+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-22639 | |||||||||
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Title | Cryo-EM structure of pyrene-labeled ADP-Pi-actin filaments | |||||||||
Map data | Pyrene-labeled ADP-Pi-actin filaments | |||||||||
Sample |
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Function / homology | Function and homology information Striated Muscle Contraction / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle fiber development / stress fiber / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / hydrolase activity / ATP binding Similarity search - Function | |||||||||
Biological species | Gallus gallus (chicken) / Chicken (chicken) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Chou SZ / Pollard TD | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2020 Title: Cryo-electron microscopy structures of pyrene-labeled ADP-P- and ADP-actin filaments. Authors: Steven Z Chou / Thomas D Pollard / Abstract: Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the ...Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_22639.map.gz | 7.5 MB | EMDB map data format | |
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Header (meta data) | emd-22639-v30.xml emd-22639.xml | 12.4 KB 12.4 KB | Display Display | EMDB header |
Images | emd_22639.png | 139 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-22639 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-22639 | HTTPS FTP |
-Validation report
Summary document | emd_22639_validation.pdf.gz | 323.6 KB | Display | EMDB validaton report |
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Full document | emd_22639_full_validation.pdf.gz | 323.1 KB | Display | |
Data in XML | emd_22639_validation.xml.gz | 6.7 KB | Display | |
Data in CIF | emd_22639_validation.cif.gz | 7.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22639 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-22639 | HTTPS FTP |
-Related structure data
Related structure data | 7k21MC 7k20C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_22639.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Pyrene-labeled ADP-Pi-actin filaments | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.045 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Pyrene-labeled ADP-Pi-actin filaments
Entire | Name: Pyrene-labeled ADP-Pi-actin filaments |
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Components |
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-Supramolecule #1: Pyrene-labeled ADP-Pi-actin filaments
Supramolecule | Name: Pyrene-labeled ADP-Pi-actin filaments / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Pyene is chemically conjugated to the side chain of actin C374 |
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Source (natural) | Organism: Gallus gallus (chicken) |
-Macromolecule #1: Actin, alpha skeletal muscle
Macromolecule | Name: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 Details: The C19 atom of pyrene (1T4) is chemically conjugated to the SG atom of actin C374 Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Chicken (chicken) |
Molecular weight | Theoretical: 41.875633 KDa |
Sequence | String: 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 ITIGNERFRC PETLFQPSFI GMESAGIHET TYNSIMKCDI DIRKDLYANN VMSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ITKQEYDEAG PSIVHRKCF |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Macromolecule #4: PHOSPHATE ION
Macromolecule | Name: PHOSPHATE ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: PO4 |
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Molecular weight | Theoretical: 94.971 Da |
Chemical component information | ChemComp-PO4: |
-Macromolecule #5: N-(pyren-1-yl)acetamide
Macromolecule | Name: N-(pyren-1-yl)acetamide / type: ligand / ID: 5 / Number of copies: 4 / Formula: 1T4 |
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Molecular weight | Theoretical: 259.302 Da |
Chemical component information | ChemComp-1T4: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 7 |
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Grid | Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: unspecified |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average exposure time: 11.0 sec. / Average electron dose: 67.9 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Helical parameters - Δz: 27.369141 Å Applied symmetry - Helical parameters - Δ&Phi: -166.57062 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 462669 |
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CTF correction | Software - Name: Gctf (ver. 1.18) |
Startup model | Type of model: EMDB MAP EMDB ID: |
Final angle assignment | Type: NOT APPLICABLE |
-Atomic model buiding 1
Initial model | PDB ID: |
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Details | Restraints for pyrene were generated with eLBOW in Phenix. The coordinates of actin and pyrene were joined together manually in a text editor, and then fitted into the map in Coot. |
Refinement | Space: REAL / Protocol: OTHER |
Output model | PDB-7k21: |