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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 / homologyEF-hand domain / Class VI myosin, motor domain / Actins signature 2. / Actins signature 1. / EF-hand calcium-binding domain. / Myosin VI cargo binding domain / EF-hand domain pair / Myosin head (motor domain) / Actin / Kinesin motor domain superfamily ...EF-hand domain / Class VI myosin, motor domain / Actins signature 2. / Actins signature 1. / EF-hand calcium-binding domain. / Myosin VI cargo binding domain / EF-hand domain pair / Myosin head (motor domain) / Actin / Kinesin motor domain superfamily / Myosin VI, cargo binding domain / EF-hand calcium-binding domain profile. / 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, conserved site / Actin family / Myosin head, motor domain / Actins and actin-related proteins signature. / Myosin motor domain profile. / Ion homeostasis / Stimuli-sensing channels / Smooth Muscle Contraction / Ras activation upon Ca2+ influx through NMDA receptor / Activation of CaMK IV / CREB phosphorylation through the activation of CaMKII / CREB phosphorylation through the activation of CaMKK / Sodium/Calcium exchangers / Reduction of cytosolic Ca++ levels / Ca2+ pathway / FCERI mediated Ca+2 mobilization / Inactivation, recovery and regulation of the phototransduction cascade / Myosin N-terminal SH3-like domain profile. / eNOS activation / rt:r-gga-2025928: / Synthesis of IP3 and IP4 in the cytosol / PKA activation / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Platelet degranulation / Cam-PDE 1 activation / Calmodulin induced events / CaMK IV-mediated phosphorylation of CREB / VEGFR2 mediated vascular permeability / Activation of Ca-permeable Kainate Receptor / RHO GTPases activate IQGAPs / RAF/MAP kinase cascade / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / Gap junction degradation / Protein methylation / Ion transport by P-type ATPases / Trafficking of AMPA receptors / RHO GTPases activate PAKs / CH domain binding / regulation of secretion / RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / myosin binding / positive regulation of actin-dependent ATPase activity / myosin complex / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / filamentous actin / motor activity / skeletal muscle fiber development / actin filament bundle assembly / actin monomer binding / DNA damage response, signal transduction by p53 class mediator / titin binding / skeletal muscle myofibril / stress fiber / ADP binding / actin filament polymerization / microtubule motor activity / actin filament / microtubule-based movement / filopodium / calcium-dependent protein binding / intracellular protein transport / ruffle / sensory perception of sound / cell body / cell cortex / endocytosis / actin filament binding / lamellipodium / disordered domain specific binding / cytoplasmic vesicle / protein C-terminus binding / nuclear membrane / microtubule binding
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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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 mm / 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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