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- EMDB-13993: In situ structure of myosin neck domain in skeletal sarcomere (ce... -

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Basic information

Entry
Database: EMDB / ID: EMD-13993
TitleIn situ structure of myosin neck domain in skeletal sarcomere (centered on regulatory light chain)
Map data
Sample
  • Organelle or cellular component: Mouse psoas muscle myofibrils
    • Protein or peptide: Myosin-4
    • Protein or peptide: Myosin light chain 1/3, skeletal muscle isoform
    • Protein or peptide: Myosin regulatory light chain 2, skeletal muscle isoform
Function / homology
Function and homology information


Striated Muscle Contraction / Smooth Muscle Contraction / contractile muscle fiber / myosin filament / myosin II complex / myosin complex / structural constituent of muscle / microfilament motor activity / myofibril / skeletal muscle tissue development ...Striated Muscle Contraction / Smooth Muscle Contraction / contractile muscle fiber / myosin filament / myosin II complex / myosin complex / structural constituent of muscle / microfilament motor activity / myofibril / skeletal muscle tissue development / cardiac muscle contraction / response to activity / muscle contraction / actin filament binding / double-stranded RNA binding / calmodulin binding / immune response / calcium ion binding / ATP binding / 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. / Myosin head, motor domain / 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 / Myosin N-terminal SH3-like domain profile. / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / 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 light chain 1/3, skeletal muscle isoform / Myosin regulatory light chain 11 / Myosin-4
Similarity search - Component
Biological speciesMus musculus (house mouse) / house mouse (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 9.0 Å
AuthorsWang Z / Grange M / Pospich S / Wagner T / Kho AL / Gautel M / Raunser S
Funding supportEuropean Union, 5 items
OrganizationGrant numberCountry
Max Planck SocietyEuropean Union
Wellcome Trust201543/Z/16/ZEuropean Union
European Research Council (ERC)856118European Union
Medical Research Council (MRC, United Kingdom)MR/R003106/1European Union
European Molecular Biology Organization (EMBO)European Union
CitationJournal: Science / Year: 2022
Title: Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.
Authors: Zhexin Wang / Michael Grange / Sabrina Pospich / Thorsten Wagner / Ay Lin Kho / Mathias Gautel / Stefan Raunser /
Abstract: In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram ...In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.
History
DepositionDec 15, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7qio
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13993.png

Downloads & links

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Map

FileDownload / File: emd_13993.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.46 Å/pix.
x 80 pix.
= 276.8 Å		3.46 Å/pix.
x 80 pix.
= 276.8 Å		3.46 Å/pix.
x 80 pix.
= 276.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.46 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-1.8774239 - 2.4783409
Average (Standard dev.)0.0037818782 (±0.06730451)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 276.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.463.463.46
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z276.800276.800276.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-1.8772.4780.004

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Supplemental data

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Mask #1

Fileemd_13993_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13993_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13993_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse psoas muscle myofibrils

EntireName: Mouse psoas muscle myofibrils
Components
  • Organelle or cellular component: Mouse psoas muscle myofibrils
    • Protein or peptide: Myosin-4
    • Protein or peptide: Myosin light chain 1/3, skeletal muscle isoform
    • Protein or peptide: Myosin regulatory light chain 2, skeletal muscle isoform

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Supramolecule #1: Mouse psoas muscle myofibrils

SupramoleculeName: Mouse psoas muscle myofibrils / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Myosin-4

MacromoleculeName: Myosin-4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse)
Molecular weightTheoretical: 96.405609 KDa
SequenceString: MSSDAEMAVF GEAAPYLRKS EKERIEAQNK PFDAKSSVFV VDAKESYVKA TVQSREGGKV TAKTEGGATV TVKDDQVFSM NPPKYDKIE DMAMMTHLHE PAVLYNLKER YAAWMIYTYS GLFCVTVNPY KWLPVYNPEV VAAYRGKKRQ EAPPHIFSIS D NAYQFMLT ...String:
MSSDAEMAVF GEAAPYLRKS EKERIEAQNK PFDAKSSVFV VDAKESYVKA TVQSREGGKV TAKTEGGATV TVKDDQVFSM NPPKYDKIE DMAMMTHLHE PAVLYNLKER YAAWMIYTYS GLFCVTVNPY KWLPVYNPEV VAAYRGKKRQ EAPPHIFSIS D NAYQFMLT DRENQSILIT GESGAGKTVN TKRVIQYFAT IAVTGDKKKE EATSGKMQGT LEDQIISANP LLEAFGNAKT VR NDNSSRF GKFIRIHFGA TGKLASADIE TYLLEKSRVT FQLKAERSYH IFYQIMSNKK PELIEMLLIT TNPYDFAYVS QGE ITVPSI DDQEELMATD TAVDILGFSA DEKVAIYKLT GAVMHYGNMK FKQKQREEQA EPDGTEVADK AAYLTSLNSA DLLK ALCYP RVKVGNEYVT KGQTVQQVYN SVGALAKSMY EKMFLWMVTR INQQLDTKQP RQYFIGVLDI AGFEIFDFNT LEQLC INFT NEKLQQFFNH HMFVLEQEEY KKEGIDWEFI DFGMDLAACI ELIEKPMGIF SILEEECMFP KATDTSFKNK LYEQHL GKS NNFQKPKPAK GKAEAHFSLV HYAGTVDYNI IGWLDKNKDP LNETVVGLYQ KSGLKTLAFL FSGGQAAEAE GGGGKKG GK KKGSSFQTVS ALFRENLNKL MTNLKSTHPH FVRCLIPNET KTPGAMEHEL VLHQLRCNGV LEGIRICRKG FPSRILYA D FKQRYKVLNA SAIPEGQFID SKKASEKLLG SIDIDHTQYK FGHTKVFFKA GLLGTLEEMR DEKLAQLITR TQAVCRGYL MRVEFKKMME RRESIFCIQY NVRAFMNVKH WPWMKLYFKI KPLLKSAE

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Macromolecule #2: Myosin light chain 1/3, skeletal muscle isoform

MacromoleculeName: Myosin light chain 1/3, skeletal muscle isoform / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse)
Molecular weightTheoretical: 20.62049 KDa
SequenceString:
MAPKKDVKKP AAAPAPAPAP APAPAKPKEE KIDLSAIKIE FSKEQQEDFK EAFLLFDRTG ECKITLSQVG DVLRALGTNP TNAEVKKVL GNPSNEEMNA KKIEFEQFLP MMQAISNNKD QGGYEDFVEG LRVFDKEGNG TVMGAELRHV LATLGEKMKE E EVEALLAG QEDSNGCINY EAFVKHIMSV

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Macromolecule #3: Myosin regulatory light chain 2, skeletal muscle isoform

MacromoleculeName: Myosin regulatory light chain 2, skeletal muscle isoform
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse)
Molecular weightTheoretical: 18.978445 KDa
SequenceString:
MAPKKAKRRA GAEGSSNVFS MFDQTQIQEF KEAFTVIDQN RDGIIDKEDL RDTFAAMGRL NVKNEELDAM MKEASGPINF TVFLTMFGE KLKGADPEDV ITGAFKVLDP EGKGTIKKQF LEELLTTQCD RFSQEEIKNM WAAFPPDVGG NVDYKNICYV I THGDAKDQ E

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statetissue

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 2.4 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.4 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 3.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 48 / Number images used: 183260
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 44001
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7qin

chain_id: L

3i5g

chain_id: B

3i5g

chain_id: C
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 300
Output model

PDB-7qio:
Homology model of myosin neck domain in skeletal sarcomere

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