.E. / SEIDMAN, J.G. / SEIDMAN, C.E. / PADRON, R.
Funding support United States, France, United Kingdom, 7items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Leducq Foundation France
Wellcome Trust United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)NHLBI-HL084553 United States
National Science Foundation (NSF, United States)DMR-1332208 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM-103485 United States
Medical Research Council (United Kingdom) United Kingdom
Citation
Journal: Elife / Year: 2017
Title: Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes.
Authors: Lorenzo Alamo / James S Ware / Antonio Pinto / Richard E Gillilan / Jonathan G Seidman / Christine E Seidman / Raúl Padrón /
Abstract: Cardiac β-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures ...Cardiac β-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures predict contractility effects but not the prominent relaxation and energetic deficits that characterize HCM. During relaxation, pairs of myosins form interacting-heads motif (IHM) structures that with other sarcomere proteins establish an energy-saving, super-relaxed (SRX) state. Using a human β-cardiac myosin IHM quasi-atomic model, we defined interactions sites between adjacent myosin heads and associated protein partners, and then analyzed rare variants from 6112 HCM and 1315 DCM patients and 33,370 ExAC controls. HCM variants, 72% that changed electrostatic charges, disproportionately altered IHM interaction residues (expected 23%; HCM 54%, p=2.6×10; DCM 26%, p=0.66; controls 20%, p=0.23). HCM variant locations predict impaired IHM formation and stability, and attenuation of the SRX state - accounting for altered contractility, reduced diastolic relaxation, and increased energy consumption, that fully characterizes HCM pathogenesis.
#original_data_1: Journal: J. Mol. Biol. / Year: 2016
Title: Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.
Authors: Lorenzo Alamo / Dan Qi / Willy Wriggers / Antonio Pinto / Jingui Zhu / Aivett Bilbao / Richard E Gillilan / Songnian Hu / Raúl Padrón /
Abstract: Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and ...Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles.
#1: Journal: J. Mol. Biol. / Year: 2008
Title: Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity.
Authors: Lorenzo Alamo / Willy Wriggers / Antonio Pinto / Fulvia Bártoli / Leiria Salazar / Fa-Qing Zhao / Roger Craig / Raúl Padrón /
Abstract: Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular ...Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.
#2: Journal: J. Mol. Biol. / Year: 2016
Title: Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.
Authors: Lorenzo Alamo / Dan Qi / Willy Wriggers / Antonio Pinto / Jingui Zhu / Aivett Bilbao / Richard E Gillilan / Songnian Hu / Raúl Padrón /
Abstract: Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and ...Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles.
#3: Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2013
Title: Atomic model of the human cardiac muscle myosin filament.
Authors: Hind A Al-Khayat / Robert W Kensler / John M Squire / Steven B Marston / Edward P Morris /
Abstract: 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 ...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.
#4: Journal: Electrophoresis / Year: 1997
Title: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.
Authors: N Guex / M C Peitsch /
Abstract: Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting ...Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS-MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.
#5: Journal: Nucleic Acids Res. / Year: 2003
Title: SWISS-MODEL: An automated protein homology-modeling server.
Authors: Torsten Schwede / Jürgen Kopp / Nicolas Guex / Manuel C Peitsch /
Abstract: SWISS-MODEL (http://swissmodel.expasy.org) is a server for automated comparative modeling of three-dimensional (3D) protein structures. It pioneered the field of automated modeling starting in 1993 ...SWISS-MODEL (http://swissmodel.expasy.org) is a server for automated comparative modeling of three-dimensional (3D) protein structures. It pioneered the field of automated modeling starting in 1993 and is the most widely-used free web-based automated modeling facility today. In 2002 the server computed 120 000 user requests for 3D protein models. SWISS-MODEL provides several levels of user interaction through its World Wide Web interface: in the 'first approach mode' only an amino acid sequence of a protein is submitted to build a 3D model. Template selection, alignment and model building are done completely automated by the server. In the 'alignment mode', the modeling process is based on a user-defined target-template alignment. Complex modeling tasks can be handled with the 'project mode' using DeepView (Swiss-PdbViewer), an integrated sequence-to-structure workbench. All models are sent back via email with a detailed modeling report. WhatCheck analyses and ANOLEA evaluations are provided optionally. The reliability of SWISS-MODEL is continuously evaluated in the EVA-CM project. The SWISS-MODEL server is under constant development to improve the successful implementation of expert knowledge into an easy-to-use server.
#6: Journal: Bioinformatics / Year: 2006
Title: The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling.
Authors: Konstantin Arnold / Lorenza Bordoli / Jürgen Kopp / Torsten Schwede
Abstract: MOTIVATION: Homology models of proteins are of great interest for planning and analysing biological experiments when no experimental three-dimensional structures are available. Building homology ...MOTIVATION: Homology models of proteins are of great interest for planning and analysing biological experiments when no experimental three-dimensional structures are available. Building homology models requires specialized programs and up-to-date sequence and structural databases. Integrating all required tools, programs and databases into a single web-based workspace facilitates access to homology modelling from a computer with web connection without the need of downloading and installing large program packages and databases.
RESULTS: SWISS-MODEL workspace is a web-based integrated service dedicated to protein structure homology modelling. It assists and guides the user in building protein homology models at different levels of complexity. A personal working environment is provided for each user where several modelling projects can be carried out in parallel. Protein sequence and structure databases necessary for modelling are accessible from the workspace and are updated in regular intervals. Tools for template selection, model building and structure quality evaluation can be invoked from within the workspace. Workflow and usage of the workspace are illustrated by modelling human Cyclin A1 and human Transmembrane Protease 3.
AVAILABILITY: The SWISS-MODEL workspace can be accessed freely at http://swissmodel.expasy.org/workspace/
Validation Report
SummaryFull reportAbout validation report
History
DepositionSep 13, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 7, 2017Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Aug 9, 2017Group: Database references / Category: pdbx_related_exp_data_set
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Nov 8, 2017Group: Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.5Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.6Oct 3, 2018Group: Data collection / Refinement description / Category: refine / Item: _refine.pdbx_refine_id
Revision 1.7Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Remark 0This entry reflects an alternative modeling of the original data in 3JBH, determined by ALAMO, L., ...This entry reflects an alternative modeling of the original data in 3JBH, determined by ALAMO, L., QI, D., WRIGGERS, W., PINTO, A., ZHU, J., BILBAO, A., GILLILAN, R.E., HU, S., PADRON, R.

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  • Simplified surface model + fitted atomic model
  • EMDB-2240
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  • Superimposition on EM map
  • EMDB-2240
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Assembly

Deposited unit
A: Myosin-7
B: Myosin-7
C: Myosin light chain 3
D: Myosin light chain 3
E: Myosin regulatory light chain 2, ventricular/cardiac muscle isoform
F: Myosin regulatory light chain 2, ventricular/cardiac muscle isoform


Theoretical massNumber of molelcules
Total (without water)528,4436
Polymers528,4436
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29570 Å2
ΔGint-132 kcal/mol
Surface area137070 Å2
SymmetryHelical symmetry: (Num. of operations: 3 / Rise per n subunits: 145 Å / Rotation per n subunits: 30 °)

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Components

#1: Protein/peptide Myosin-7 / / Myosin heavy chain 7 / Myosin heavy chain slow isoform / MyHC-slow / Myosin heavy chain / cardiac muscle beta isoform / MyHC-beta


Mass: 223445.984 Da / Num. of mol.: 2
Details: MYOSIN 2 HEAVY CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE HEAD HOMOLOGY MODEL OF HUMAN BETA CARDIAC ...MYOSIN 2 HEAVY CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE HEAD HOMOLOGY MODEL OF HUMAN BETA CARDIAC HEAVY MEROMYOSIN INTERACTING-HEADS MOTIF
Fragment: SUBFRAGMENT 1(S1) / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P12883
#2: Protein/peptide Myosin light chain 3 / / Cardiac myosin light chain 1 / CMLC1 / Myosin light chain 1 / slow-twitch muscle B/ventricular isoform / MLC1SB / Ventricular myosin alkali light chain / Ventricular myosin light chain 1 / VLCl / Ventricular/slow twitch myosin alkali light chain / MLC-lV/sb


Mass: 21962.068 Da / Num. of mol.: 2
Details: MYOSIN 2 ESSENTIAL LIGHT CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE (CHAIN C) AND BLOCKED (CHAIN D) ...MYOSIN 2 ESSENTIAL LIGHT CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE (CHAIN C) AND BLOCKED (CHAIN D) HEAD HOMOLOGY MODEL OF HUMAN BETA CARDIAC HEAVY MEROMYOSIN INTERACTING-HEADS MOTIF
Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P08590
#3: Protein/peptide Myosin regulatory light chain 2, ventricular/cardiac muscle isoform / MLC-2v / Cardiac myosin light chain 2 / Myosin light chain 2 / slow skeletal/ventricular muscle isoform / MLC-2s/v / Ventricular myosin light chain 2


Mass: 18813.273 Da / Num. of mol.: 2
Details: MYOSIN 2 REGULATORY LIGHT CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE (CHAIN E) AND BLOCKED (CHAIN F) ...MYOSIN 2 REGULATORY LIGHT CHAIN, VENTRICULAR CARDIAC MUSCLE, FREE (CHAIN E) AND BLOCKED (CHAIN F) HEAD HOMOLOGY MODEL OF HUMAN BETA CARDIAC HEAVY MEROMYOSIN INTERACTING-HEADS MOTIF
Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P10916

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MYOSIN THICK FILAMENTS TARANTULA STRIATED MUSCLE / Type: TISSUE
Details: THE ATOMIC MODEL CONSIST OF ONE INTERACTING-HEADS MOTIF, FORMED BY TWO HEAVY MEROMYOSIN (S1 MYOSIN HEAD WITH A SEGMENT OF S2 AND ESSENTIAL AND REGULATORY LIGHT CHAINS) SO CALLED BLOCKED AND FREE HEADS, THE HOMOLOGY MODEL IS BASED ON TARANTULA STRIATED MUSCLE MODEL STRUCTURE 3JBH. THIS MODEL WAS RIGIDLY DOCKED TO A TARANTULA 3D MAP (EMD-1950) AND ALSO AGAINST HUMAN BETA-CARDIAC NEGATIVELY STAINED THICK FILAMENT 2.8 NM RESOLUTION 3D-RECONSTRUCTION (EMD-2240)
Entity ID: 1, 2, 3 / Source: NATURAL
Source (natural)Organism: Aphonopelma (spider) / Cellular location: SARCOPLASM / Organ: LEG / Organelle: THICK FILAMENTS / Tissue: MUSCLE
Buffer solutionpH: 7
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1100 MMSodium ChlorideNaClSodium chloride
23 MMMagnesium ChlorideMgCl2
31 MMTriethylene glycol diamine tetraacetic acid (EGTA)C14H24N2O10
45 MMPIPESC8H18N2O6S2
55 MMSodium dihydrogen phosphateNAH2PO4
63 MMSdium AzideNAN3
SpecimenDetails: A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001).
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Holey carbon grids had been rendered hydrophilic by glow discharge in n-amylamine vapor for 3 minutes before use.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 296 K
Details: PLUNGING IN A LIQUID ETHANE COOLED BY LIQUID NITROGEN. BLOTTING WAS PERFORMED FROM ONE SIDE OF THE GRID TILL A THIN SAMPLE FILM ON IT USING WHATMAN NO. 42 FILTER PAPER, THEN THE GRID WAS IMMEDIATELY PLUNGED UNDER GRAVITY INTO LIQUID ETHANE COOLED BY LIQUID NITROGEN. GRIDS WERE STORED UNDER LIQUID NITROGEN.

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM120T
Details: Holey carbon grids Cryopreserved in Liquid ethane were observed in a Philips CM120 electron microscope under low dose conditions. Only filaments on thin carbon over holes were photographed
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 35000 X / Calibrated magnification: 35000 X / Nominal defocus max: 1950 nm / Nominal defocus min: 1950 nm / Calibrated defocus min: 1950 nm / Calibrated defocus max: 1950 nm / Cs: 2 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Model: GATAN LIQUID NITROGEN / Temperature (max): 90 K / Temperature (min): 88 K
Image recordingElectron dose: 9 e/Å2 / Film or detector model: KODAK SO-163 FILM / Num. of real images: 500
Details: Low-dose electron micrographs of 1008 frozen-hydrated thick filaments halves ere digitized at 0.248 nm per pixel using a Nikon Super Coolscan 8000 ED scanner.
Image scansSampling size: 8.47 µm / Width: 250 / Height: 250

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectionHELIXBOXER
7UCSF Chimera10model fittingTHE "FIT IN MAP" TOOL WAS USED WITH DEFAULT OPTIONS
13ITERATIVE HELICAL REAL SPACE RECONSTRUCTION (EGELMAN, 2000)SPIDER 143D reconstructionmodification of the IHRSR method
Image processingDetails: Three-dimensional single particle reconstruction was carried out by a modification of the IHRSR method, using SPIDER. Low-dose electron micrographs of 1008 frozen-hydrated thick filaments halves where digitized at 0.248 nm per pixel using a Nikon Super Coolscan 8000 ED scanner. Filaments were aligned with the bare zone at the top, to ensure correct polarity in subsequent steps.
CTF correctionType: NONE
Particle selectionNum. of particles selected: 15504
Details: A total of 15,504 segments, each 62 nm long, with an overlap of 55.8 nm, and containing aprox. 40,000 unique pairs of interacting myosin heads went into the reconstruction.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 20 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 10700 / Algorithm: BACK PROJECTION
Details: For projection matching, giving a total of 4,095 projections (13 tilted projections plus-minus 12 deg. every 2deg., 45 reference rotated projections (0-90 degrees, 2deg. rotation angle), and 7 image axial shifts of 2.2 nm. The resulting 3D-map combines about 10,700 out of 15,504 filament segments, a yield of 69 percent of included segments. There are 4 helices of myosin heads, rotated 30 degrees, every 145 Angstroms. The filament segments were selected based on visual judgement of good helical order.
Num. of class averages: 4095 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: CORRELATION COEFFICIENT
Atomic model building

3D fitting-ID: 1 / PDB-ID: 3JBH

IDPdb chain-IDPdb chain residue range
1A1-962
2B1-962
3C1-156
4D1-156
5E1-196
6F1-196
RefinementHighest resolution: 20 Å

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Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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