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- PDB-3j8a: Structure of the F-actin-tropomyosin complex -

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Basic information

Entry
Database: PDB / ID: 3j8a
TitleStructure of the F-actin-tropomyosin complex
Components
  • Actin, alpha skeletal muscle
  • tropomyosin alpha-1
KeywordsSTRUCTURAL PROTEIN/HYDROLASE / contractile filament / muscle / thin filament / cytoskeleton / structural protein-hydrolase complex
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
Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. ...Actin; Chain A, domain 2 / Actin; Chain A, domain 2 / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / 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 / Nucleotidyltransferase; domain 5 / Roll / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesMus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.7 Å
Authorsvon der Ecken, J. / Mueller, M. / Lehman, W. / Manstein, J.M. / Penczek, A.P. / Raunser, S.
CitationJournal: 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
DepositionOct 8, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 10, 2014Provider: repository / Type: Initial release
Revision 1.1Dec 17, 2014Group: Database references
Revision 1.2Mar 11, 2015Group: Database references
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Remark 700SHEET DETERMINATION METHOD: AUTHOR DETERMINED

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
F: tropomyosin alpha-1
G: tropomyosin alpha-1
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,65017
Polymers232,3937
Non-polymers2,25810
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 27.5 Å / Rotation per n subunits: -166.4 °)
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C
41chain D
51chain E

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA5 - 371
211chain BB5 - 371
311chain CC5 - 371
411chain DD5 - 371
511chain EE5 - 371
DetailsThe full filament can be generated using the provided helical parameters from a small repeating subunit comprising chain A and residues 145-184 of chains F and G.

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Components

#1: Protein tropomyosin alpha-1


Mass: 11507.176 Da / Num. of mol.: 2 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli) / Keywords: SEE REMARK 999
#2: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
Sequence detailsMOUSE TROPOMYSIN WAS USED (UNP P58771, RESIDUES 97-231). DUE TO THE LIMITED RESOLUTION OF THE CRYO- ...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).

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1F-actin-tropomyosin complexCOMPLEXFilament0
2F-actin (alpha)1
3tropomyosin (alpha)1
Buffer solutionName: 5 mM Tris-HCl, pH 7.5, 1 mM DTT, 100 mM KCl, 2 mM MgCl2
pH: 7.5
Details: 5 mM Tris-HCl, pH 7.5, 1 mM DTT, 100 mM KCl, 2 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: C-flats 2/1 copper 300 mesh, Protochips, glow-discharged
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temp: 106 K / Humidity: 90 %
Details: Sample was applied to grid, incubated for 10 seconds, and manually blotted for 3 seconds from the backside with filter paper before plunging into liquid ethane (GATAN CRYOPLUNGE 3)
Method: Sample was applied to grid, incubated for 10 seconds, and manually blotted for 3 seconds from the backside with filter paper.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 17, 2013 / Details: Cs-corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 0 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 14.6 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansNum. digital images: 1311
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Cootmodel fitting
2DireXmodel fitting
3PHENIXmodel fitting
4SPARX3D reconstruction
CTF correctionDetails: each micrograph
Helical symmertyAngular rotation/subunit: 166.4 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1 / Details: none
3D reconstructionMethod: helicon, cter / Resolution: 3.7 Å / Resolution method: FSC 0.5 CUT-OFF / Nominal pixel size: 1.14 Å / Actual pixel size: 1.12 Å
Details: The tropomyosin map filtered to 6.5 Angstrom was merged with the final F-actin map (3.7 Angstrom) to obtain a map of the entire F-actin tropomyosin complex. Coordinates must be shifted by (0. ...Details: The tropomyosin map filtered to 6.5 Angstrom was merged with the final F-actin map (3.7 Angstrom) to obtain a map of the entire F-actin tropomyosin complex. Coordinates must be shifted by (0.123 -0.381 0.273) to match coordinates to fibre diffraction standards (filament axis = z axis passing through x=0 and y=0).
Symmetry type: HELICAL
Atomic model building
IDB valueProtocolTarget criteriaDetailsSpace
155.4FLEXIBLE FITR-factorMETHOD--maximum likelihood, flexible fitting
2FLEXIBLE FITcross-correlationMETHOD--flexible fittingREAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14A7N

4a7n

A1
24A7N

4a7n

B1
34A7N

4a7n

C1
44A7N

4a7n

D1
54A7N

4a7n

E1
RefinementResolution: 3.7→95.2 Å / SU ML: 0.2 / σ(F): 100 / Phase error: 26.02 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.2708 29 0.01 %
Rwork0.2771 285222 -
obs0.2771 285251 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 129.39 Å2 / Biso mean: 50.7811 Å2 / Biso min: 0 Å2
Refinement stepCycle: LAST / Resolution: 3.7→95.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15660 0 140 0 15800
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00414760
ELECTRON MICROSCOPYf_angle_d1.09720040
ELECTRON MICROSCOPYf_chiral_restr0.042235
ELECTRON MICROSCOPYf_plane_restr0.0052560
ELECTRON MICROSCOPYf_dihedral_angle_d14.7375485
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A8809ELECTRON MICROSCOPY5.287TORSIONAL
12B8809ELECTRON MICROSCOPY5.287TORSIONAL
13C8809ELECTRON MICROSCOPY5.287TORSIONAL
14D8809ELECTRON MICROSCOPY5.287TORSIONAL
15E8809ELECTRON MICROSCOPY5.287TORSIONAL
LS refinement shellResolution: 3.7→95.2 Å / Total num. of bins used: 1
RfactorNum. reflection% reflection
Rfree0.2708 29 -
Rwork0.2771 285222 -
all-285251 -
obs--100 %

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