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- PDB-7kon: Structure of upper Tn Ca2+ free (rotated) and lower Tn Ca2+ bound... -

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Basic information

Entry
Database: PDB / ID: 7kon
TitleStructure of upper Tn Ca2+ free (rotated) and lower Tn Ca2+ bound cardiac native thin filament at pCa=5.8
Components
  • Actin, alpha skeletal muscle
  • Tropomyosin alpha-1 chain
  • Troponin C
  • Troponin I, cardiac muscle
  • Troponin T, cardiac muscle
KeywordsCONTRACTILE PROTEIN / actin / tropomyosin / troponin / TF
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsGalkin, V.E. / Risi, C.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL140925 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116790 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116788 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: The structure of the native cardiac thin filament at systolic Ca levels.
Authors: Cristina M Risi / Ian Pepper / Betty Belknap / Maicon Landim-Vieira / Howard D White / Kelly Dryden / Jose R Pinto / P Bryant Chase / Vitold E Galkin /
Abstract: Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca levels. Thin filaments are comprised of two actin strands, each ...Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca levels revealed the transition between the Ca-free and Ca-bound states. However, in working cardiac muscle, Ca levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca-free, Ca-bound, or mixed (e.g., Ca free on one side and Ca bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca fluctuations.
History
DepositionNov 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 24, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 31, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-22978
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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: Actin, alpha skeletal muscle
G: Actin, alpha skeletal muscle
H: Actin, alpha skeletal muscle
I: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
M: Actin, alpha skeletal muscle
N: Actin, alpha skeletal muscle
O: Actin, alpha skeletal muscle
P: Tropomyosin alpha-1 chain
Q: Tropomyosin alpha-1 chain
R: Tropomyosin alpha-1 chain
S: Tropomyosin alpha-1 chain
T: Troponin T, cardiac muscle
U: Troponin I, cardiac muscle
V: Troponin C
W: Tropomyosin alpha-1 chain
X: Tropomyosin alpha-1 chain
Y: Tropomyosin alpha-1 chain
Z: Tropomyosin alpha-1 chain
a: Troponin T, cardiac muscle
b: Troponin I, cardiac muscle
c: Troponin C


Theoretical massNumber of molelcules
Total (without water)1,013,21529
Polymers1,013,21529
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area110360 Å2
ΔGint-741 kcal/mol
Surface area317910 Å2

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Components

#1: Protein
Actin, alpha skeletal muscle /


Mass: 41862.613 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
#2: Protein
Tropomyosin alpha-1 chain


Mass: 32921.773 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
#3: Protein Troponin T, cardiac muscle /


Mass: 23023.039 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
#4: Protein Troponin I, cardiac muscle /


Mass: 19639.691 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
#5: Protein Troponin C /


Mass: 18288.287 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig)
Sequence detailsThe resolution within the thin filament is not enough to rebuild the model using the porcine ...The resolution within the thin filament is not enough to rebuild the model using the porcine sequence, so previously determined structures of equivalent proteins from different organisms were fit to the map

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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: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament at pCa=5.8 mode 2
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: non-helical filament
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
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)
Details: Images were collected in movie-mode - 40 subframes at 0.85e/A^2

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
4RELION3.08CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3.08initial Euler assignment
11RELION3.08final Euler assignment
12RELION3.08classification
13RELION3.083D reconstruction
Image processingDetails: images with energy filter enabled at super-resolution mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10593 / Algorithm: BACK PROJECTION / Details: The map was filtered to 11A / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16KN7

6kn7

1
26KN8

6kn8

1

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