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- EMDB-47180: Cryo-EM structure of recombinant R254H ACTA1 phalloidin-stabilize... -

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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


skeletal muscle fiber adaptation / Formation of the dystrophin-glycoprotein complex (DGC) / cellular response to potassium ion / Striated Muscle Contraction / response to steroid hormone / myosin binding / mesenchyme migration / striated muscle thin filament / skeletal muscle thin filament assembly / response to mechanical stimulus ...skeletal muscle fiber adaptation / Formation of the dystrophin-glycoprotein complex (DGC) / cellular response to potassium ion / Striated Muscle Contraction / response to steroid hormone / myosin binding / mesenchyme migration / striated muscle thin filament / skeletal muscle thin filament assembly / response to mechanical stimulus / stress fiber / skeletal muscle fiber development / muscle contraction / sarcomere / filopodium / actin filament / ADP binding / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / 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
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

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Structure visualization

Supplemental images

emd_47180.png

Downloads & links

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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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.18 Å/pix.
x 400 pix.
= 473.6 Å		1.18 Å/pix.
x 400 pix.
= 473.6 Å		1.18 Å/pix.
x 400 pix.
= 473.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.184 Å
Density

Histogram

Histogram (log scale)
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 °

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Supplemental data

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Additional map: original map of R256H mutant actin filament

Fileemd_47180_additional_1.map
Annotationoriginal map of R256H mutant actin filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map2 of R256H mutant actin filament

Fileemd_47180_half_map_1.map
Annotationhalf map2 of R256H mutant actin filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map1 of R256H mutant actin filament

Fileemd_47180_half_map_2.map
Annotationhalf map1 of R256H mutant actin filament
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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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

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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

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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)

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Supramolecule #3: phalloidin

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

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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

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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

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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

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / 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
Processinghelical reconstruction
Aggregation statehelical array

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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

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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

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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)
FSC plot (resolution estimation)

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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

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