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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 / homology
Function and homology information


CH domain binding / Trafficking of AMPA receptors / regulation of secretion / Gap junction degradation / RNA polymerase II, holoenzyme / actin filament-based movement / inner ear auditory receptor cell differentiation / vesicle transport along actin filament / inner ear morphogenesis / microfilament motor activity ...CH domain binding / Trafficking of AMPA receptors / regulation of secretion / Gap junction degradation / RNA polymerase II, holoenzyme / actin filament-based movement / inner ear auditory receptor cell differentiation / vesicle transport along actin filament / inner ear morphogenesis / microfilament motor activity / myosin binding / microfilament motor activity => GO:0000146 / positive regulation of microfilament motor activity => GO:0120081 / myosin complex / mesenchyme migration / clathrin-coated vesicle / tropomyosin binding / myosin heavy chain binding / troponin I binding / DNA damage response, signal transduction by p53 class mediator / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle / filamentous actin / actin filament bundle assembly / actin monomer binding / motor activity / skeletal muscle fiber development / skeletal muscle myofibril / endocytic vesicle / stress fiber / clathrin-coated pit / titin binding / actin filament polymerization / enzyme regulator activity / ruffle / filopodium / actin filament / actin filament organization / ADP binding / sensory perception of sound / ruffle membrane / intracellular protein transport / cell cortex / microvillus / cell body / protein transport / calcium-dependent protein binding / actin filament binding / endocytosis / actin cytoskeleton / lamellipodium / disordered domain specific binding / cytoplasmic vesicle / nuclear membrane / vesicle / calmodulin binding / protein C-terminus binding / protein domain specific binding / positive regulation of gene expression / calcium ion binding / magnesium ion binding / perinuclear region of cytoplasm / Golgi apparatus / positive regulation of transcription by RNA polymerase II / protein-containing complex / nucleoplasm / ATP binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Myosin VI cargo binding domain / Myosin VI, cargo binding domain / Class VI myosin, motor domain / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Myosin S1 fragment, N-terminal / Myosin head, motor domain / Myosin. Large ATPases. / Myosin head (motor domain) / Myosin motor domain profile. ...Myosin VI cargo binding domain / Myosin VI, cargo binding domain / Class VI myosin, motor domain / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Myosin S1 fragment, N-terminal / Myosin head, motor domain / Myosin. Large ATPases. / Myosin head (motor domain) / Myosin motor domain profile. / Actins signature 1. / Actins signature 2. / Actin, conserved site / Actins and actin-related proteins signature. / Actin/actin-like conserved site / Actin / Actin / Actin family / Kinesin motor domain superfamily / EF-hand domain pair / ATPase, nucleotide binding domain / EF-hand, calcium binding motif / EF-hand calcium-binding domain profile. / EF-Hand 1, calcium-binding site / EF-hand domain / EF-hand calcium-binding domain. / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Unconventional myosin-6 / Calmodulin / Actin, alpha skeletal muscle / Unconventional myosin-VI
Similarity search - Component
Biological speciesSus scrofa (pig)
Gallus gallus (chicken)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsGurel, P.S. / Alushin, G.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director5DP5OD017885 United States
Citation
Journal: Elife / Year: 2017
Title: Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity.
Authors: Pinar S Gurel / Laura Y Kim / Paul V Ruijgrok / Tosan Omabegho / Zev Bryant / Gregory M Alushin /
Abstract: Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited ...Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Å) and Mg-ADP (5.5 Å) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.
#1: 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.
History
DepositionNov 17, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2018Group: Author supporting evidence / Category: pdbx_audit_support / Item: _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
Polymers990,16220
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Unconventional myosin-VI


Mass: 93207.297 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Tissue: skeletal muscle / Gene: MYO6 / Plasmid: pBiex1 / Cell line (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F1RQI7, UniProt: Q29122*PLUS
#2: Protein
Actin, alpha skeletal muscle / / Alpha-actin-1


Mass: 41560.266 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: skeletal muscle / References: UniProt: P68135
#3: Protein
Calmodulin / / CaM


Mass: 16406.004 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: CALM, CAM, RCJMB04_24e7 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62149

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1myosin VI-actin in the rigor (nucleotide-free) stateCOMPLEX#1-#30MULTIPLE SOURCES
2myosin VICOMPLEX#11RECOMBINANT
3actinCOMPLEX#21NATURAL
4calmodulinCOMPLEX#31RECOMBINANT
Molecular weightValue: 0.139 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa (pig)9823
23Oryctolagus cuniculus (rabbit)9986
34Gallus gallus (chicken)9031
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
24Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.5
Details: Buffer was filtered through 0.44 um filter and degassed.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMpotassium chlorideKCl1
21 mMmagnesium chlorideMgCl21
31 mMEGTAC14H24N2O101
410 mMimidazoleC3H4N21
52 mMTris hydrochlorideC4H11NO31
60.5 mMdithiothreitolC4H10O2S21
7200 mMadenosine triphosphateC10H16N5O13P31
80.01 %sodium azideNaN31
910 U/mLapyraseapyrase1
SpecimenConc.: 0.045 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.45 mg/mL myosin VI was added to 0.025 mg/mL actin
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K
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 ...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

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
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) / Num. of grids imaged: 3 / Num. of real images: 778
Image scansSampling size: 5 µm / Width: 3838 / 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
16FREALIGN9.113D reconstruction
17MDFFmodel refinement
18PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.73 ° / Axial rise/subunit: 28.06 Å / Axial symmetry: C1
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56116 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 150 / Protocol: FLEXIBLE FIT / Space: REAL
Details: 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 ...Details: 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.

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