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- PDB-6bnv: CryoEM structure of MyosinVI-actin complex in the rigor (nucleoti... -

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Basic information

Entry
Database: PDB / ID: 6bnv
TitleCryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state, backbone-averaged with side chains truncated to alanine
Components
  • Actin, alpha skeletal muscle
  • Calmodulin
  • Unconventional myosin-VI
KeywordsCONTRACTILE PROTEIN / Cytoskeleton / Filament / complex
Function/homologyActivation of Ca-permeable Kainate Receptor / Ion transport by P-type ATPases / CaMK IV-mediated phosphorylation of CREB / Calmodulin induced events / Cam-PDE 1 activation / Ion homeostasis / Phase 0 - rapid depolarisation / Sodium/Calcium exchangers / Glycogen breakdown (glycogenolysis) / Protein methylation ...Activation of Ca-permeable Kainate Receptor / Ion transport by P-type ATPases / CaMK IV-mediated phosphorylation of CREB / Calmodulin induced events / Cam-PDE 1 activation / Ion homeostasis / Phase 0 - rapid depolarisation / Sodium/Calcium exchangers / Glycogen breakdown (glycogenolysis) / Protein methylation / CREB phosphorylation through the activation of CaMKK / CLEC7A (Dectin-1) induces NFAT activation / CREB phosphorylation through the activation of CaMKII / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Activation of CaMK IV / PKA activation / Synthesis of IP3 and IP4 in the cytosol / Calcineurin activates NFAT / eNOS activation / Ca2+ pathway / Ras activation upon Ca2+ influx through NMDA receptor / Reduction of cytosolic Ca++ levels / VEGFR2 mediated vascular permeability / FCERI mediated Ca+2 mobilization / Inactivation, recovery and regulation of the phototransduction cascade / CH domain binding / Trafficking of AMPA receptors / RHO GTPases activate IQGAPs / RHO GTPases activate PAKs / Gap junction degradation / Stimuli-sensing channels / Platelet degranulation / Class VI myosin, motor domain / Myosin VI, cargo binding domain / Myosin VI cargo binding domain / regulation of secretion / RAF/MAP kinase cascade / Smooth Muscle Contraction / DNA-directed RNA polymerase II, holoenzyme / actin filament-based movement / clathrin-coated endocytic vesicle / myosin binding / N-terminal myristoylation domain binding / positive regulation of actin-dependent ATPase activity / mesenchyme migration / Myosin N-terminal SH3-like domain profile. / myosin complex / calcium channel complex / tropomyosin binding / protein phosphatase activator activity / myosin heavy chain binding / Myosin S1 fragment, N-terminal / positive regulation of cyclic-nucleotide phosphodiesterase activity / skeletal muscle thin filament assembly / troponin I binding / striated muscle thin filament / positive regulation of ryanodine-sensitive calcium-release channel activity / actin filament bundle / catalytic complex / Actins signature 1. / Actins signature 2. / Actin, conserved site / adenylate cyclase binding / filamentous actin / positive regulation of cAMP biosynthetic process / detection of calcium ion / Myosin motor domain profile. / Myosin head, motor domain / Actins and actin-related proteins signature. / Actin/actin-like conserved site / Actin family / regulation of cardiac muscle contraction / actin monomer binding / skeletal muscle fiber development / voltage-gated potassium channel complex / negative regulation of ryanodine-sensitive calcium-release channel activity / DNA damage response, signal transduction by p53 class mediator / positive regulation of phosphoprotein phosphatase activity / actin filament bundle assembly / calcium channel inhibitor activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / motor activity / skeletal muscle myofibril / Myosin head (motor domain) / sarcomere / stress fiber / ADP binding / titin binding / actin filament polymerization / regulation of heart rate / Kinesin motor domain superfamily / actin filament / positive regulation of DNA binding / Actin / regulation of cytokinesis / filopodium / spindle microtubule / ruffle / calcium-dependent protein binding / intracellular protein transport
Function and homology information
Specimen sourceSus scrofa / mammal / image: Sus scrofa domestica
Gallus gallus / bird / Chicken /
Oryctolagus cuniculus / mammal / European rabbit /
MethodElectron microscopy (4.6 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
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
O: Calmodulin
P: Calmodulin
Q: Calmodulin
R: Calmodulin
S: Calmodulin
T: Calmodulin


Theoretical massNumber of molelcules
Total (without water)990,16220
Polyers990,16220
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Unconventional myosin-VI


Mass: 93207.297 Da / Num. of mol.: 6
Source: (gene. exp.) Sus scrofa / mammal / image: Sus scrofa domestica
Tissue: skeletal muscle / Gene: MYO6 / Plasmid name: pBiex1 / Cell line (production host): sf9 / Production host: Spodoptera frugiperda / References: UniProt:F1RQI7, UniProt:Q29122*PLUS
#2: Protein/peptide
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41560.266 Da / Num. of mol.: 8
Source: (natural) Oryctolagus cuniculus / mammal / European rabbit /
Tissue: skeletal muscle / References: UniProt:P68135
#3: Protein/peptide
Calmodulin / CaM


Mass: 16406.004 Da / Num. of mol.: 6 / Source: (gene. exp.) Gallus gallus / bird / Chicken / / Gene: CALM, CAM, RCJMB04_24e7 / Production host: Spodoptera frugiperda / References: UniProt:P62149

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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

Component
IDNameTypeEntity IDParent IDSource
1myosin VI-actin in the rigor (nucleotide-free) stateCOMPLEX1, 2, 30MULTIPLE SOURCES
2myosin VICOMPLEX11RECOMBINANT
3actinCOMPLEX21NATURAL
4calmodulinCOMPLEX31RECOMBINANT
Molecular weightValue: 0.139 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
129823Sus scrofa
239986Oryctolagus cuniculus
349031Gallus gallus
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
127108Spodoptera frugiperda
247108Spodoptera frugiperda
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
910U/mLapyraseapyrase1
SpecimenConc.: 0.045 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 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. 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.

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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: 3 / Number of real images: 778
Image scansSampling size: 5 microns / Dimension width: 3838 / Dimension height: 3711 / 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
8DireXMODEL FITTING
9MDFFMODEL FITTING
10NAMDMODEL FITTING
12EMAN2INITIAL EULER ASSIGNMENT
13SPARXINITIAL EULER ASSIGNMENT
14FREALIGN9.11FINAL EULER ASSIGNMENT
16FREALIGN9.11RECONSTRUCTION
17MDFFMODEL REFINEMENT
18PHENIXMODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.73 deg. / Axial rise/subunit: 28.06 Å / Axial symmetry: C1
3D reconstructionResolution: 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 buildingDetails: 2BKI chain B was grafted onto Chains A-F of 6BNP, then assembled by rigid body docking into the 7.5 A low pass-filtered density map, followed by flexible fitting with DireX. The resulting model was subjected to MDFF using the 7.5 A low pass-filtered density map. Atomistic models were backbone-averaged in Phenix and side chains were truncated to alanine. This treatment has resulted in the errors noted in the associated caveats.
Overall b value: 150 / Ref protocol: FLEXIBLE FIT / Ref space: REAL

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