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- EMDB-7100: The Structure of the Actin-Smooth Muscle Myosin Motor Domain Comp... -

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Entry
Database: EMDB / ID: EMD-7100
TitleThe Structure of the Actin-Smooth Muscle Myosin Motor Domain Complex in the Rigor State
Map dataMuscle alpha-actin decorated with the motor domain of chicken gizzard smooth muscle myosin II
Sample
  • Complex: Filamentous muscle alpha-actin decorated with the motor domain of recombinant smooth muscle myosin II motor domain expressed in Sf9 cells
    • Complex: alpha-actin
      • Protein or peptide: Myosin-11
    • Complex: myosin II
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsADP-F-actin / apo-myosin / helix muscle / MOTOR PROTEIN
Function / homology
Function and homology information


myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / myosin filament / actomyosin structure organization / myosin II complex / cardiac muscle cell development ...myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / myosin filament / actomyosin structure organization / myosin II complex / cardiac muscle cell development / structural constituent of muscle / cytoskeletal motor activator activity / microfilament motor activity / myofibril / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / smooth muscle contraction / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / ADP binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / actin filament binding / lamellipodium / actin binding / cell body / calmodulin binding / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / 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 / Myosin head, motor 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 / 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
Myosin-11 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Gallus gallus (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsTaylor KA / Banerjee C
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM30598 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01 HL110869 United States
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR53975 United States
American Heart Association15PRE25090150 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10 RR25080 United States
National Institutes of Health/Office of the DirectorS10 OD018142 United States
CitationJournal: J Struct Biol / Year: 2017
Title: The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.
Authors: Chaity Banerjee / Zhongjun Hu / Zhong Huang / J Anthony Warrington / Dianne W Taylor / Kathleen M Trybus / Susan Lowey / Kenneth A Taylor /
Abstract: Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography ...Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.
History
DepositionNov 2, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseSep 19, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bih
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7100.png

Downloads & links

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Map

FileDownload / File: emd_7100.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMuscle alpha-actin decorated with the motor domain of chicken gizzard smooth muscle myosin II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 256 pix.
= 252.442 Å		0.99 Å/pix.
x 256 pix.
= 252.442 Å		0.99 Å/pix.
x 256 pix.
= 252.442 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9861 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.029346576 - 0.050385498
Average (Standard dev.)0.00007721186 (±0.005672934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin636363
Dimensions256256256
Spacing256256256
CellA=B=C: 252.4416 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.98610156250.98610156250.9861015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z252.442252.442252.442
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS636363
NC/NR/NS256256256
D min/max/mean-0.0290.0500.000

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Supplemental data

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Sample components

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Entire : Filamentous muscle alpha-actin decorated with the motor domain of...

EntireName: Filamentous muscle alpha-actin decorated with the motor domain of recombinant smooth muscle myosin II motor domain expressed in Sf9 cells
Components
  • Complex: Filamentous muscle alpha-actin decorated with the motor domain of recombinant smooth muscle myosin II motor domain expressed in Sf9 cells
    • Complex: alpha-actin
      • Protein or peptide: Myosin-11
    • Complex: myosin II
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Filamentous muscle alpha-actin decorated with the motor domain of...

SupramoleculeName: Filamentous muscle alpha-actin decorated with the motor domain of recombinant smooth muscle myosin II motor domain expressed in Sf9 cells
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: alpha-actin

SupramoleculeName: alpha-actin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Supramolecule #3: myosin II

SupramoleculeName: myosin II / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Myosin-11

MacromoleculeName: Myosin-11 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Tissue: skeletal muscle
Molecular weightTheoretical: 91.343227 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSQKPLSDDE KFLFVDKNFV NNPLAQADWS AKKLVWVPSE KHGFEAASIK EEKGDEVTVE LQENGKKVTL SKDDIQKMNP PKFSKVEDM AELTCLNEAS VLHNLRERYF SGLIYTYSGL FCVVINPYKQ LPIYSEKIID MYKGKKRHEM PPHIYAIADT A YRSMLQDR ...String:
MSQKPLSDDE KFLFVDKNFV NNPLAQADWS AKKLVWVPSE KHGFEAASIK EEKGDEVTVE LQENGKKVTL SKDDIQKMNP PKFSKVEDM AELTCLNEAS VLHNLRERYF SGLIYTYSGL FCVVINPYKQ LPIYSEKIID MYKGKKRHEM PPHIYAIADT A YRSMLQDR EDQSILCTGE SGAGKTENTK KVIQYLAVVA SSHKGKKDTS ITQGPSFSYG ELEKQLLQAN PILEAFGNAK TV KNDNSSR FGKFIRINFD VTGYIVGANI ETYLLEKSRA IRQAKDERTF HIFYYLIAGA SEQMRNDLLL EGFNNYTFLS NGH VPIPAQ QDDEMFQETL EAMTIMGFTE EEQTSILRVV SSVLQLGNIV FKKERNTDQA SMPDNTAAQK VCHLMGINVT DFTR SILTP RIKVGRDVVQ KAQTKEQADF AIEALAKAKF ERLFRWILTR VNKALDKTKR QGASFLGILD IAGFEIFEIN SFEQL CINY TNEKLQQLFN HTMFILEQEE YQREGIEWNF IDFGLDLQPC IELIERPTNP PGVLALLDEE CWFPKATDTS FVEKLI QEQ GNHAKFQKSK QLKDKTEFCI LHYAGKVTYN ASAWLTKNMD PLNDNVTSLL NQSSDKFVAD LWKDVDRIVG LDQMAKM TE SSLPSASKTK KGMFRTVGQL YKEQLTKLMT TLRNTNPNFV RCIIPNHEKR AGKLDAHLVL EQLRCNGVLE GIRICRQG F PNRIVFQEFR QRYEILAANA IPKGFMDGKQ ACILMIKALE LDPNLYRIGQ SKIFFRTGVL AHLEEERDLD YKDDDDK

UniProtKB: Myosin-11

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 42.096953 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL 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 NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4
Details: actin buffer: 10 mM imidazole, 10 mM KCl, 1.0 mM MgCl2, 1.0 mM EGTA, 0.5 mM DTT, pH 7.4, myosin buffer: 10 mM imidazole, 10 mM KCl, 1.0 mM MgCl2, 1.0 mM EGTA, 0.5 mM DTT, pH 7.0
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 276 K / Instrument: GATAN CRYOPLUNGE 3
Details: Some specimens were frozen manually using a homemade plunger..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-43 / Number real images: 4000 / Average exposure time: 1.34 sec. / Average electron dose: 60.0 e/Å2 / Details: Only 1417 of the 4000 recorded images were used.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.2 µm
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: 4
Applied symmetry - Helical parameters - Δz: 28.18685 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.779310 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.2) / Number images used: 85000
Segment selectionNumber selected: 346395
Details: Each "particle" consisted of a filament segment masked to a length of 21.0 nm, or slightly more than 7 actin subunits with 2.76 nm separation. Adjacent filament segments overlapped by about ...Details: Each "particle" consisted of a filament segment masked to a length of 21.0 nm, or slightly more than 7 actin subunits with 2.76 nm separation. Adjacent filament segments overlapped by about 6 subunit repeats (approximately 84% overlap).
Startup modelType of model: EMDB MAP
EMDB ID:

1987


Details: Reference map was low-pass filtered to 10 nm resolution.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 390.86
Output model

PDB-6bih:
The Structure of the Actin-Smooth Muscle Myosin Motor Domain Complex in the Rigor State

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