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- PDB-8v30: Smooth Muscle Gamma Actin (ACTG2) Filament Mutant R40C -

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Basic information

Entry
Database: PDB / ID: 8v30
TitleSmooth Muscle Gamma Actin (ACTG2) Filament Mutant R40C
Components
  • Actin, gamma-enteric smooth muscle
  • Phalloidin
KeywordsSTRUCTURAL PROTEIN / Filament / Actin / Smooth Muscle / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


Formation of the dystrophin-glycoprotein complex (DGC) / myosin filament / mesenchyme migration / Smooth Muscle Contraction / cell periphery / filopodium / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / lamellipodium / actin cytoskeleton / cell body ...Formation of the dystrophin-glycoprotein complex (DGC) / myosin filament / mesenchyme migration / Smooth Muscle Contraction / cell periphery / filopodium / 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 / cytoplasm / 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, gamma-enteric smooth muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsPalmer, N.J. / Carman, P.J. / Ceron, R.H. / Dominguez, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: Sci Adv / Year: 2024
Title: Molecular mechanisms linking missense ACTG2 mutations to visceral myopathy.
Authors: Rachel H Ceron / Faviolla A Báez-Cruz / Nicholas J Palmer / Peter J Carman / Malgorzata Boczkowska / Robert O Heuckeroth / E Michael Ostap / Roberto Dominguez /
Abstract: Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the ...Visceral myopathy is a life-threatening disease characterized by muscle weakness in the bowel, bladder, and uterus. Mutations in smooth muscle γ-actin (ACTG2) are the most common cause of the disease, but the mechanisms by which the mutations alter muscle function are unknown. Here, we examined four prevalent ACTG2 mutations (R40C, R148C, R178C, and R257C) that cause different disease severity and are spread throughout the actin fold. R178C displayed premature degradation, R148C disrupted interactions with actin-binding proteins, R40C inhibited polymerization, and R257C destabilized filaments. Because these mutations are heterozygous, we also analyzed 50/50 mixtures with wild-type (WT) ACTG2. The WT/R40C mixture impaired filament nucleation by leiomodin 1, and WT/R257C produced filaments that were easily fragmented by smooth muscle myosin. Smooth muscle tropomyosin isoform Tpm1.4 partially rescued the defects of R40C and R257C. Cryo-electron microscopy structures of filaments formed by R40C and R257C revealed disrupted intersubunit contacts. The biochemical and structural properties of the mutants correlate with their genotype-specific disease severity.
History
DepositionNov 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin, gamma-enteric smooth muscle
B: Actin, gamma-enteric smooth muscle
C: Actin, gamma-enteric smooth muscle
D: Actin, gamma-enteric smooth muscle
E: Actin, gamma-enteric smooth muscle
F: Phalloidin
G: Phalloidin
H: Phalloidin
I: Phalloidin
J: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,71120
Polymers213,45310
Non-polymers2,25810
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, gamma-enteric smooth muscle / Alpha-actin-3 / Gamma-2-actin / Smooth muscle gamma-actin


Mass: 41881.766 Da / Num. of mol.: 5 / Mutation: R40C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Tissue: Smooth Muscle / Gene: ACTG2, ACTA3, ACTL3, ACTSG / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: P63267, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein/peptide
Phalloidin


Type: Peptide-like / Class: Toxin / Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: BIRD: PRD_002366
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: ACTG2 R40C Filament / Type: COMPLEX
Details: ACTG2 filaments, purified from Expi293 cells R40C mutation
Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Tissue: Smooth Muscle
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F
Buffer solutionpH: 8 / Details: Actin F-buffer
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMTrisC4H11NO31
2100 mMPotassium ChlorideKCl1
30.2 mMCalcium ChlorideCaCl21
41 mMEGTAC14H24N2O101
51 mMMagnesium ChlorideMgCl21
60.2 mMATPC10H16N5O13P31
75 uMPhalloidinC35H48N8O11S1
SpecimenConc.: 0.168 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: ACTG2 F-actin in the ADP state. R40C mutation
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot Force: 0 Blot Time: 2.5 s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4461

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selectionFilament tracer job
2EPUimage acquisition
4cryoSPARC4CTF correctionCTF correction job
7Coot0.9.8.3model fitting
12cryoSPARC43D reconstructionHelix Refinement
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.6 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3400116 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8F8P

8f8p


Accession code: 8F8P / Source name: PDB / Type: experimental model

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