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- EMDB-7115: Structure of bare actin filament -

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Basic information

Entry
Database: EMDB / ID: EMD-7115
TitleStructure of bare actin filament
Map datacryoEM density map of actin alone, sharpened with a -350 B factor
Sample
  • Complex: bare actin filament
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsCytoskeleton / Filament / CONTRACTILE PROTEIN
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / 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, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsGurel PS / Alushin GA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director5DP5OD017885 United States
CitationJournal: Nat Nanotechnol / Year: 2018
Title: Controllable molecular motors engineered from myosin and RNA.
Authors: Tosan Omabegho / Pinar S Gurel / Clarence Y Cheng / Laura Y Kim / Paul V Ruijgrok / Rhiju Das / Gregory M Alushin / Zev Bryant /
Abstract: Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . ...Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . Previously, synthetic nucleic acid motors and modified natural protein motors have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors . Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure . We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing . Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.
History
DepositionNov 17, 2017-
Header (metadata) releaseJan 10, 2018-
Map releaseJan 10, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 12
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6bno
  • Surface level: 12
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6bnu
  • Surface level: 12
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bno
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bnu
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7115_1.png

emd_7115_2.png

Downloads & links

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Map

FileDownload / File: emd_7115.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM density map of actin alone, sharpened with a -350 B factor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.27 Å/pix.
x 512 pix.
= 650.24 Å		1.27 Å/pix.
x 512 pix.
= 650.24 Å		1.27 Å/pix.
x 512 pix.
= 650.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.27 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 12.0 / Movie #1: 12
Minimum - Maximum-14.238341999999999 - 35.019120000000001
Average (Standard dev.)-0.000000005170885 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 650.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.271.271.27
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z650.240650.240650.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-14.23835.019-0.000

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Supplemental data

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Additional map: Un-sharpened density map of actin

Fileemd_7115_additional.map
AnnotationUn-sharpened density map of actin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full reconstruction of actin

Fileemd_7115_half_map_1.map
AnnotationHalf map of the full reconstruction of actin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map of the full reconstruction of actin

Fileemd_7115_half_map_2.map
AnnotationHalf map of the full reconstruction of actin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : bare actin filament

EntireName: bare actin filament
Components
  • Complex: bare actin filament
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: bare actin filament

SupramoleculeName: bare actin filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Actin filament in the ADP state
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 42 KDa

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Skeletal Muscle
Molecular weightTheoretical: 41.560266 KDa
SequenceString: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVH

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 8 / 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: 8 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.025 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMKClpotassium chloride
1.0 mMMgCl2magnesium chloride
1.0 mMC14H24N2O10EGTA
10.0 mMC3H4N2imidazole
2.0 mMC4H11NO3Tris hydrochloride
0.5 mMC4H10O2S2dithiothreitol
200.0 mMC10H16N5O13P3adenosine triphosphate
0.01 %NaN3sodium azide

Details: Buffer was filtered through 0.44 um filter and degassed.
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details: Sample was applied to a glow-discharged holey carbon grid, incubated for 60 seconds and blotted for 3 seconds from the backside with filter paper..
DetailsFilamentous bare actin

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-24 / Number grids imaged: 1 / Number real images: 442 / Average exposure time: 0.25 sec. / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 28.11 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. 9.11) / Number images used: 63139
Startup modelType of model: EMDB MAP
EMDB ID:

1990


Details: low-pass filtered to 35 A
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial models were assembled from 8 actins (3J8A) through rigid body docking in Chimera, followed by flexible fitting with DireX. Resulting models were subjected to MDFF.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 350
Output model

PDB-6bno:
Structure of bare actin filament

PDB-6bnu:
Structure of bare actin filament, backbone-averaged with sidechains truncated to alanine

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