|Entry||Database: PDB / ID: 6bnp|
|Title||CryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state|
|Keywords||CONTRACTILE PROTEIN / Cytoskeleton / Filament / complex|
|Function/homology||Trafficking of AMPA receptors / Gap junction degradation / Class VI myosin, motor domain / Myosin VI, cargo binding domain / Myosin VI cargo binding domain / regulation of secretion / DNA-directed RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / positive regulation of actin-dependent ATPase activity ...Trafficking of AMPA receptors / Gap junction degradation / Class VI myosin, motor domain / Myosin VI, cargo binding domain / Myosin VI cargo binding domain / regulation of secretion / DNA-directed RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / positive regulation of actin-dependent ATPase activity / mesenchyme migration / Myosin N-terminal SH3-like domain profile. / myosin complex / tropomyosin binding / myosin heavy chain binding / Myosin S1 fragment, N-terminal / troponin I binding / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / Actins signature 1. / Actin, conserved site / Actins signature 2. / filamentous actin / Myosin motor domain profile. / Myosin head, motor domain / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin family / actin monomer binding / skeletal muscle fiber development / DNA damage response, signal transduction by p53 class mediator / actin filament bundle assembly / motor activity / skeletal muscle myofibril / Myosin head (motor domain) / stress fiber / ADP binding / titin binding / actin filament polymerization / Kinesin motor domain superfamily / actin filament / Actin / filopodium / ruffle / calcium-dependent protein binding / intracellular protein transport / sensory perception of sound / cell body / cell cortex / endocytosis / actin filament binding / lamellipodium / cytoplasmic vesicle / nuclear membrane / calmodulin binding / cadherin binding / protein domain specific binding / positive regulation of gene expression / magnesium ion binding / calcium ion binding / perinuclear region of cytoplasm / Golgi apparatus / positive regulation of transcription by RNA polymerase II / P-loop containing nucleoside triphosphate hydrolase / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm / Unconventional myosin-VI / Actin, alpha skeletal muscle / Unconventional myosin-VI|
Function and homology information
|Specimen source||Sus scrofa / Wild boar / mammal / image: Sus scrofa domestica|
Oryctolagus cuniculus / European rabbit / mammal /
|Method||Electron microscopy (4.6 Å resolution / Filament / Helical) / Transmission electron microscopy|
|Authors||Gurel, P.S. / Alushin, G.A.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Date||Deposition: Nov 17, 2017 / Release: Jan 10, 2018|
Downloads & links
I: Unconventional myosin-VI
J: Unconventional myosin-VI
K: Unconventional myosin-VI
L: Unconventional myosin-VI
M: Unconventional myosin-VI
N: Unconventional myosin-VI
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
Mass: 79837.367 Da / Num. of mol.: 6
Source: (gene. exp.) Sus scrofa / Wild boar / mammal / image: Sus scrofa domestica
Gene: MYO6 / Plasmid name: pBiex-1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda / References: UniProt:F1RQI7, UniProt:Q29122*PLUS
Mass: 41560.266 Da / Num. of mol.: 8
Source: (natural) Oryctolagus cuniculus / European rabbit / mammal /
Tissue: Skeletal Muscle / References: UniProt:P68135
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: FILAMENT / Reconstruction method: HELICAL|
|Source (recombinant)||Organism: Spodoptera frugiperda|
|Buffer solution||Details: Buffer was filtered through 0.44 um filter and degassed.|
|Specimen||Conc.: 0.45 mg/ml|
Details: 0.45 mg/mL myosin VI was added to 0.025 mg/mL actin
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
|Specimen support||Grid material: COPPER / Grid mesh size: 200 / Grid type: C-flat-1.2/1.3|
|Vitrification||Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 kelvins|
Details: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An additional 3 uL of myosin VI was applied. After 60 seconds, 3 uL solution was removed, and the grid was blotted for 3 seconds from the backside with filter paper.
-Electron microscopy imaging
|Microscopy||Microscope model: FEI TECNAI 20|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: 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 holder||Cryogen: NITROGEN|
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
|Image recording||Average 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: 3 / Number of real images: 778|
|Image scans||Sampling size: 5 microns / Dimension width: 3838 / Dimension height: 3710 / Movie frames/image: 24 / Used frames/image: 1-24|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Helical symmerty||Angular rotation/subunit: -166.73 deg. / Axial rise/subunit: 28.06 Å / Axial symmetry: C1|
|3D reconstruction||Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 56116 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: HELICAL|
|Atomic model building||Details: Initial models were assembled from 8 actins (3J8A) and 6 myosins (2BKI) through rigid body docking in Chimera, followed by flexible fitting with DireX. Resulting models were subjected to MDFF.|
Overall b value: 150 / Ref protocol: FLEXIBLE FIT
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