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- PDB-5jlf: Structure of the F-actin-tropomyosin complex (Reprocessed) -

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Basic information

Entry
Database: PDB / ID: 5jlf
TitleStructure of the F-actin-tropomyosin complex (Reprocessed)
Components
  • Actin, alpha skeletal muscle
  • Tropomyosin Alpha-1
KeywordsCONTRACTILE 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 / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / actin filament bundle / filamentous actin / actin filament bundle assembly / skeletal muscle thin filament assembly / striated muscle thin filament / 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 / single particle reconstruction / cryo EM / Resolution: 3.6 Å
Authorsvon der Ecken, J. / Raunser, S.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
Behrens-Weise foundation Germany
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
DepositionApr 27, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 15, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2016Group: Database references
Revision 1.2Jul 13, 2016Group: Database references
Revision 1.3Aug 2, 2017Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Experimental preparation
Category: em_sample_support / em_software ...em_sample_support / em_software / pdbx_database_related / struct_conn
Item: _em_sample_support.grid_type / _em_software.name / _em_software.version
Revision 1.4Oct 17, 2018Group: Data collection / Database references ...Data collection / Database references / Other / Refinement description / Source and taxonomy / Structure summary
Category: cell / em_entity_assembly ...cell / em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / pdbx_database_related / refine / refine_hist / refine_ls_restr / refine_ls_restr_ncs / refine_ls_shell
Item: _cell.Z_PDB / _pdbx_database_related.content_type ..._cell.Z_PDB / _pdbx_database_related.content_type / _refine.pdbx_refine_id / _refine_hist.pdbx_refine_id / _refine_ls_restr.pdbx_refine_id / _refine_ls_restr_ncs.pdbx_refine_id / _refine_ls_shell.pdbx_refine_id
Revision 1.5Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.6Feb 12, 2020Group: Structure summary / Category: struct / Item: _struct.title

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Assembly

Deposited unit
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
F: Tropomyosin Alpha-1
G: Tropomyosin Alpha-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,65017
Polymers232,3937
Non-polymers2,25810
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D
51E

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A5 - 377
2111B5 - 377
3111C5 - 377
4111D5 - 377
5111E5 - 377

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(0.890287, -0.455397, 0.001282), (0.455394, 0.890287, 0.002208), (-0.002147, -0.001382, 0.999997)26.49284, -12.38326, 54.54871
3given(0.890178, 0.455605, -0.002526), (-0.455608, 0.89018, -0.000493), (0.002023, 0.00159, 0.999997)-17.81378, 23.05453, -54.59245
4given(-0.972489, 0.232903, -0.004542), (-0.232947, -0.972346, 0.016695), (-0.000528, 0.017293, 0.99985)66.68998, 105.3926, 26.41459
5given(-0.971725, -0.236111, -0.00152), (0.236056, -0.971605, 0.016131), (-0.005286, 0.015316, 0.999869)89.6367, 86.37128, -27.74693

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Components

#1: 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
#2: Protein Tropomyosin Alpha-1


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 / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Sequence detailsMOUSE TROPOMYSIN WAS USED (UNP P58771, TPM1_MOUSE, RESIDUES 97-231). DUE TO THE LIMITED RESOLUTION ...MOUSE TROPOMYSIN WAS USED (UNP P58771, TPM1_MOUSE, RESIDUES 97-231). DUE TO THE LIMITED RESOLUTION OF THE CRYO-EM DENSITY IN THE REGION O 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: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1F-actin-tropomyosin complexCOMPLEXFilament#1-#20MULTIPLE SOURCES
2F-actinActinCOMPLEX#11NATURAL
3tropomyosinCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Oryctolagus cuniculus (rabbit)9986
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 5 mM Tris-HCl pH 7.5, 1 mM DTT, 100 mM KCl, and 2 mM MgCl2
Buffer component
IDConc.FormulaBuffer-ID
15 mMTris-HClTris1
21 mMDTT1
3100 mMKCl1
42 mMMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: 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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 2600 nm
Image recordingAverage exposure time: 0.475 sec. / Electron dose: 16 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k)
Image scansMovie frames/image: 8 / Used frames/image: 2-8

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Processing

SoftwareName: REFMAC / Version: 5.8.0088 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1SPARXparticle selectionsxhelixboxer.py
2EPUimage acquisition
4CTFFIND4CTF correction
5RELION1.3CTF correction
8UCSF Chimeramodel fittingrigid-body fitting
9iMODFITmodel fittingflexible fitting
11Cootmodel refinementmanual building, refinement
12REFMACmodel refinementrefinement
13RELION1.3initial Euler assignment
14RELION1.3final Euler assignment
15RELION1.3classification2D classification
16SPARXclassificationsorting out of outliers within the same filament
17RELION1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 166.9 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 119000
3D reconstructionResolution: 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
IDB valueProtocolSpaceDetails
198BACKBONE TRACERECIPROCAL
2RIGID BODY FITtropomyosin fitting
Atomic model buildingPDB-ID: 3J8A

3j8a

RefinementResolution: 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.
RfactorNum. reflection% reflection
Rwork0.33314 --
obs0.33314 64792 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 97.923 Å2
Baniso -1Baniso -2Baniso -3
1-1.04 Å20.58 Å2-0.1 Å2
2--2.71 Å2-0.27 Å2
3----3.75 Å2
Refinement stepCycle: 1 / Total: 14450
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.01914765
ELECTRON MICROSCOPYr_bond_other_d0.0030.0213920
ELECTRON MICROSCOPYr_angle_refined_deg1.7391.97920050
ELECTRON MICROSCOPYr_angle_other_deg1.112332135
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.7551830
ELECTRON MICROSCOPYr_dihedral_angle_2_deg22.30324.16625
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.457152515
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.0581585
ELECTRON MICROSCOPYr_chiral_restr0.1130.22235
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.02116505
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023220
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it4.6189.3487335
ELECTRON MICROSCOPYr_mcbond_other4.6149.3487334
ELECTRON MICROSCOPYr_mcangle_it7.74814.0329160
ELECTRON MICROSCOPYr_mcangle_other7.74814.0329161
ELECTRON MICROSCOPYr_scbond_it5.7410.1777430
ELECTRON MICROSCOPYr_scbond_other5.7410.1777431
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.90214.91310891
ELECTRON MICROSCOPYr_long_range_B_refined18.46296.52259272
ELECTRON MICROSCOPYr_long_range_B_other18.46296.52259273
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Dom-ID: 1 / Ens-ID: 1 / Number: 5674 / Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.5

Auth asym-IDRms dev position (Å)
A10.23
B15.28
C15.97
D8.49
E8.24
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.558 4788 -
Rfree-0 -
obs--100 %

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