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- EMDB-5257: Phosphorylated smooth muscle heavy meromyosin shows an open confo... -

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Basic information

Entry
Database: EMDB / ID: EMD-5257
TitlePhosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation
Map dataThis is an image of the 2D crystal looking from the top down
Sample
  • Sample: Heavy meromyosin subfragment of chicken gizzard smooth muscle myosin with the regulatory light chain in the phosphoryated state
  • Organelle or cellular component: heavy meromyosin
KeywordssmHMM / heavy meromyosin / S1 / phosphorylation / smooth muscle / activation
Function / homology
Function and homology information


RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / elastic fiber assembly / myofibril assembly / skeletal muscle myosin thick filament assembly / myosin light chain binding / myosin II binding / muscle myosin complex / actomyosin ...RHO GTPases activate PAKs / myosin II filament / Smooth Muscle Contraction / elastic fiber assembly / myofibril assembly / skeletal muscle myosin thick filament assembly / myosin light chain binding / 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 / myosin heavy chain binding / myofibril / 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
: / 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 ...: / 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 crystallography / cryo EM / Resolution: 20.0 Å
AuthorsBaumann BAJ / Taylor D / Huang Z / Tama F / Fagnant PM / Trybus KM / Taylor K
CitationJournal: J Cell Biol / Year: 1999
Title: Visualization of head-head interactions in the inhibited state of smooth muscle myosin.
Authors: T Wendt / D Taylor / T Messier / K M Trybus / K A Taylor /
Abstract: The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed ...The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.
History
DepositionJan 21, 2011-
Header (metadata) releaseMay 5, 2011-
Map releaseNov 9, 2011-
UpdateMar 19, 2012-
Current statusMar 19, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.609
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.609
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j04
  • Surface level: 0.609
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5257_1.png

Downloads & links

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Map

FileDownload / File: emd_5257.map.gz / Format: CCP4 / Size: 1.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of the 2D crystal looking from the top down
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.46 Å/pix.
x 35 pix.
= 85.998 Å		2.45 Å/pix.
x 81 pix.
= 428.995 Å		2.48 Å/pix.
x 169 pix.
= 419.069 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 2.4797 Å / Y: 2.4514 Å / Z: 2.4571 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.609 / Movie #1: 0.609
Minimum - Maximum-4.67003298 - 4.40441799
Average (Standard dev.)-0.03437874 (±1.01000309)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-84-17
Dimensions8116935
Spacing17516935
CellA: 419.0693 Å / B: 428.995 Å / C: 85.9985 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.47969822485212.45142.4570857142857
M x/y/z16917535
origin x/y/z0.0000.0000.000
length x/y/z419.069428.99585.998
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-84-40-17
NC/NR/NS1698135
D min/max/mean-4.6704.404-0.034

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Supplemental data

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Sample components

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Entire : Heavy meromyosin subfragment of chicken gizzard smooth muscle myo...

EntireName: Heavy meromyosin subfragment of chicken gizzard smooth muscle myosin with the regulatory light chain in the phosphoryated state
Components
  • Sample: Heavy meromyosin subfragment of chicken gizzard smooth muscle myosin with the regulatory light chain in the phosphoryated state
  • Organelle or cellular component: heavy meromyosin

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Supramolecule #1000: Heavy meromyosin subfragment of chicken gizzard smooth muscle myo...

SupramoleculeName: Heavy meromyosin subfragment of chicken gizzard smooth muscle myosin with the regulatory light chain in the phosphoryated state
type: sample / ID: 1000
Details: Chicken gizzard smooth muscle heavy meromyosin was expressed, isolated and thiophosphorylated as per Wendt et al. 1999.
Number unique components: 1
Molecular weightTheoretical: 350 KDa

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Supramolecule #1: heavy meromyosin

SupramoleculeName: heavy meromyosin / type: organelle_or_cellular_component / ID: 1 / Name.synonym: heavy meromyosin / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Gallus gallus (chicken) / synonym: Chicken / Tissue: smooth muscle / Cell: Baculovirus / Location in cell: sarcomere
Molecular weightTheoretical: 350 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: PVL1392

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.8
Details: 1 mM Mg, 20 mM phosphate, 1 mM ATP, 1 mM EGTA, 7-10% polyethylene glycol 6000, 90-120 mM NaCl
GridDetails: 200 mesh carbon coated grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: homemade solenoid activated plunge freezer. Carried out in cold room at 4 degrees C
Method: Blot for 4 sec before plunging
Detailscrystals grown on a lipid monolayer
Crystal formationDetails: crystals grown on a lipid monolayer

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Electron microscopy

MicroscopeFEI/PHILIPS CM300FEG/T
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism corrected at 250 times magnification
DateOct 10, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 85 / Average electron dose: 40 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus min: 4.0 µm / Nominal magnification: 24000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle min: -60 / Tilt angle max: 60 / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Software - Name: CCP4
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
Crystal parametersUnit cell - A: 219.3 Å / Unit cell - B: 174.8 Å / Unit cell - C: 94.4 Å / Unit cell - γ: 94.4 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Plane group: P 2
CTF correctionDetails: determined using ICE and corrected with CTFAPPPLY

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Atomic model buiding 1

Initial modelPDB ID:

1br1

SoftwareName: NMFF
DetailsProtocol: rigid body. The model was roughly fit into the density map using O the refined using NMFF. The entire structure was then minimized using minCHARMM.pl
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j04:
EM structure of the heavy meromyosin subfragment of Chick smooth muscle Myosin with regulatory light chain in phosphorylated state

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Atomic model buiding 2

Initial modelPDB ID:

2mys

SoftwareName: NMFF
DetailsProtocol: rigid body. The model was roughly fit into the density map using O the refined using NMFF. The entire structure was then minimized using minCHARMM.pl
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j04:
EM structure of the heavy meromyosin subfragment of Chick smooth muscle Myosin with regulatory light chain in phosphorylated state

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