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- EMDB-28082: Helical reconstruction of the human cardiac actin-tropomyosin-myo... -

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Entry
Database: EMDB / ID: EMD-28082
TitleHelical reconstruction of the human cardiac actin-tropomyosin-myosin complex in complex with ADP-Mg2+
Map dataPrimary map used for model building of the helical reconstruction of the actin-tropomyosin-B-cardiac myosin II complex with ADP-Mg2 bound.
Sample
  • Complex: Human cardiac actin-tropomyosin-beta-myosin II complex bound to ADPMg2+.
    • Complex: Cardiac F-actin complex
      • Protein or peptide: Actin, alpha cardiac muscle 1
    • Complex: Human beta-cardiac myosin II
      • Protein or peptide: beta-cardiac myosin II
    • Complex: Human cardiac tropomyosin
      • Protein or peptide: Tropomyosin alpha-1 chain
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordsactin / tropomyosin / myosin / cardiac / MOTOR PROTEIN
Function / homology
Function and homology information


regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / actin-myosin filament sliding / muscle myosin complex / regulation of muscle contraction / regulation of the force of heart contraction / transition between fast and slow fiber ...regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / bleb / actin-myosin filament sliding / muscle myosin complex / regulation of muscle contraction / regulation of the force of heart contraction / transition between fast and slow fiber / myosin filament / ruffle organization / adult heart development / Striated Muscle Contraction / cardiac muscle hypertrophy in response to stress / muscle filament sliding / myosin complex / myosin II complex / structural constituent of muscle / sarcomere organization / ventricular cardiac muscle tissue morphogenesis / microfilament motor activity / heart contraction / regulation of heart contraction / myosin binding / negative regulation of vascular associated smooth muscle cell migration / myofibril / mesenchyme migration / negative regulation of vascular associated smooth muscle cell proliferation / Smooth Muscle Contraction / skeletal muscle contraction / striated muscle contraction / ATP metabolic process / cytoskeletal protein binding / cardiac muscle contraction / stress fiber / positive regulation of stress fiber assembly / cytoskeleton organization / positive regulation of cell adhesion / muscle contraction / regulation of heart rate / negative regulation of cell migration / actin filament organization / sarcomere / cellular response to reactive oxygen species / filopodium / actin filament / wound healing / structural constituent of cytoskeleton / ruffle membrane / Z disc / actin filament binding / regulation of cell shape / lamellipodium / actin cytoskeleton / actin binding / cell body / cytoskeleton / calmodulin binding / protein heterodimerization activity / positive regulation of gene expression / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Tropomyosins signature. / Tropomyosin / Tropomyosin / 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. ...Tropomyosins signature. / Tropomyosin / Tropomyosin / 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 / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Actin alpha cardiac muscle 1 / Tropomyosin alpha-1 chain / Myosin-7
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDoran MH / Lehman W / Rynkiewicz MJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL036153 United States
CitationJournal: J Gen Physiol / Year: 2023
Title: Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin.
Authors: Matthew H Doran / Michael J Rynkiewicz / David Rasicci / Skylar M L Bodt / Meaghan E Barry / Esther Bullitt / Christopher M Yengo / Jeffrey R Moore / William Lehman /
Abstract: Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular ...Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.
History
DepositionSep 8, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28082.png

Downloads & links

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Map

FileDownload / File: emd_28082.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map used for model building of the helical reconstruction of the actin-tropomyosin-B-cardiac myosin II complex with ADP-Mg2 bound.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 440 pix.
= 474.32 Å		1.08 Å/pix.
x 440 pix.
= 474.32 Å		1.08 Å/pix.
x 440 pix.
= 474.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.078 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0238
Minimum - Maximum-0.10581913 - 0.19441107
Average (Standard dev.)0.0004102688 (±0.0044674864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 474.31998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28082_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Non-locally-filtered map associated with primary map.

Fileemd_28082_additional_1.map
AnnotationNon-locally-filtered map associated with primary map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1.

Fileemd_28082_half_map_1.map
AnnotationHalf map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2.

Fileemd_28082_half_map_2.map
AnnotationHalf map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human cardiac actin-tropomyosin-beta-myosin II complex bound to A...

EntireName: Human cardiac actin-tropomyosin-beta-myosin II complex bound to ADPMg2+.
Components
  • Complex: Human cardiac actin-tropomyosin-beta-myosin II complex bound to ADPMg2+.
    • Complex: Cardiac F-actin complex
      • Protein or peptide: Actin, alpha cardiac muscle 1
    • Complex: Human beta-cardiac myosin II
      • Protein or peptide: beta-cardiac myosin II
    • Complex: Human cardiac tropomyosin
      • Protein or peptide: Tropomyosin alpha-1 chain
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Human cardiac actin-tropomyosin-beta-myosin II complex bound to A...

SupramoleculeName: Human cardiac actin-tropomyosin-beta-myosin II complex bound to ADPMg2+.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Cardiac actomyosin-tropomyosin complex with ADPMg2+ bound to the myosin motor head.

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Supramolecule #2: Cardiac F-actin complex

SupramoleculeName: Cardiac F-actin complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2 / Details: F-actin forms the backbone of the complex
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: Human beta-cardiac myosin II

SupramoleculeName: Human beta-cardiac myosin II / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Details: The motor domain of the myosin saturates the actin filament.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Human cardiac tropomyosin

SupramoleculeName: Human cardiac tropomyosin / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 / Details: Tropomyosin wraps around the F-actin core.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: beta-cardiac myosin II

MacromoleculeName: beta-cardiac myosin II / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.157695 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MGDSEMAVFG AAAPYLRKSE KERLEAQTRP FDLKKDVFVP DDKQEFVKAK IVSREGGKVT AETEYGKTVT VKEDQVMQQN PPKFDKIED MAMLTFLHEP AVLYNLKDRY GSWMIYTYSG LFCVTVNPYK WLPVYTPEVV AAYRGKKRSE APPHIFSISD N AYQYMLTD ...String:
MGDSEMAVFG AAAPYLRKSE KERLEAQTRP FDLKKDVFVP DDKQEFVKAK IVSREGGKVT AETEYGKTVT VKEDQVMQQN PPKFDKIED MAMLTFLHEP AVLYNLKDRY GSWMIYTYSG LFCVTVNPYK WLPVYTPEVV AAYRGKKRSE APPHIFSISD N AYQYMLTD RENQSILITG ESGAGKTVNT KRVIQYFAVI AAIGDRSKKD QSPGKGTLED QIIQANPALE AFGNAKTVRN DN SSRFGKF IRIHFGATGK LASADIETYL LEKSRVIFQL KAERDYHIFY QILSNKKPEL LDMLLITNNP YDYAFISQGE TTV ASIDDA EELMATDNAF DVLGFTSEEK NSMYKLTGAI MHFGNMKFKL KQREEQAEPD GTEEADKSAY LMGLNSADLL KGLC HPRVK VGNEYVTKGQ NVQQVIYATG ALAKAVYERM FNWMVTRINA TLETKQPRQY FIGVLDIAGF EIFDFNSFEQ LCINF TNEK LQQFFNHHMF VLEQEEYKKE GIEWTFIDFG MDLQACIDLI EKPMGIMSIL EEECMFPKAT DMTFKAKLFD NHLGKS ANF QKPRNIKGKP EAHFSLIHYA GIVDYNIIGW LQKNKDPLNE TVVGLYQKSS LKLLSTLFAN YAGADAPIEK GKGKAKK GS SFQTVSALHR ENLNKLMTNL RSTHPHFVRC IIPNETKSPG VMDNPLVMHQ LRCNGVLEGI RICRKGFPNR ILYGDFRQ R YRILNPAAIP EGQFIDSRKG AEKLLSSLDI DHNQYKFGHT KVFFKAGLLG LLEEMRDERL SRIITRIQAQ SRGVLARME YKKLLERRDS LLVIQWNIRA FMGVKNWPWM KLYFKIKPLL KSGLNDIFEA QKIEWHEDYK DDDDK

UniProtKB: Myosin-7

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Macromolecule #2: Actin, alpha cardiac muscle 1

MacromoleculeName: Actin, alpha cardiac muscle 1 / type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 42.064891 KDa
SequenceString: MCDDEETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDDEETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVLSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWISKQEYDE AGPSIVHRKC F

UniProtKB: Actin alpha cardiac muscle 1

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Macromolecule #3: Tropomyosin alpha-1 chain

MacromoleculeName: Tropomyosin alpha-1 chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.763621 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDAIKKKMQM LKLDKENALD RAEQAEADKK AAEDRSKQLE DELVSLQKKL KGTEDELDKY SEALKDAQEK LELAEKKATD AEADVASLN RRIQLVEEEL DRAQERLATA LQKLEEAEKA ADESERGMKV IESRAQKDEE KMEIQEIQLK EAKHIAEDAD R KYEEVARK ...String:
MDAIKKKMQM LKLDKENALD RAEQAEADKK AAEDRSKQLE DELVSLQKKL KGTEDELDKY SEALKDAQEK LELAEKKATD AEADVASLN RRIQLVEEEL DRAQERLATA LQKLEEAEKA ADESERGMKV IESRAQKDEE KMEIQEIQLK EAKHIAEDAD R KYEEVARK LVIIESDLER AEERAELSEG KCAELEEELK TVTNNLKSLE AQAEKYSQKE DRYEEEIKVL SDKLKEAETR AE FAERSVT KLEKSIDDLE DELYAQKLKY KAISEELDHA LNDMTSI

UniProtKB: Tropomyosin alpha-1 chain

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.13 mg/mL
BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: 15 mA was used in the Pelco Easiglow glow discharge machine
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 4 / Number real images: 3961 / Average exposure time: 3.12 sec. / Average electron dose: 53.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 27.9 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.8 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 131194
Segment selectionNumber selected: 628787 / Software - Name: RELION (ver. 3.1.1)
Startup modelType of model: OTHER / Details: Featureless Cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6x5z


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8efh:
Helical reconstruction of the human cardiac actin-tropomyosin-myosin complex in complex with ADP-Mg2+

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