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- PDB-9mou: Structure of native murine cardiac thin filament variant I79N in ... -

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Basic information

Entry
Database: PDB / ID: 9mou
TitleStructure of native murine cardiac thin filament variant I79N in troponin T at pCa=5.8 in Ca2+-bound partially activated state (upper strand)
Components
  • Actin, alpha cardiac muscle 1
  • Isoform 6 of Troponin T, cardiac muscle
  • Tropomyosin alpha-1 chain
  • Troponin C, slow skeletal and cardiac muscles
  • Troponin I, cardiac muscle
KeywordsMOTOR PROTEIN / thin filament / cryo-EM / troponin / tropomyosin / muscle structure
Function / homology
Function and homology information


positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / RHOB GTPase cycle / regulation of systemic arterial blood pressure by ischemic conditions / Formation of the dystrophin-glycoprotein complex (DGC) / Striated Muscle Contraction / troponin C binding / cytoplasmic actin-based contraction involved in cell motility / RHOA GTPase cycle / diaphragm contraction ...positive regulation of heart rate by epinephrine / muscle thin filament tropomyosin / RHOB GTPase cycle / regulation of systemic arterial blood pressure by ischemic conditions / Formation of the dystrophin-glycoprotein complex (DGC) / Striated Muscle Contraction / troponin C binding / cytoplasmic actin-based contraction involved in cell motility / RHOA GTPase cycle / diaphragm contraction / regulation of ATP-dependent activity / regulation of muscle filament sliding speed / troponin T binding / cardiac Troponin complex / cardiac myofibril / Smooth Muscle Contraction / troponin complex / actin-myosin filament sliding / cardiac myofibril assembly / regulation of muscle contraction / regulation of smooth muscle contraction / negative regulation of ATP-dependent activity / transition between fast and slow fiber / positive regulation of ATP-dependent activity / Ion homeostasis / cardiac muscle tissue morphogenesis / Striated Muscle Contraction / actomyosin structure organization / muscle filament sliding / I band / response to metal ion / regulation of cardiac muscle contraction by calcium ion signaling / ventricular cardiac muscle tissue morphogenesis / microfilament motor activity / tropomyosin binding / myosin binding / regulation of heart contraction / troponin I binding / myofibril / mesenchyme migration / striated muscle thin filament / skeletal muscle thin filament assembly / vasculogenesis / striated muscle contraction / calcium channel inhibitor activity / cardiac muscle contraction / sarcomere / filopodium / actin filament / response to calcium ion / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / intracellular calcium ion homeostasis / calcium-dependent protein binding / actin filament binding / lamellipodium / actin cytoskeleton / heart development / actin binding / cell body / response to ethanol / in utero embryonic development / hydrolase activity / response to xenobiotic stimulus / protein heterodimerization activity / protein domain specific binding / calcium ion binding / synapse / positive regulation of gene expression / protein kinase binding / negative regulation of apoptotic process / glutamatergic synapse / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Troponin T / Troponin I residues 1-32 / Troponin I residues 1-32 / : / Troponin / Troponin domain superfamily / Troponin / Tropomyosins signature. / Tropomyosin / Tropomyosin ...Troponin T / Troponin I residues 1-32 / Troponin I residues 1-32 / : / Troponin / Troponin domain superfamily / Troponin / Tropomyosins signature. / Tropomyosin / Tropomyosin / EF-hand domain pair / : / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / ATPase, nucleotide binding domain / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Troponin C, slow skeletal and cardiac muscles / Troponin T, cardiac muscle / Troponin I, cardiac muscle / Tropomyosin alpha-1 chain / Actin, alpha cardiac muscle 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsRisi, C.M. / Galkin, V.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL160966 United States
CitationJournal: J Mol Cell Cardiol / Year: 2025
Title: The role of the troponin T interactions with actin in regulation of cardiac thin filament revealed by the troponin T pathogenic variant Ile79Asn.
Authors: Cristina M Risi / Maicon Landim-Vieira / Betty Belknap / P Bryant Chase / Jose R Pinto / Vitold E Galkin /
Abstract: Cardiac muscle contraction/relaxation cycle depends on the rising and falling Ca levels in sarcomeres that control the extent of interactions between myosin-based thick and actin-based thin filaments. ...Cardiac muscle contraction/relaxation cycle depends on the rising and falling Ca levels in sarcomeres that control the extent of interactions between myosin-based thick and actin-based thin filaments. Cardiac thin filament (cTF) consists of actin, tropomyosin (Tm) that regulates myosin binding to actin, and troponin complex that governs Tm position upon Ca-binding. Troponin has three subunits - Ca-binding troponin C (TnC), Tm stabilizing troponin T (TnT), and inhibitory troponin I (TnI). TnT N-terminus (TnT1) interactions with actin stabilize the inhibited state of cTF. TnC, TnI, and Tm work in concert to control actomyosin interactions. Cryo-electron microscopy (cryo-EM) provided factual structures of healthy cTF, but structures of cTF carrying missense mutations linked to human cardiomyopathy are unknown. Variant Ile79Asn in human cardiac TnT (TnT-I79N) increases myofilament Ca sensitivity and slows cross-bridge kinetics, leading to severe hypertrophic/restrictive cardiomyopathy. Here, we used TnT-I79N mutation as a tool to examine the role of TnT1 in the complex mechanism of cTF regulation. Comparison of the cryo-EM structures of murine wild type and TnT-I79N native cTFs at systolic Ca levels (pCa = 5.8) demonstrates that TnT-I79N causes 1) dissociation of the TnT1 loop from its actin interface that results in Tm release to a more activated position, 2) reduced interaction of TnI C-terminus with actin-Tm, and 3) increased frequency of Ca-bound regulatory units. Our data indicate that the TnT1 loop is a crucial element of the allosteric regulatory network that couples Tn subunits and Tm to maintain adequate cTF response to physiological Ca levels during a heartbeat.
History
DepositionDec 27, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 4, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 2, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, alpha cardiac muscle 1
B: Actin, alpha cardiac muscle 1
C: Actin, alpha cardiac muscle 1
D: Actin, alpha cardiac muscle 1
E: Actin, alpha cardiac muscle 1
F: Actin, alpha cardiac muscle 1
G: Actin, alpha cardiac muscle 1
H: Troponin C, slow skeletal and cardiac muscles
I: Troponin I, cardiac muscle
J: Isoform 6 of Troponin T, cardiac muscle
K: Isoform 6 of Troponin T, cardiac muscle
L: Tropomyosin alpha-1 chain
M: Tropomyosin alpha-1 chain
N: Tropomyosin alpha-1 chain
O: Tropomyosin alpha-1 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)540,74532
Polymers537,46415
Non-polymers3,28117
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 15 molecules ABCDEFGHIJKLMNO

#1: Protein
Actin, alpha cardiac muscle 1 / Alpha-cardiac actin


Mass: 42064.891 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
References: UniProt: P68033, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein Troponin C, slow skeletal and cardiac muscles / TN-C


Mass: 18437.520 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / References: UniProt: P19123
#3: Protein Troponin I, cardiac muscle / Cardiac troponin I


Mass: 24308.908 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / References: UniProt: P48787
#4: Protein Isoform 6 of Troponin T, cardiac muscle / TnTc / Cardiac muscle troponin T / cTnT


Mass: 34660.332 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / References: UniProt: P45379
#5: Protein
Tropomyosin alpha-1 chain / Alpha-tropomyosin / Tropomyosin-1


Mass: 32735.609 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / References: UniProt: P58771

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Non-polymers , 3 types, 17 molecules

#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestN
Has protein modificationN

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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

ComponentName: Native murine cardiac thin filament variant I79N in troponin T at pCa=5.8
Type: COMPLEX / Entity ID: #1-#5 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 34 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 17361

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7PHENIXmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7615631
3D reconstructionResolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58913 / Algorithm: BACK PROJECTION / Details: filtered to 7 Angstroms / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17KO5

7ko5

17KO51PDBexperimental model
27UTI

7uti

17UTI2PDBexperimental model
38UZY

8uzy

18UZY3PDBexperimental model
41AF-P50752-F1-v44AlphaFoldin silico model

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