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- EMDB-22978: Structure of upper Tn Ca2+ free (rotated) and lower Tn Ca2+ bound... -

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Basic information

Entry
Database: EMDB / ID: EMD-22978
TitleStructure of upper Tn Ca2+ free (rotated) and lower Tn Ca2+ bound cardiac native thin filament at pCa=5.8
Map dataStructure of Ca2 free / Ca2 bound cardiac native thin filament at pCa=5.8 mode 1
Sample
  • Complex: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament at pCa=5.8 mode 2
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Tropomyosin alpha-1 chain
    • Protein or peptide: Troponin T, cardiac muscle
    • Protein or peptide: Troponin I, cardiac muscle
    • Protein or peptide: Troponin C
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsGalkin VE / Risi CM
Funding support United States, 3 items
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, 2020-
Header (metadata) releaseMar 24, 2021-
Map releaseMar 24, 2021-
UpdateMar 31, 2021-
Current statusMar 31, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kon
  • Surface level: 0.031
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22978.png

Downloads & links

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Map

FileDownload / File: emd_22978.map.gz / Format: CCP4 / Size: 16.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Ca2 free / Ca2 bound cardiac native thin filament at pCa=5.8 mode 1
Voxel sizeX=Y=Z: 2.712 Å
Density
Contour LevelBy AUTHOR: 0.031 / Movie #1: 0.031
Minimum - Maximum-0.026170654 - 0.11553131
Average (Standard dev.)0.0006107877 (±0.007851753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions162162162
Spacing162162162
CellA=B=C: 439.344 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.7122.7122.712
M x/y/z162162162
origin x/y/z0.0000.0000.000
length x/y/z439.344439.344439.344
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS162162162
D min/max/mean-0.0260.1160.001

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Supplemental data

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Sample components

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Entire : Structure of Ca2+ free / Ca2+ bound cardiac native thin filament ...

EntireName: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament at pCa=5.8 mode 2
Components
  • Complex: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament at pCa=5.8 mode 2
    • Protein or peptide: Actin, alpha skeletal muscle
    • Protein or peptide: Tropomyosin alpha-1 chain
    • Protein or peptide: Troponin T, cardiac muscle
    • Protein or peptide: Troponin I, cardiac muscle
    • Protein or peptide: Troponin C

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Supramolecule #1: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament ...

SupramoleculeName: Structure of Ca2+ free / Ca2+ bound cardiac native thin filament at pCa=5.8 mode 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 41.862613 KDa
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIITNWD DMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSGDGV T HNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

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Macromolecule #2: Tropomyosin alpha-1 chain

MacromoleculeName: Tropomyosin alpha-1 chain / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 32.921773 KDa
SequenceString: ASMDAIKKKM QMLKLDKENA LDRAEQAEAD KKAAEDRSKQ LEDELVSLQK KLKGTEDELD KYSEALKDAQ EKLELAEKKA TDAEADVAS LNRRIQLVEE ELDRAQERLA TALQKLEEAE KAADESERGM KVIESRAQKD EEKMEIQEIQ LKEAKHIAED A DRKYEEVA ...String:
ASMDAIKKKM QMLKLDKENA LDRAEQAEAD KKAAEDRSKQ LEDELVSLQK KLKGTEDELD KYSEALKDAQ EKLELAEKKA TDAEADVAS LNRRIQLVEE ELDRAQERLA TALQKLEEAE KAADESERGM KVIESRAQKD EEKMEIQEIQ LKEAKHIAED A DRKYEEVA RKLVIIESDL ERAEERAELS EGKCAELEEE LKTVTNNLKS LEAQAEKYSQ KEDRYEEEIK VLSDKLKEAE TR AEFAERS VTKLEKSIDD LEDELYAQKL KYKAISEELD HALNDMTSI

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Macromolecule #3: Troponin T, cardiac muscle

MacromoleculeName: Troponin T, cardiac muscle / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 23.023039 KDa
SequenceString:
FDDIHRKRME KDLNELQALI EAHFENRKKE EEELVSLKDR IERRRAERAE QQRIRNEREK ERQNRLAEER ARREEEENRR KAEDEARKK KALSNMMHFG GYIQKQAQTE RKSGKRQTER EKKKKILAER RKVLAIDHLN EDQLREKAKE LWQSIYNLEA E KFDLQEKF KQQKYEINVL RNRINDNQ

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Macromolecule #4: Troponin I, cardiac muscle

MacromoleculeName: Troponin I, cardiac muscle / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 19.639691 KDa
SequenceString:
ISASRKLQLK TLLLQIAKQE LEREAEERRG EKGRALSTRC QPLELAGLGF AELQDLCRQL HARVDKVDEE RYDIEAKVTK NITEIADLT QKIFDLRGKF KRPTLRRVRI SADAMMQALL GARAKESLDL RAHLKQVKKE DTEKENREVG DWRKNIDALS G MEGRKKKF ES

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Macromolecule #5: Troponin C

MacromoleculeName: Troponin C / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 18.288287 KDa
SequenceString:
DDIYKAAVEQ LTEEQKNEFK AAFDIFVLGA EDGCISTKEL GKVMRMLGQN PTPEELQEMI DEVDEDGSGT VDFDEFLVMM VRCMKDDSK GKSEEELSDL FRMFDKNADG YIDLDELKIM LQATGETITE DDIEELMKDG DKNNDGRIDY DEFLEFMKGV E

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Detailsnon-helical filament

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 34.0 e/Å2
Details: Images were collected in movie-mode - 40 subframes at 0.85e/A^2

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.08)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6kn7


Details: filtered to 100A resolution
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.08)
Final 3D classificationSoftware - Name: RELION (ver. 3.08)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.08)
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Details: The map was filtered to 11A / Number images used: 10593
Detailsimages with energy filter enabled at super-resolution mode
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6kn7

,

6kn8

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7kon:
Structure of upper Tn Ca2+ free (rotated) and lower Tn Ca2+ bound cardiac native thin filament at pCa=5.8

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