+Open data
-Basic information
Entry | Database: PDB / ID: 5jlf | |||||||||
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Title | Structure of the F-actin-tropomyosin complex (Reprocessed) | |||||||||
Components |
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Keywords | CONTRACTILE PROTEIN / CONTRACTILE FILAMENT / MUSCLE / THIN FILAMENT / CYTOSKELETON / STRUCTURAL PROTEIN / HYDROLASE COMPLEX / F-actin / tropomyosin / filament / protein polymers / cryo EM | |||||||||
Function / homology | Function and homology information cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly ...cytoskeletal motor activator activity / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / 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 | |||||||||
Biological species | Mus musculus (house mouse) Oryctolagus cuniculus (rabbit) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | von der Ecken, J. / Raunser, S. | |||||||||
Funding support | Germany, 2items
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Citation | Journal: Nature / Year: 2016 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. #1: Journal: Nature / Year: 2015 Title: Structure of the F-actin-tropomyosin complex. Authors: Julian von der Ecken / Mirco Müller / William Lehman / Dietmar J Manstein / Pawel A Penczek / Stefan Raunser / Abstract: Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead ...Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5jlf.cif.gz | 390.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5jlf.ent.gz | 323.9 KB | Display | PDB format |
PDBx/mmJSON format | 5jlf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5jlf_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 5jlf_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 5jlf_validation.xml.gz | 58.8 KB | Display | |
Data in CIF | 5jlf_validation.cif.gz | 87.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/jl/5jlf ftp://data.pdbj.org/pub/pdb/validation_reports/jl/5jlf | HTTPS FTP |
-Related structure data
Related structure data | 8162MC 8163MC 8164C 8165C 5jlhC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS oper:
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-Components
#1: Protein | Mass: 41875.633 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135 #2: Protein | Mass: 11507.176 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: MOUSE TROPOMYSIN WAS USED (UNP P58771, RESIDUES 97-231). 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.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli) #3: Chemical | ChemComp-ADP / #4: Chemical | ChemComp-MG / Sequence details | MOUSE TROPOMYSIN WAS USED (UNP P58771, TPM1_MOUSE, RESIDUES 97-231). DUE TO THE LIMITED RESOLUTION ...MOUSE 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 |
-Sample preparation
Component |
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Molecular weight | Value: 0.2 MDa / Experimental value: NO | ||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||||||
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 | ||||||||||||||||||||||||||||
Vitrification | Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % Details: Sample was applied to a glow-discharged holey carbon grid, incubated for 10 s and manually blotted for 3 s from the backside with filter paper. |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 2600 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) |
Image scans | Movie frames/image: 8 / Used frames/image: 2-8 |
-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: 119000 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91000 Details: THE TROPOMYOSIN MAP FILTERED TO 6.5 ANGSTROM WAS MERGED WITH THE FINAL F-ACTIN MAP (3.6 ANGSTROM) TO OBTAIN A MAP OF THE ENTIRE F-ACTIN-TROPOMYOSIN COMPLEX. Symmetry type: HELICAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Atomic model building | PDB-ID: 3J8A | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 3.6→193.6 Å / Cor.coef. Fo:Fc: 0.857 / SU B: 33.198 / SU ML: 0.463 / ESU R: 1.724 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS MERGED WITH THE FINAL F-ACTIN MAP (3.6 ANGSTROM) TO OBTAIN A 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: 97.923 Å2
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Refinement step | Cycle: 1 / Total: 14450 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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