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- PDB-7k20: Cryo-EM structure of pyrene-labeled ADP-actin filaments -

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Basic information

Entry
Database: PDB / ID: 7k20
TitleCryo-EM structure of pyrene-labeled ADP-actin filaments
ComponentsActin, alpha skeletal muscle
KeywordsCYTOSOLIC PROTEIN / actin / pyrene / fluorescence / ADP
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
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
N-(pyren-1-yl)acetamide / ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChou, S.Z. / Pollard, T.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM026338 United States
CitationJournal: 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
DepositionSep 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: 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

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,34616
Polymers167,5034
Non-polymers2,84312
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41875.633 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Details: The C19 atom of pyrene (1T4) is chemically conjugated to the SG atom of actin C374
Source: (natural) Gallus gallus (chicken) / References: UniProt: P68139
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-1T4 / N-(pyren-1-yl)acetamide


Mass: 259.302 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H13NO / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Pyrene-labeled ADP-actin filaments / Type: COMPLEX
Details: Pyene is chemically conjugated to the side chain of actin C374
Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 11 sec. / Electron dose: 67.9 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
4Gctf1.18CTF correction
7Coot1.3.0model fitting
12RELION3.083D reconstruction
13PHENIX3.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.57869 ° / Axial rise/subunit: 27.385195 Å / Axial symmetry: C1
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 268618 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
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.
Atomic model buildingPDB-ID: 6DJN

6djn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01312100
ELECTRON MICROSCOPYf_angle_d0.95316432
ELECTRON MICROSCOPYf_dihedral_angle_d11.087224
ELECTRON MICROSCOPYf_chiral_restr0.0641808
ELECTRON MICROSCOPYf_plane_restr0.0072092

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