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- PDB-7mf3: Structure of the autoinhibited state of smooth muscle myosin-2 -

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Basic information

Entry
Database: PDB / ID: 7mf3
TitleStructure of the autoinhibited state of smooth muscle myosin-2
Components
  • Myosin light polypeptide 6
  • Myosin regulatory light chain 2, smooth muscle major isoform
  • Myosin-11
KeywordsCONTRACTILE PROTEIN / Muscle contraction / ATPase / autoinhibition / 10S
Function / homology
Function and homology information


RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / myofibril assembly / myosin light chain binding / elastic fiber assembly / skeletal muscle myosin thick filament assembly / muscle myosin complex / myosin II binding / myosin filament ...RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / myofibril assembly / myosin light chain binding / elastic fiber assembly / skeletal muscle myosin thick filament assembly / muscle myosin complex / myosin II binding / myosin filament / myosin II complex / actomyosin structure organization / actomyosin / smooth muscle contraction / cardiac muscle cell development / microfilament motor activity / structural constituent of muscle / myofibril / myosin heavy chain binding / ADP binding / actin filament binding / actin binding / calmodulin binding / calcium ion binding / magnesium ion binding / ATP binding / cytosol
Similarity search - Function
EF-hand domain / DNA repair protein XRCC4-like, C-terminal / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...EF-hand domain / DNA repair protein XRCC4-like, C-terminal / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) / Myosin motor domain profile. / Myosin head, motor domain / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / Kinesin motor domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Myosin regulatory light chain 2, smooth muscle major isoform / ADENOSINE-5'-DIPHOSPHATE / Myosin light polypeptide 6 / PHOSPHATE ION / Myosin-11
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHeissler, S.M. / Arora, A.S. / Billington, N. / Sellers, J.R. / Chinthalapudi, K.
CitationJournal: Sci Adv / Year: 2021
Title: Cryo-EM structure of the autoinhibited state of myosin-2.
Authors: Sarah M Heissler / Amandeep S Arora / Neil Billington / James R Sellers / Krishna Chinthalapudi /
Abstract: We solved the near-atomic resolution structure of smooth muscle myosin-2 in the autoinhibited state (10) using single-particle cryo–electron microscopy. The 3.4-Å structure reveals the precise ...We solved the near-atomic resolution structure of smooth muscle myosin-2 in the autoinhibited state (10) using single-particle cryo–electron microscopy. The 3.4-Å structure reveals the precise molecular architecture of 10 and the structural basis for myosin-2 regulation. We reveal the position of the phosphorylation sites that control myosin autoinhibition and activation by phosphorylation of the regulatory light chain. Further, we present a previously unidentified conformational state in myosin-2 that traps ADP and P produced by the hydrolysis of ATP in the active site. This noncanonical state represents a branch of the myosin enzyme cycle and explains the autoinhibition of the enzyme function of 10 along with its reduced affinity for actin. Together, our structure defines the molecular mechanisms that drive 10 formation, stabilization, and relief by phosphorylation of the regulatory light chain.
History
DepositionApr 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 5, 2022Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Myosin-11
B: Myosin-11
C: Myosin light polypeptide 6
D: Myosin regulatory light chain 2, smooth muscle major isoform
E: Myosin regulatory light chain 2, smooth muscle major isoform
F: Myosin light polypeptide 6
G: Myosin-11
H: Myosin-11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)990,44616
Polymers989,3048
Non-polymers1,1428
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, These chains forms a hexamer complex. This is called myosin-2 monomer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 8 molecules ABGHCFDE

#1: Protein
Myosin-11 / / Myosin heavy chain 11 / Myosin heavy chain / gizzard smooth muscle


Mass: 229018.953 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Organ: gizzard / References: UniProt: P10587
#2: Protein Myosin light polypeptide 6 / G2 catalytic / LC17-GI / LC17-NM / Myosin light chain alkali smooth-muscle/non-muscle isoforms


Mass: 16873.025 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Organ: Gizzard / References: UniProt: P02607
#3: Protein Myosin regulatory light chain 2, smooth muscle major isoform / MLC-2 / DTNB / G1 / Isoform L20-A


Mass: 19741.047 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Organ: Gizzard / References: UniProt: P02612

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Non-polymers , 3 types, 8 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Smooth muscle myosin-2 10S complex / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.531256 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken) / Organ: Gizzard
Buffer solutionpH: 7.3 / Details: 150 mM NaCl, 1mM EGTA, 2mM MgCl2
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 1 mg/ml
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 292 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Cs: 0.01 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 234464 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5I4E
Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323284
ELECTRON MICROSCOPYf_angle_d0.66331278
ELECTRON MICROSCOPYf_dihedral_angle_d5.1943057
ELECTRON MICROSCOPYf_chiral_restr0.0413400
ELECTRON MICROSCOPYf_plane_restr0.0044121

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