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- PDB-6bnu: Structure of bare actin filament, backbone-averaged with sidechai... -

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Basic information

Entry
Database: PDB / ID: 6bnu
TitleStructure of bare actin filament, backbone-averaged with sidechains truncated to alanine
ComponentsActin, alpha skeletal muscle
KeywordsCONTRACTILE PROTEIN / Cytoskeleton / Filament
Function/homologypositive regulation of actin-dependent ATPase activity / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / skeletal muscle thin filament assembly / troponin I binding / striated muscle thin filament / actin filament bundle / Actins signature 1. / Actins signature 2. ...positive regulation of actin-dependent ATPase activity / mesenchyme migration / tropomyosin binding / myosin heavy chain binding / skeletal muscle thin filament assembly / troponin I binding / striated muscle thin filament / actin filament bundle / Actins signature 1. / Actins signature 2. / Actin, conserved site / filamentous actin / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin family / actin monomer binding / skeletal muscle fiber development / actin filament bundle assembly / skeletal muscle myofibril / stress fiber / titin binding / actin filament polymerization / actin filament / Actin / filopodium / calcium-dependent protein binding / cell body / lamellipodium / protein domain specific binding / positive regulation of gene expression / magnesium ion binding / calcium ion binding / ATP binding / identical protein binding / cytoplasm / Actin, alpha skeletal muscle
Function and homology information
Specimen sourceOryctolagus cuniculus / mammal / European rabbit /
MethodElectron microscopy (7.5 Å resolution / Filament / Helical) / Transmission electron microscopy
AuthorsGurel, P.S. / Alushin, G.A.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 17, 2017 / Release: Jan 10, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 10, 2018Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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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
E: Actin, alpha skeletal muscle
F: Actin, alpha skeletal muscle
G: Actin, alpha skeletal muscle
H: Actin, alpha skeletal muscle


Theoretical massNumber of molelcules
Total (without water)332,4828
Polyers332,4828
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 41560.266 Da / Num. of mol.: 8
Source: (natural) Oryctolagus cuniculus / mammal / European rabbit /
References: UniProt:P68135

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: HELICAL

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Sample preparation

ComponentName: Bare actin filament / Type: COMPLEX / Entity ID: 1 / Source: NATURAL
Molecular weightValue: 0.042 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus
Buffer solutionDetails: Buffer was filtered through 0.44 um filter and degassed.
pH: 7.5
Buffer component
IDConc.UnitsNameFormulaBuffer ID
150mMpotassium chlorideKCl1
21mMmagnesium chlorideMgCl21
31mMEGTAC14H24N2O101
410mMimidazoleC3H4N21
52mMTris hydrochlorideC4H11NO31
60.5mMdithiothreitolC4H10O2S21
7200mMadenosine triphosphateC10H16N5O13P31
80.01%sodium azideNaN31
SpecimenConc.: 0.025 mg/ml / Details: filamentous bare actin / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 kelvins
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.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 100 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 442
Image scansSampling size: 5 microns / Dimension width: 3838 / Dimension height: 3710 / Movie frames/image: 24 / Used frames/image: 1-24

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Processing

EM software
IDNameVersionCategory
1AppionPARTICLE SELECTION
2LeginonIMAGE ACQUISITION
4CTFFIND3CTF CORRECTION
5FREALIGN9.11CTF CORRECTION
10EMAN2INITIAL EULER ASSIGNMENT
11SPARXINITIAL EULER ASSIGNMENT
12FREALIGN9.11FINAL EULER ASSIGNMENT
14FREALIGN9.11RECONSTRUCTION
15PHENIXMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.65 deg. / Axial rise/subunit: 28.11 Å / Axial symmetry: C1
3D reconstructionResolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 63139 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingDetails: Atomistic model was backbone-averaged in Phenix; side chains were truncated to alanine. This treatment has resulted in the errors noted in the associated caveats.
Overall b value: 350 / Ref protocol: FLEXIBLE FIT / Ref space: REAL

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