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- PDB-9duv: Cryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilize... -

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Basic information

Entry
Database: PDB / ID: 9duv
TitleCryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilized F-actin
Components
  • Actin, alpha skeletal muscle
  • phalloidin
KeywordsSTRUCTURAL PROTEIN / Actin / cardiomyopathy / contractility / muscle / sarcomere
Function / homology
Function and homology information


Formation of the dystrophin-glycoprotein complex (DGC) / Striated Muscle Contraction / myosin binding / mesenchyme migration / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber / muscle contraction / sarcomere ...Formation of the dystrophin-glycoprotein complex (DGC) / Striated Muscle Contraction / myosin binding / mesenchyme migration / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber / muscle contraction / sarcomere / filopodium / actin filament / ADP binding / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / lamellipodium / actin cytoskeleton / cell body / blood microparticle / hydrolase activity / positive regulation of gene expression / extracellular space / extracellular exosome / ATP binding / cytosol
Similarity search - Function
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
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGarg, A. / Greenberg, M.J. / Zhang, R.
Funding support United States, France, 16items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL007081 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL007227 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL173569 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138448 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138854 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL141086 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL141086 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL138466 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL139714 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL151078 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL161185 United States
Leducq Foundation20CVD02 France
Burroughs Wellcome Fund1014782 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalCH-II-2015-462 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalCH-II-2017-628 United States
Childrens Discovery Institute of Washington University and St. Louis Childrens HospitalPM-LI-2019-829 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility.
Authors: Ankit Garg / Silvia Jansen / Lina Greenberg / Rui Zhang / Kory J Lavine / Michael J Greenberg /
Abstract: Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with ...Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 variant, R256H. We previously identified this variant in a family with dilated cardiomyopathy, who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using cryoelectron microscopy, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that human-induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric organization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.
History
DepositionOct 4, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _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 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
M: phalloidin
N: phalloidin
O: phalloidin
P: phalloidin
Q: phalloidin
R: phalloidin
S: phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,51125
Polymers256,80213
Non-polymers2,70912
Water00
1


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

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Components

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


Mass: 41856.582 Da / Num. of mol.: 6 / Mutation: R254H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTA1, ACTA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P68133, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein/peptide
phalloidin


Mass: 808.899 Da / Num. of mol.: 7 / Source method: obtained synthetically / Source: (synth.) Amanita phalloides (death cap)
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilized F-actinCOMPLEX#1-#20MULTIPLE SOURCES
2R256H ACTA1 F-actinCOMPLEX#11RECOMBINANT
3phalloidinCOMPLEX#21SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Amanita phalloides (death cap)67723
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Details: 25 mM KCl, 2 mM EGTA, 60 mM MOPS (pH7), 1 mM DTT, and 4 mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMpotassium chlorideKCl1
22 mMEGTAC14H24N2O101
360 mMMOPSC7H15NO4S1
41 mMDTTC4H10O2S21
54 mMmagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11.63 sec. / Electron dose: 55.3 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 2593

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.4particle selection
2EPUimage acquisition
4cryoSPARC3.4CTF correction
7Coot0.9.8.92model fitting
9cryoSPARC3.4initial Euler assignment
10cryoSPARC3.4final Euler assignment
11cryoSPARC3.4classification
12cryoSPARC3.43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -167.181 ° / Axial rise/subunit: 27.557 Å / Axial symmetry: C1
Particle selectionDetails: Actin filaments were automatically picked from the micrographs using template based 'Filament tracer' in CryoSPARC
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 810328 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
Atomic model buildingPDB-ID: 6T1Y

6t1y


Accession code: 6T1Y / Source name: PDB / Type: experimental model

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