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- EMDB-22638: Cryo-EM structure of pyrene-labeled ADP-actin filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-22638
TitleCryo-EM structure of pyrene-labeled ADP-actin filaments
Map dataMg-ADP-pyrenyl-actin filament
Sample
  • Complex: Pyrene-labeled ADP-actin filaments
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: N-(pyren-1-yl)acetamide
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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken) / Chicken (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChou SZ / Pollard TD
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseNov 4, 2020-
Map releaseNov 4, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
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  • Surface view with fitted model
  • Atomic models: PDB-7k20
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7k20
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22638.png

Downloads & links

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Map

FileDownload / File: emd_22638.map.gz / Format: CCP4 / Size: 134.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMg-ADP-pyrenyl-actin filament
Voxel sizeX=Y=Z: 1.045 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.031317536 - 0.08841074
Average (Standard dev.)0.000116744806 (±0.0017867439)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions328328328
Spacing328328328
CellA=B=C: 342.75998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0451.0451.045
M x/y/z328328328
origin x/y/z0.0000.0000.000
length x/y/z342.760342.760342.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS328328328
D min/max/mean-0.0310.0880.000

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Supplemental data

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Sample components

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Entire : Pyrene-labeled ADP-actin filaments

EntireName: Pyrene-labeled ADP-actin filaments
Components
  • Complex: Pyrene-labeled ADP-actin filaments
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: N-(pyren-1-yl)acetamide

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Supramolecule #1: Pyrene-labeled ADP-actin filaments

SupramoleculeName: Pyrene-labeled ADP-actin filaments / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Pyene is chemically conjugated to the side chain of actin C374
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: 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
Source (natural)Organism: Chicken (chicken)
Molecular weightTheoretical: 41.875633 KDa
SequenceString: 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

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / 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 #4: N-(pyren-1-yl)acetamide

MacromoleculeName: N-(pyren-1-yl)acetamide / type: ligand / ID: 4 / Number of copies: 4 / Formula: 1T4
Molecular weightTheoretical: 259.302 Da
Chemical component information

ChemComp-1T4:
N-(pyren-1-yl)acetamide

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
GridPretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: EMDB MAP
EMDB ID:

7937

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.385195 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.57869 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 268618

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Atomic model buiding 1

Initial modelPDB ID:

6djn

DetailsRestraints 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.
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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