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- EMDB-42938: Cryo-EM Structure of Smooth Muscle Gamma Actin (ACTG2) Mutant R257C -

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Basic information

Entry
Database: EMDB / ID: EMD-42938
TitleCryo-EM Structure of Smooth Muscle Gamma Actin (ACTG2) Mutant R257C
Map data
Sample
  • Complex: ACTG2 R257C Filament
    • Protein or peptide: Actin, gamma-enteric smooth muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsFilament / Actin / Smooth Muscle / CYTOSOLIC PROTEIN / STRUCTURAL 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
Actin, gamma-enteric smooth muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 2.72 Å
AuthorsPalmer NJ / Carman PJ / Ceron RH / Dominguez R
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42938.png

Downloads & links

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Map

FileDownload / File: emd_42938.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163
Minimum - Maximum-0.22858354 - 0.9803461
Average (Standard dev.)0.0031954448 (±0.036359396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42938_msk_1.map
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Half map: #2

Fileemd_42938_half_map_1.map
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Half map: #1

Fileemd_42938_half_map_2.map
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Sample components

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Entire : ACTG2 R257C Filament

EntireName: ACTG2 R257C Filament
Components
  • Complex: ACTG2 R257C Filament
    • Protein or peptide: Actin, gamma-enteric smooth muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ACTG2 R257C Filament

SupramoleculeName: ACTG2 R257C Filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: ACTG2 filaments, purified from Expi293 cells
Source (natural)Organism: Homo sapiens (human) / Tissue: Smooth

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Macromolecule #1: Actin, gamma-enteric smooth muscle

MacromoleculeName: Actin, gamma-enteric smooth muscle / type: protein_or_peptide / ID: 1 / Number of copies: 5 / 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) / Tissue: Smooth Muscle
Molecular weightTheoretical: 41.881762 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MCEEETTALV CDNGSGLCKA GFAGDDAPRA VFPSIVGRPR HQGVMVGMGQ KDSYVGDEAQ SKRGILTLKY PIE(HIC)GI ITN WDDMEKIWHH SFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDS GD GVTHNVPIYE ...String:
MCEEETTALV CDNGSGLCKA GFAGDDAPRA VFPSIVGRPR HQGVMVGMGQ KDSYVGDEAQ SKRGILTLKY PIE(HIC)GI ITN WDDMEKIWHH SFYNELRVAP EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT TGIVLDS GD GVTHNVPIYE GYALPHAIMR LDLAGRDLTD YLMKILTERG YSFVTTAERE IVRDIKEKLC YVALDFENEM ATAASSSS L EKSYELPDGQ VITIGNERFC CPETLFQPSF IGMESAGIHE TTYNSIMKCD IDIRKDLYAN NVLSGGTTMY PGIADRMQK EITALAPSTM KIKIIAPPER KYSVWIGGSI LASLSTFQQM WISKPEYDEA GPSIVHRKCF

UniProtKB: Actin, gamma-enteric smooth muscle

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / 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 #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.168 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
5.0 mMC4H11NO3Tris
100.0 mMKClPotassium Chloride
0.2 mMCaCl2Calcium Chloride
1.0 mMC14H24N2O10EGTA
1.0 mMMgCl2Magnesium Chloride
0.2 mMC10H15N5O10P2ATP

Details: Actin F-buffer
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blot Force: 0 Blot Time: 2.5 s.
DetailsACTG2 F-actin in the ADP state. R257C mutation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 1021 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.5 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Software - details: Helix Refinement / Number images used: 473627
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8f8p


Details: Individually mutated residues within structure of skeletal muscle actin until each subunit had the sequence of gamma smooth muscle actin with R257C.
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8f8p


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8v2z:
Cryo-EM Structure of Smooth Muscle Gamma Actin (ACTG2) Mutant R257C

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