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- PDB-3j04: EM structure of the heavy meromyosin subfragment of Chick smooth ... -

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Basic information

Entry
Database: PDB / ID: 3j04
TitleEM structure of the heavy meromyosin subfragment of Chick smooth muscle Myosin with regulatory light chain in phosphorylated state
Components
  • Myosin light polypeptide 6
  • Myosin regulatory light chain 2, smooth muscle major isoform
  • Myosin-11
KeywordsSTRUCTURAL PROTEIN / PHOSPHORYLATION / 2D CRYSTALLINE ARRAYS / MYOSIN REGULATION / MYOSIN LIGHT CHAINS
Function / homology
Function and homology information


RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / actomyosin ...RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / actomyosin / myosin filament / actomyosin structure organization / myosin II complex / cardiac muscle cell development / structural constituent of muscle / microfilament motor activity / myofibril / myosin heavy chain binding / smooth muscle contraction / stress fiber / ADP binding / actin filament binding / actin binding / calmodulin binding / calcium ion binding / magnesium ion binding / ATP binding / cytosol / cytoplasm
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 / : / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. ...: / 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 / : / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / Kinesin motor domain superfamily / EF-hand domain pair / 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 light polypeptide 6 / Myosin regulatory light chain 2, smooth muscle major isoform / Myosin-11
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / electron crystallography / cryo EM / Resolution: 20 Å
AuthorsBaumann, B.A.J. / Taylor, D. / Huang, Z. / Tama, F. / Fagnant, P.M. / Trybus, K. / Taylor, K.
CitationJournal: J Mol Biol / Year: 2012
Title: Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation.
Authors: Bruce A J Baumann / Dianne W Taylor / Zhong Huang / Florence Tama / Patricia M Fagnant / Kathleen M Trybus / Kenneth A Taylor /
Abstract: Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a ...Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.
History
DepositionFeb 18, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2011Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2011Group: Database references
Revision 1.2Feb 1, 2012Group: Database references
Revision 1.3Feb 21, 2024Group: Author supporting evidence / Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_single_particle_entity
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)275,7926
Polymers275,7926
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 104657.781 Da / Num. of mol.: 2 / Fragment: MEROMYOSIN SUBFRAGMENT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: PVL1392 / Cell (production host): sarcomere / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): smooth muscle / References: UniProt: P10587
#2: Protein Myosin regulatory light chain 2, smooth muscle major isoform / MLC-2 / DTNB / G1 / Isoform L20-A


Mass: 16583.412 Da / Num. of mol.: 2 / Fragment: S-1 SUBFRAGMENT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: PVL1392 / Cell (production host): sarcomere / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): smooth muscle / References: UniProt: P02612
#3: Protein Myosin light polypeptide 6 / G2 catalytic / LC17-GI / LC17-NM / Myosin light chain alkali smooth-muscle/non-muscle isoforms


Mass: 16654.791 Da / Num. of mol.: 2 / Fragment: S-1 SUBFRAGMENT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: PVL1392 / Cell (production host): sarcomere / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): smooth muscle / References: UniProt: P02607

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: heavy meromyosin subfragment of Chick smooth muscle Myosin with regulatory light chain in phosphorylated state
Type: COMPLEX
Buffer solutionpH: 7.8
Details: 1 mM Mg, 20 mM phosphate, 1 mM ATP, 1 mM EGTA, 7-10% polyethylene glycol 6000, 90-120 mM NaCl
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh carbon coated grid
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Details: Carried out in cold room at 4 degrees C / Method: Blot for 4 sec before plunging
Crystal growpH: 7.8 / Details: pH 7.8

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM300FEG/T / Date: Oct 10, 2004
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 24000 X / Nominal defocus min: 4000 nm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: eucentic / Tilt angle max: 60 ° / Tilt angle min: -60 °
Image recordingElectron dose: 40 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 85
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1CCP4model fitting
2NMFFmodel fitting
CTF correctionDetails: determined using ICE anc corrected with CTFAPPPLY
3D reconstructionMethod: Cross-common lines / Resolution: 20 Å
Details: A total of 85 unique averaged structure factors were obtained and had an average phase residual of 17.9 degrees with a resolution to approx 2.1 nm
Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: REFINEMENT PROTOCOL--RIGID BODY DETAILS--The model was roughly fit into the density map using O the refined using NMFF. The entire structure was then minimized using minCHARMM.pl
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms19065 0 0 0 19065

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