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- EMDB-27331: The structure of the native cardiac thin filament junction region -

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Basic information

Entry
Database: EMDB / ID: EMD-27331
TitleThe structure of the native cardiac thin filament junction region
Map dataEven half-map
Sample
  • Complex: Junction region of the native cardiac thin filament
    • Protein or peptide: Actin, alpha cardiac muscle 1
    • Protein or peptide: Tropomyosin alpha-1 chain
    • Protein or peptide: Troponin T2, cardiac type
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


RHOB GTPase cycle / Striated Muscle Contraction / RHOA GTPase cycle / troponin C binding / cardiac Troponin complex / cardiac myofibril / actin-myosin filament sliding / troponin complex / regulation of muscle contraction / muscle filament sliding ...RHOB GTPase cycle / Striated Muscle Contraction / RHOA GTPase cycle / troponin C binding / cardiac Troponin complex / cardiac myofibril / actin-myosin filament sliding / troponin complex / regulation of muscle contraction / muscle filament sliding / negative regulation of ATP-dependent activity / positive regulation of ATP-dependent activity / regulation of heart contraction / sarcomere organization / ventricular cardiac muscle tissue morphogenesis / myosin binding / tropomyosin binding / heart contraction / mesenchyme migration / troponin I binding / cardiac muscle contraction / sarcomere / muscle contraction / filopodium / actin filament organization / actin filament / response to calcium ion / actin filament binding / actin cytoskeleton / lamellipodium / cell body / actin binding / protein heterodimerization activity / positive regulation of gene expression / protein homodimerization activity / identical protein binding / cytoplasm
Similarity search - Function
Troponin T / Tropomyosins signature. / Tropomyosin / Troponin / Troponin domain superfamily / Tropomyosin / Troponin / Actins signature 1. / Actin, conserved site / Actins signature 2. ...Troponin T / Tropomyosins signature. / Tropomyosin / Troponin / Troponin domain superfamily / Tropomyosin / Troponin / 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
Similarity search - Domain/homology
Actin, alpha cardiac muscle 1 / Troponin T2, cardiac type / Tropomyosin alpha-1 chain
Similarity search - Component
Biological speciesSus scrofa (pig) / pig (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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)HL160966 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM116790 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL140925 United States
CitationJournal: PNAS Nexus / Year: 2023
Title: High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.
Authors: Cristina M Risi / Betty Belknap / Howard D White / Kelly Dryden / Jose R Pinto / P Bryant Chase / Vitold E Galkin /
Abstract: Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca levels. Cardiac thin filament (cTF) consists of two strands ...Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at (pCa 8) Ca conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.
History
DepositionJun 17, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27331.png

Downloads & links

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Map

FileDownload / File: emd_27331.map.gz / Format: CCP4 / Size: 3.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEven half-map
Voxel sizeX=Y=Z: 1.356 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-2.8214474 - 10.090697
Average (Standard dev.)-1.23495e-05 (±0.9999923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions8072166
Spacing7280166
CellA: 97.631996 Å / B: 108.479996 Å / C: 225.096 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Even half-map

Fileemd_27331_half_map_1.map
AnnotationEven half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half-map

Fileemd_27331_half_map_2.map
AnnotationEven half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Junction region of the native cardiac thin filament

EntireName: Junction region of the native cardiac thin filament
Components
  • Complex: Junction region of the native cardiac thin filament
    • Protein or peptide: Actin, alpha cardiac muscle 1
    • Protein or peptide: Tropomyosin alpha-1 chain
    • Protein or peptide: Troponin T2, cardiac type
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Junction region of the native cardiac thin filament

SupramoleculeName: Junction region of the native cardiac thin filament / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Sus scrofa (pig) / Organ: heart

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Macromolecule #1: Actin, alpha cardiac muscle 1

MacromoleculeName: Actin, alpha cardiac muscle 1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: pig (pig) / Organ: heart
Molecular weightTheoretical: 42.064891 KDa
SequenceString: MCDDEETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDDEETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVLSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWISKQEYDE AGPSIVHRKC F

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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: pig (pig) / Organ: heart
Molecular weightTheoretical: 32.762656 KDa
SequenceString: MDAIKKKMQM LKLDKENALD RAEQAEADKK AAEDRSKRLE DELVSLQKKL KATEDELDKY SEAPKDAQEK LELAEKKATD AEADVASLN RRIQLVEEEL DRAQERLATA LQKLEEAEKA ADESERGMKV IESRAQKDEE KMEIQEIQLK EAKHIAEDAD R KYEEVARK ...String:
MDAIKKKMQM LKLDKENALD RAEQAEADKK AAEDRSKRLE DELVSLQKKL KATEDELDKY SEAPKDAQEK LELAEKKATD AEADVASLN RRIQLVEEEL DRAQERLATA LQKLEEAEKA ADESERGMKV IESRAQKDEE KMEIQEIQLK EAKHIAEDAD R KYEEVARK LVIIESDLER AEERAELSEG KCAELEEELK TVTNNLKSLE AQAEKYSQKE DKYEEEIKVL SDKLKEAETR AE FAERSVT KLEKSIDDLE DELYAQKLKY KAISEELDHA LNDMTSI

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Macromolecule #3: Troponin T2, cardiac type

MacromoleculeName: Troponin T2, cardiac type / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: pig (pig) / Organ: heart
Molecular weightTheoretical: 35.158918 KDa
SequenceString: MSDVEETVDE YEEEQEEGAA EEEEAWRQDG DEQEEAVEEE AGGEAEAEEA NAEEAGQEED GREAEDGPME ESKPKPRLFM PNLVPPKIP DGERVDFDDI HRKRMKDLNE LQTLIEAHFE NRKKEEELVS LKDRIEKRRA ERAEQQRIRT EREKERQTRL A EERARREE ...String:
MSDVEETVDE YEEEQEEGAA EEEEAWRQDG DEQEEAVEEE AGGEAEAEEA NAEEAGQEED GREAEDGPME ESKPKPRLFM PNLVPPKIP DGERVDFDDI HRKRMKDLNE LQTLIEAHFE NRKKEEELVS LKDRIEKRRA ERAEQQRIRT EREKERQTRL A EERARREE EENRRKAEDE ARKKKALSNM MHFGGYIQKQ AQTERKSGKR QTEREKKKKI LAERRKVLAI DHLNEDQLRE KA KELWQSI YNLEAEKFDL QEKFKQQKYE INVLRNRIND NQKVSKTRGK AKVTGRWK

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 5 / Number real images: 24133 / Average electron dose: 34.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7379204
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7ko4


Details: Filtered to 40A resolution
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.08)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.08)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.08)
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Details: Filtered to 3.8 A resolution / Number images used: 236359
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ko4

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8dd0:
The structure of the native cardiac thin filament junction region

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