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- PDB-1i84: CRYO-EM STRUCTURE OF THE HEAVY MEROMYOSIN SUBFRAGMENT OF CHICKEN ... -

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Entry
Database: PDB / ID: 1i84
TitleCRYO-EM STRUCTURE OF THE HEAVY MEROMYOSIN SUBFRAGMENT OF CHICKEN GIZZARD SMOOTH MUSCLE MYOSIN WITH REGULATORY LIGHT CHAIN IN THE DEPHOSPHORYLATED STATE. ONLY C ALPHAS PROVIDED FOR REGULATORY LIGHT CHAIN. ONLY BACKBONE ATOMS PROVIDED FOR S2 FRAGMENT.
Components
  • SMOOTH MUSCLE MYOSIN ESSENTIAL LIGHT CHAIN
  • SMOOTH MUSCLE MYOSIN HEAVY CHAIN
  • SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN
KeywordsCONTRACTILE PROTEIN / muscle protein / smooth muscle / myosin subfragment 2 / heavy meromyosin / essential light chain / regulatory light chain / motor protein / coiled-coil
Function / homology
Function and homology information


myosin II filament / 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 ...myosin II filament / 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 / myosin complex / microfilament motor activity / structural constituent of muscle / myofibril / myosin heavy chain binding / skeletal muscle tissue development / 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 domain profile. / Myosin, N-terminal, SH3-like / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) ...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 domain profile. / Myosin, N-terminal, SH3-like / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / Myosin head, motor domain / 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, skeletal muscle isoform / : / Myosin light polypeptide 6 / Myosin-11
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 20 Å
AuthorsWendt, T. / Taylor, D. / Trybus, K.M. / Taylor, K.
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2001
Title: Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2.
Authors: T Wendt / D Taylor / K M Trybus / K Taylor /
Abstract: Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. ...Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically "blocked" because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.
#1: Journal: J.Cell Biol. / Year: 1999
Title: Visualization of Head-head Interactions in the Inhibited State of Smooth Muscle Myosin
Authors: Wendt, T. / Taylor, D. / Messier, T. / Trybus, K.M. / Taylor, K.A.
#2: Journal: Cell(Cambridge,Mass.) / Year: 1998
Title: Crystal Structure of a Vertebrate Smooth Muscle Myosin Motor Domain and its Complex with the Essential Light Chain: Visualization of the Pre-power Stroke State
Authors: Dominguez, R. / Freyzon, Y. / Trybus, K.M. / Cohen, C.
#3: Journal: Science / Year: 1993
Title: Three-dimensional Structure of Myosin Subfragment-1: a Molecular Motor
Authors: Rayment, I. / Rypniewsky, W.R. / Schmidt-Base, K. / Smith, R. / Tomchick, D.R. / Benning, M.M. / Winkelmann, D.A. / Wesenberg, G. / Holden, H.M.
#4: Journal: J.Mol.Biol. / Year: 1996
Title: The Structure of the Head-tail Junction of the Myosin Molecule
Authors: Offer, G. / Knight, P.
History
DepositionMar 12, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 28, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance

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Structure visualization

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Assembly

Deposited unit
S: SMOOTH MUSCLE MYOSIN HEAVY CHAIN
T: SMOOTH MUSCLE MYOSIN ESSENTIAL LIGHT CHAIN
U: SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN
V: SMOOTH MUSCLE MYOSIN HEAVY CHAIN
W: SMOOTH MUSCLE MYOSIN ESSENTIAL LIGHT CHAIN
Z: SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN


Theoretical massNumber of molelcules
Total (without water)344,5516
Polymers344,5516
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)133.0, 304.0, 200.0
Angle α, β, γ (deg.)90.00, 90.00, 91.5
Int Tables number3
Space group name H-MP112

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Components

#1: Antibody SMOOTH MUSCLE MYOSIN HEAVY CHAIN / MYOSIN HEAVY CHAIN / GIZZARD SMOOTH MUSCLE


Mass: 137072.938 Da / Num. of mol.: 2 / Fragment: MEROMYOSIN SUBFRAGMENT. S1 AND S2 FRAGMENTS.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Description: FLAG-TAG AT C-TERMINUS; / Plasmid: PVL1392 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: P10587, EC: 3.6.1.32
#2: Protein SMOOTH MUSCLE MYOSIN ESSENTIAL LIGHT CHAIN / ELC / MYOSIN ALKALI LIGHT CHAIN


Mass: 16873.025 Da / Num. of mol.: 2 / Fragment: S1 FRAGMENT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: PVL1392 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: GenBank: 212393, UniProt: P02607*PLUS
#3: Protein SMOOTH MUSCLE MYOSIN REGULATORY LIGHT CHAIN / RLC / MYOSIN REGULATORY LIGHT CHAIN 2 / SMOOTH MUSCLE (MAJOR) / Coordinate model: Cα atoms only


Mass: 18329.598 Da / Num. of mol.: 2 / Fragment: S1 FRAGMENT
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Plasmid: PVL1392 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): SF9 / References: UniProt: P02609

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 101
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
Crystal symmetry∠γ: 91.5 ° / A: 133 Å / B: 304 Å / C: 90 Å / Space group name H-M: P2

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Sample preparation

ComponentName: HEAVY MEROMYOSIN SUBFRAGMENT OF CHICKEN GIZZARD SMOOTH MUSCLE MYOSIN WITH REGULATORY LIGHT CHAIN IN THE DEPHOSPHORYLATED STATE
Type: COMPLEX
EM crystal formationDetails: 2-D arrays were grown on a positively charged lipid monolayer system consisting of didodecyldimethylammonium and dilauryl phosphatidyl choline (Taylor, K. A. & Taylor, D. W. (1992) J. Struct. ...Details: 2-D arrays were grown on a positively charged lipid monolayer system consisting of didodecyldimethylammonium and dilauryl phosphatidyl choline (Taylor, K. A. & Taylor, D. W. (1992) J. Struct. Biol. 108, 140-147). Crystallization buffers consisted of 1 mM Mg2+, 20 mM phosphate, pH range 7-8, 1 mM ATP, 1 mM EGTA and PEG 6000 (7-10%) and NaCl (90-120 mM). Specimens were recovered on 200 mesh copper grids coated with a reticulated carbon film. The specimen was picked up from the grid bar side and blotted from that side.
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Specimens were frozen in liquid ethane using a drop freezing apparatus.
Crystal growTemperature: 277 K / Method: lipid monolayer / pH: 7.5
Details: MgCl, sodium phosphate, ATP, EGTA, PEG 6000, pH 7.5, Lipid monolayer, temperature 277K
Crystal grow
*PLUS
Method: other / Details: data none

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Data collection

MicroscopyModel: FEI/PHILIPS CM300FEG/T / Date: Jul 1, 1999
Electron gunAccelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 24000 X
Specimen holderTemperature: 93 K
Image recordingElectron dose: 5 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 101 / Scanner model: PERKIN ELMER
Radiation wavelengthRelative weight: 1

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Processing

Image processingDetails: Micrographs were processed using the ICE processing software
Crystal symmetry∠γ: 91.5 ° / A: 133 Å / B: 304 Å / C: 90 Å / Space group name H-M: P2
3D reconstructionResolution: 20 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: VISUAL AGREEMENT
Details: REFINEMENT PROTOCOL--MANUAL DETAILS--The atomic model consists of two S1 heads and a segment of S2 containing 91 residues. Each S1 consists of a heavy chain and two light chains, the ELC and ...Details: REFINEMENT PROTOCOL--MANUAL DETAILS--The atomic model consists of two S1 heads and a segment of S2 containing 91 residues. Each S1 consists of a heavy chain and two light chains, the ELC and the RLC. The atomic model of the S1 subfragment was constructed using two X-ray crystallography models. The heavy chain residues from ala2 to leu819 as well as the essential light chain were obtained from the chicken smooth muscle structure, PDB entry 1BR1, chains A and B. The remainder of S1 including heavy chain residues from glu820 to lys852 (smooth muscle numbering but skeletal myosin sequence) were obtained from residues glu811-lys843 of chicken skeletal muscle myosin, PDB entry 2MYS, since the structure of the entire smooth muscle S1 fragment is not available. Note that the residue numbers for the chain segment from 2MYS that were spliced onto 1BR1 have been renumbered to run consecutively from 819 but the residues themselves are the chicken skeletal muscle myosin heavy chain residues. The regulatory light chain coordinates were also obtained from 2MYS (skeletal) because a smooth muscle myosin model is not available. This regulatory light chain consisted only of C alphas. In fitting the model to the molecular envelope, the lever arm of the free head, i.e. ELC, RLC and heavy chain residues from ile796-lys852, were rotated as a rigid body about the Calpha of ile796. The amount of rotation was about 20 degrees. The blocked head was not modified. The alignment of 2MYS to 1BR1 was done using the conserved residues on the essential light chain. This alignment was carried out using O. The rms of the fit using 123 C alpha atoms of the conserved residues was 1.632 Angstroms. The C alpha distance between leu819 and glu811 (2MYS residue number) after the transformation was 4.23 Angstroms. This distance has not been altered in the model. The splice site at 819-820 is also slightly long and was left that way so the splice site would be easily visible. The other long bonds are at locations where the chain was bent to fit the structure. No minimization or refinement was done to fix them. The following residues have long peptide bond distances- chain S- LEU 819 and GLU 820 THR 854 and ARG 855 MET 860 and GLN 861 GLN 903 and ALA 904 chain V- LEU 819 and GLU 820 LYS 852 and VAL 853 MET 860 and GLN 861 GLN 903 and ALA 904 The 91 residues of the S2 fragment built into the model were calculated using a program written by Dr. Gerald Offer (reference 4). These coordinates consist only of backbone atoms. The S2 sequence begins at val853 which is the correct residue number for smooth muscle myosin. Peptide chain designations- Blocked myosin S1 head myosin heavy chain (with S2 segment) is chain V ELC is chain W RLC is chain Z Free myosin S1 head myosin heavy chain (with S2 fragment) is chain S ELC is chain T RLC is chain U The terms blocked and free refer to the conformations of the two S1 heads. SEQUENCE GAPS IN THE MOLECULAR MODEL- Myosin heavy chain- 205-210, 452-457, 635-655, 944-1185. ELC- 1-2. RLC (skeletal)- 1-18, 127, 142-147, 164-166.
RefinementHighest resolution: 20 Å
Refinement stepCycle: LAST / Highest resolution: 20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16580 0 0 0 16580

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