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

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Basic information

Entry
Database: PDB / ID: 6bnp
TitleCryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state
Components
  • Actin, alpha skeletal muscle
  • Unconventional myosin-VI
KeywordsCONTRACTILE PROTEIN / Cytoskeleton / Filament / complex
Function / homologyKinesin motor domain superfamily / Myosin motor domain profile. / Actins signature 1. / Myosin VI cargo binding domain / Myosin head (motor domain) / Myosin head, motor domain / Actin family / Myosin, N-terminal, SH3-like / Actins signature 2. / Actins and actin-related proteins signature. ...Kinesin motor domain superfamily / Myosin motor domain profile. / Actins signature 1. / Myosin VI cargo binding domain / Myosin head (motor domain) / Myosin head, motor domain / Actin family / Myosin, N-terminal, SH3-like / Actins signature 2. / Actins and actin-related proteins signature. / Actin, conserved site / Myosin N-terminal SH3-like domain profile. / Actin/actin-like conserved site / Class VI myosin, motor domain / Myosin VI, cargo binding domain / P-loop containing nucleoside triphosphate hydrolase / Gap junction degradation / Actin / Trafficking of AMPA receptors / Myosin S1 fragment, N-terminal / regulation of secretion / RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / 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 / cytoplasmic vesicle / nuclear membrane / microtubule binding / calmodulin binding / protein domain specific binding / positive regulation of gene expression / magnesium ion binding / calcium ion binding / Golgi apparatus / perinuclear region of cytoplasm / positive regulation of transcription by RNA polymerase II / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm / Unconventional myosin-VI / Actin, alpha skeletal muscle / Unconventional myosin-VI
Function and homology information
Specimen sourceSus scrofa (pig)
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 visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-7116
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  • Superimposition on EM map
  • EMDB-7116
  • Imaged by UCSF Chimera
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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)815,11830
Polyers811,50614
Non-polymers3,61216
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)45690
ΔGint (kcal/M)-289
Surface area (Å2)310990

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Components

#1: Protein/peptide
Unconventional myosin-VI


Mass: 79837.367 Da / Num. of mol.: 6 / Source: (gene. exp.) Sus scrofa (pig) / Gene: MYO6 / Plasmid name: pBiex-1 / 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: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Formula: Mg / Magnesium
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 8 / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM

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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, 20MULTIPLE SOURCES
2myosin VICOMPLEX11RECOMBINANT
3actinCOMPLEX21NATURAL
Molecular weight
IDValueEntity assembly IDExperimental value
10.122 MDa1NO
20.08 MDa1NO
30.042 MDa1NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129823Sus scrofa (pig)
239986Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Spodoptera 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.: 0.45 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: 3710 / Movie frames/image: 24 / Used frames/image: 1-24

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Processing

EM software
IDNameVersionCategory
1Appionparticle selection
2Leginonimage acquisition
4CTFFINDCTF correction
5FREALIGN9.11CTF correction
8DireXmodel fitting
9MDFFmodel fitting
10NAMDmodel fitting
12MDFFmodel refinement
13EMAN2initial Euler assignment
14SPARXinitial Euler assignment
15FREALIGN9.11final Euler assignment
17FREALIGN9.113D reconstruction
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: Initial models were assembled from 8 actins (3J8A) and 6 myosins (2BKI) through rigid body docking in Chimera, followed by flexible fitting with DireX. Resulting models were subjected to MDFF.
Overall b value: 150 / Ref protocol: FLEXIBLE FIT

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