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- PDB-5jlh: Cryo-EM structure of a human cytoplasmic actomyosin complex at ne... -
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Basic information
Entry | Database: PDB / ID: 5jlh | |||||||||
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Title | Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution | |||||||||
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![]() | CONTRACTILE PROTEIN / CONTRACTILE FILAMENT / MUSCLE / THIN FILAMENT / CYTOSKELETON / STRUCTURAL PROTEIN / HYDROLASE COMPLEX / F-actin / tropomyosin / filament / myosin / protein polymers / cryo EM | |||||||||
Function / homology | ![]() RHOBTB2 GTPase cycle / RHOU GTPase cycle / RHOV GTPase cycle / contractile vacuole / COPI-mediated anterograde transport / Platelet degranulation / basal body patch / sorocarp development / myosin II filament / tight junction assembly ...RHOBTB2 GTPase cycle / RHOU GTPase cycle / RHOV GTPase cycle / contractile vacuole / COPI-mediated anterograde transport / Platelet degranulation / basal body patch / sorocarp development / myosin II filament / tight junction assembly / macropinocytic cup / Neutrophil degranulation / regulation of transepithelial transport / morphogenesis of a polarized epithelium / actin crosslink formation / actin filament-based movement / structural constituent of postsynaptic actin cytoskeleton / profilin binding / protein localization to bicellular tight junction / dense body / vocalization behavior / Formation of annular gap junctions / Gap junction degradation / Cell-extracellular matrix interactions / actomyosin / myosin filament / RHO GTPases Activate ROCKs / hyperosmotic response / Adherens junctions interactions / regulation of stress fiber assembly / RHO GTPases activate CIT / actomyosin structure organization / Sema4D induced cell migration and growth-cone collapse / Sensory processing of sound by outer hair cells of the cochlea / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / regulation of synaptic vesicle endocytosis / apical junction complex / regulation of focal adhesion assembly / myosin II complex / cortical actin cytoskeleton / microfilament motor activity / positive regulation of wound healing / maintenance of blood-brain barrier / myofibril / sarcomere organization / NuA4 histone acetyltransferase complex / EPHA-mediated growth cone collapse / cell leading edge / pseudopodium / Recycling pathway of L1 / filamentous actin / RHO GTPases activate PAKs / brush border / calyx of Held / actin filament bundle assembly / skeletal muscle contraction / mitotic cytokinesis / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / neuronal action potential / RHO GTPases activate IQGAPs / RHOBTB2 GTPase cycle / phagocytosis / stress fiber / skeletal muscle tissue development / RHO GTPases activate PKNs / EPHB-mediated forward signaling / phagocytic vesicle / cellular response to starvation / mitochondrion organization / axonogenesis / extracellular matrix / cell projection / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / cell motility / actin filament / FCGR3A-mediated phagocytosis / sensory perception of sound / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Signaling by high-kinase activity BRAF mutants / Schaffer collateral - CA1 synapse / MAP2K and MAPK activation / structural constituent of cytoskeleton / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / VEGFA-VEGFR2 Pathway / cellular response to type II interferon / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by BRAF and RAF1 fusions / cell-cell junction / actin filament binding / cell junction / Clathrin-mediated endocytosis / protein-macromolecule adaptor activity / cell cortex Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
![]() | von der Ecken, J. / Heissler, S.M. / Pathan-Chhatbar, S. / Manstein, D.J. / Raunser, S. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution. Abstract: The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated ...The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated during a complex mechanochemical reaction cycle. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins, a high-resolution structure of the F-actin–myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release) states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 735.5 KB | Display | ![]() |
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PDB format | ![]() | 588.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 101.4 KB | Display | |
Data in CIF | ![]() | 155.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8164MC ![]() 8165MC ![]() 8162C ![]() 8163C ![]() 5jlfC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Assembly
Deposited unit | ![]()
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS ensembles :
NCS oper:
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Components
#1: Protein | Mass: 41707.570 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Protein | Mass: 117570.633 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: NON MUSCLE MYOSIN 2C HEAVY CHAIN, MOTOR DOMAIN RESIDUES 1-799 LINKED TO ALPHA-ACTININ 3, REPEATS 1 AND 2 RESIDUES 800-1039 FRAGMENT: UNP Q7Z406-1 RESIDUES 1-799, UNP P05095 RESIDUES 265-502 Source: (gene. exp.) ![]() ![]() ![]() Gene: MYH14, KIAA2034, FP17425, abpA, actnA, DDB_G0268632 / Production host: ![]() ![]() #3: Protein | Mass: 11507.176 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Details: Human TROPOMYSIN WAS USED (UNP P06753-2,TPM3_HUMAN, RESIDUES 62-196). DUE TO THE LIMITED RESOLUTION OF THE CRYO-EM DENSITY IN THE REGION OF TROPOMYOSIN, TROPOMYOSIN HAS BEEN REPRESENTED AS POLY(UNK). Source: (gene. exp.) ![]() ![]() ![]() #4: Chemical | ChemComp-ADP / #5: Chemical | ChemComp-MG / Sequence details | Human TROPOMYSIN WAS USED (UNP P06753-2,TPM3_HUMAN, RESIDUES 62-196) DUE TO THE LIMITED RESOLUTION ...Human TROPOMYSIN | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component |
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Molecular weight | Value: 0.44 MDa / Experimental value: NO | ||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 Details: 5 mM Tris-HCl pH 7.5, 1 mM DTT, 100 mM KCl, and 2 mM MgCl2 | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1 | ||||||||||||||||||||||||||||
EM embedding | Material: I | ||||||||||||||||||||||||||||
Vitrification | Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % Details: Sample (2 uL of F-actin-tropomyosin solution) was applied to a glow-discharged holey carbon grid, incubated for 20 s and manually blotted from the backside for less than a second with filter ...Details: Sample (2 uL of F-actin-tropomyosin solution) was applied to a glow-discharged holey carbon grid, incubated for 20 s and manually blotted from the backside for less than a second with filter paper. Afterwards 1.5 uL of myosin solution (3 uM without nucleotide) were added directly on the grid, incubated for 10 s and then manually blotted for 5 s from the backside with filter paper. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Cs corrected microscope |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated defocus min: 700 nm / Calibrated defocus max: 2800 nm |
Image recording | Average exposure time: 0.475 sec. / Electron dose: 16 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 6300 |
Image scans | Movie frames/image: 8 / Used frames/image: 2-8 |
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Processing
Software | Name: REFMAC / Version: 5.8.0088 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 166.9 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 138000 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118000 Details: THE TROPOMYOSIN MAP FILTERED TO 7.0 ANGSTROM WAS MERGED WITH THE FINAL F-ACTIN-MYOSIN MAP (3.9 ANGSTROM) TO OBTAIN A MAP OF THE ENTIRE F-ACTIN-MYOSIN-TROPOMYOSIN COMPLEX. Symmetry type: HELICAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Atomic model building | Source name: PDB / Type: experimental model
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Refinement | Resolution: 3.9→211.2 Å / Cor.coef. Fo:Fc: 0.907 / SU B: 27.204 / SU ML: 0.36 / ESU R: 0.558 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS MERGED WITH THE FINAL F-ACTIN-MUYOSIN MAP (3.9 ANGSTROM) TO OBTAIN MAP OF THE ENTIRE F-ACTIN TROPOMYOSIN COMPLEX.
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 180.474 Å2
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Refinement step | Cycle: 1 / Total: 26477 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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