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- PDB-6bnv: CryoEM structure of MyosinVI-actin complex in the rigor (nucleoti... -

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Basic information

Entry
Database: PDB / ID: 6bnv
TitleCryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state, backbone-averaged with side chains truncated to alanine
Components
  • Actin, alpha skeletal muscle
  • Calmodulin
  • Unconventional myosin-VI
KeywordsCONTRACTILE PROTEIN / Cytoskeleton / Filament / complex
Function / homologyInactivation, recovery and regulation of the phototransduction cascade / Actin, conserved site / Myosin VI, cargo binding domain / P-loop containing nucleoside triphosphate hydrolase / Actin/actin-like conserved site / EF-Hand 1, calcium-binding site / EF-hand domain pair / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like / Actin family ...Inactivation, recovery and regulation of the phototransduction cascade / Actin, conserved site / Myosin VI, cargo binding domain / P-loop containing nucleoside triphosphate hydrolase / Actin/actin-like conserved site / EF-Hand 1, calcium-binding site / EF-hand domain pair / Myosin S1 fragment, N-terminal / Myosin, N-terminal, SH3-like / Actin family / Kinesin motor domain superfamily / EF-hand domain / Myosin head, motor domain / FCERI mediated Ca+2 mobilization / Ca2+ pathway / Reduction of cytosolic Ca++ levels / Stimuli-sensing channels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Class VI myosin, motor domain / Calmodulin / rt:r-gga-442982: / Myosin N-terminal SH3-like domain profile. / Calcineurin activates NFAT / Synthesis of IP3 and IP4 in the cytosol / PKA activation / Platelet degranulation / Cam-PDE 1 activation / Calmodulin induced events / CaMK IV-mediated phosphorylation of CREB / Myosin motor domain profile. / Actin / EF-hand calcium-binding domain profile. / Actins and actin-related proteins signature. / Actins signature 2. / Actins signature 1. / EF-hand calcium-binding domain. / Myosin VI cargo binding domain / EF-hand domain pair / Myosin head (motor domain) / rt:r-gga-442745: / Sodium/Calcium exchangers / Smooth Muscle Contraction / Trafficking of AMPA receptors / Gap junction degradation / Ion transport by P-type ATPases / Protein methylation / Glycogen breakdown (glycogenolysis) / RAF/MAP kinase cascade / RHO GTPases activate PAKs / RHO GTPases activate IQGAPs / CLEC7A (Dectin-1) induces NFAT activation / Ion homeostasis / Activation of Ca-permeable Kainate Receptor / CH domain binding / regulation of secretion / RNA polymerase II, holoenzyme / actin filament-based movement / spindle pole body organization / clathrin-coated endocytic vesicle / positive regulation of actin-dependent ATPase activity / myosin complex / mesenchyme migration / myosin binding / tropomyosin binding / N-terminal myristoylation domain binding / positive regulation by host of symbiont cAMP-mediated signal transduction / myosin heavy chain binding / troponin I binding / adenylate cyclase activator activity / positive regulation of ryanodine-sensitive calcium-release channel activity / actin filament bundle / protein phosphatase activator activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / filamentous actin / catalytic complex / adenylate cyclase binding / skeletal muscle thin filament assembly / detection of calcium ion / striated muscle thin filament / skeletal muscle fiber development / actin monomer binding / DNA damage response, signal transduction by p53 class mediator / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / actin filament bundle assembly / enzyme regulator activity / skeletal muscle myofibril / motor activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / positive regulation of phosphoprotein phosphatase activity / stress fiber / calcium channel complex / voltage-gated potassium channel complex / titin binding / actin filament polymerization / regulation of heart rate / ADP binding / sarcomere / filopodium
Function and homology information
Specimen sourceSus scrofa (pig)
Gallus gallus (chicken)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 4.6 Å resolution
AuthorsGurel, P.S. / Alushin, G.A.
CitationJournal: Nat Nanotechnol / Year: 2018
Title: Controllable molecular motors engineered from myosin and RNA.
Authors: Tosan Omabegho / Pinar S Gurel / Clarence Y Cheng / Laura Y Kim / Paul V Ruijgrok / Rhiju Das / Gregory M Alushin / Zev Bryant
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 17, 2017 / Release: Jan 10, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 10, 2018Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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Structure viewerMolecule:
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Assembly

Deposited unit
I: Unconventional myosin-VI
J: Unconventional myosin-VI
K: Unconventional myosin-VI
L: Unconventional myosin-VI
M: Unconventional myosin-VI
N: Unconventional myosin-VI
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
F: Actin, alpha skeletal muscle
G: Actin, alpha skeletal muscle
H: Actin, alpha skeletal muscle
O: Calmodulin
P: Calmodulin
Q: Calmodulin
R: Calmodulin
S: Calmodulin
T: Calmodulin


Theoretical massNumber of molelcules
Total (without water)990,16220
Polyers990,16220
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Unconventional myosin-VI


Mass: 93207.297 Da / Num. of mol.: 6 / Source: (gene. exp.) Sus scrofa (pig) / Tissue: skeletal muscle / Gene: MYO6 / Plasmid name: pBiex1 / Cell line (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F1RQI7, UniProt: Q29122*PLUS
#2: Protein/peptide
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41560.266 Da / Num. of mol.: 8 / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle / References: UniProt: P68135
#3: Protein/peptide
Calmodulin / / CaM


Mass: 16406.004 Da / Num. of mol.: 6 / Source: (gene. exp.) Gallus gallus (chicken) / Gene: CALM, CAM, RCJMB04_24e7 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62149

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1myosin VI-actin in the rigor (nucleotide-free) stateCOMPLEX1, 2, 30MULTIPLE SOURCES
2myosin VICOMPLEX11RECOMBINANT
3actinCOMPLEX21NATURAL
4calmodulinCOMPLEX31RECOMBINANT
Molecular weightValue: 0.139 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129823Sus scrofa (pig)
239986Oryctolagus cuniculus (rabbit)
349031Gallus gallus (chicken)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
127108Spodoptera frugiperda (fall armyworm)
247108Spodoptera frugiperda (fall armyworm)
Buffer solutionDetails: Buffer was filtered through 0.44 um filter and degassed.
pH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
150 mMpotassium chlorideKCl1
21 mMmagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
410 mMimidazoleC3H4N21
52 mMTris hydrochlorideC4H11NO31
60.5 mMdithiothreitolC4H10O2S21
7200 mMadenosine triphosphateC10H16N5O13P31
80.01 %sodium azideNaN31
910 U/mLapyraseapyrase1
SpecimenConc.: 0.045 mg/ml
Details: 0.45 mg/mL myosin VI was added to 0.025 mg/mL actin
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 kelvins
Details: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An additional 3 uL of myosin VI was applied. After 60 seconds, 3 uL solution was removed, and the grid was blotted for 3 seconds from the backside with filter paper.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 100 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 778
Image scansSampling size: 5 microns / Width: 3838 / Height: 3711 / Movie frames/image: 24 / Used frames/image: 1-24

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Processing

EM software
IDNameVersionCategory
1Appionparticle selection
2Leginonimage acquisition
4CTFFIND3CTF correction
5FREALIGN9.11CTF correction
8DireXmodel fitting
9MDFFmodel fitting
10NAMDmodel fitting
12EMAN2initial Euler assignment
13SPARXinitial Euler assignment
14FREALIGN9.11final Euler assignment
16FREALIGN9.113D reconstruction
17MDFFmodel refinement
18PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.73 deg. / Axial rise/subunit: 28.06 Å / Axial symmetry: C1
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 56116 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingDetails: 2BKI chain B was grafted onto Chains A-F of 6BNP, then assembled by rigid body docking into the 7.5 A low pass-filtered density map, followed by flexible fitting with DireX. The resulting model was subjected to MDFF using the 7.5 A low pass-filtered density map. Atomistic models were backbone-averaged in Phenix and side chains were truncated to alanine. This treatment has resulted in the errors noted in the associated caveats.
Overall b value: 150 / Ref protocol: FLEXIBLE FIT / Ref space: REAL

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