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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


myosin filament / mesenchyme migration / Smooth Muscle Contraction / filopodium / cell periphery / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / lamellipodium / cell body / blood microparticle / cytoskeleton ...myosin filament / mesenchyme migration / Smooth Muscle Contraction / filopodium / cell periphery / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / lamellipodium / cell body / blood microparticle / cytoskeleton / hydrolase activity / positive regulation of gene expression / extracellular space / extracellular exosome / ATP binding / cytosol / cytoplasm
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: To Be Published
Title: Smooth Muscle Gamma Actin (ACTG2) Filament Mutant R257C
Authors: Ceron RH / Baez-Cruz FA / Palmer NJ / Carman PJ / Boczkowska M / Heuckeroth RO / Ostap EM / Dominguez R
History
DepositionNov 25, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 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)
Voxel sizeX=Y=Z: 1.07 Å
Density
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
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42938_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42938_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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 / Adenosine diphosphate

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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 mMC10H15N5O10P2ATPAdenosine triphosphate

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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 1021 / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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
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
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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