[English] 日本語
Yorodumi
- EMDB-47180: Cryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilize... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-47180
TitleCryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilized F-actin
Map dataB-factor sharpened map of R256H mutant actin filament
Sample
  • Complex: Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilized F-actin
    • Complex: R256H ACTA1 F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: phalloidin
      • Protein or peptide: phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin / cardiomyopathy / contractility / muscle / sarcomere / STRUCTURAL PROTEIN
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 / actin filament / filopodium / ADP binding / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / lamellipodium / 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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesHomo sapiens (human) / Amanita phalloides (death cap)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGarg A / Greenberg MJ / Zhang R
Funding support United States, France, 16 items
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, 2024-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Downloads & links

-
Map

FileDownload / File: emd_47180.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor sharpened map of R256H mutant actin filament
Voxel sizeX=Y=Z: 1.184 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-2.6892018 - 4.8925867
Average (Standard dev.)0.0032232595 (±0.10265043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 473.6 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Sample components

-
Entire : Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilize...

EntireName: Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilized F-actin
Components
  • Complex: Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilized F-actin
    • Complex: R256H ACTA1 F-actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: phalloidin
      • Protein or peptide: phalloidin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

-
Supramolecule #1: Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilize...

SupramoleculeName: Cryo-EM structure of recombinant R256H ACTA1 phalloidin-stabilized F-actin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

-
Supramolecule #2: R256H ACTA1 F-actin

SupramoleculeName: R256H ACTA1 F-actin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

-
Supramolecule #3: phalloidin

SupramoleculeName: phalloidin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Amanita phalloides (death cap) / Synthetically produced: Yes

-
Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.856582 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG ...String:
DEDETTALVC DNGSGLVKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GII TNW DDMEKIWHHT FYNELRVAPE EHPTLLTEAP LNPKANREKM TQIMFETFNV PAMYVAIQAV LSLYASGRTT GIVLDSG DG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSL E KSYELPDGQV ITIGNEHFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF

UniProtKB: Actin, alpha skeletal muscle

-
Macromolecule #2: phalloidin

MacromoleculeName: phalloidin / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Amanita phalloides (death cap)
Molecular weightTheoretical: 808.899 Da
SequenceString:
W(EEP)A(DTH)C(HYP)A

-
Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

-
Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

-
Experimental details

-
Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

-
Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
25.0 mMKClpotassium chloride
2.0 mMC14H24N2O10EGTA
60.0 mMC7H15NO4SMOPS
1.0 mMC4H10O2S2DTT
4.0 mMMgCl2magnesium chloride

Details: 25 mM KCl, 2 mM EGTA, 60 mM MOPS (pH7), 1 mM DTT, and 4 mM MgCl2
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

-
Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 2593 / Average exposure time: 11.63 sec. / Average electron dose: 55.3 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

-
Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.557 Å
Applied symmetry - Helical parameters - Δ&Phi: -167.181 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.4) / Number images used: 810328
Segment selectionSoftware - Name: cryoSPARC (ver. 3.4)
Details: Actin filaments were automatically picked from the micrographs using template based 'Filament tracer' in CryoSPARC
Startup modelType of model: OTHER / Details: 'Ab-Initio Reconstruction' in CryoSPARC
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.4)

-
Atomic model buiding 1

Initial modelPDB ID:

6t1y


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-9duv:
Cryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilized F-actin

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more