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- PDB-7rb8: cryo-EM structure of the ADP state wild type myosin-15-F-actin complex -

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Basic information

Entry
Database: PDB / ID: 7rb8
Titlecryo-EM structure of the ADP state wild type myosin-15-F-actin complex
Components
  • Actin, alpha skeletal muscle, intermediate form
  • Isoform 3 of Unconventional myosin-XV
KeywordsMOTOR PROTEIN/ATP Binding Protein / myosin motor proteins / actin cytoskeleton / stereocilia / deafness / MOTOR PROTEIN / MOTOR PROTEIN-ATP Binding Protein complex
Function / homology
Function and homology information


Striated Muscle Contraction / stereocilium bundle / stereocilium / myosin complex / inner ear morphogenesis / cytoskeletal motor activity / striated muscle thin filament / skeletal muscle thin filament assembly / response to light stimulus / stress fiber ...Striated Muscle Contraction / stereocilium bundle / stereocilium / myosin complex / inner ear morphogenesis / cytoskeletal motor activity / striated muscle thin filament / skeletal muscle thin filament assembly / response to light stimulus / stress fiber / skeletal muscle fiber development / actin filament / locomotory behavior / sensory perception of sound / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin binding / hydrolase activity / ATP binding / cytosol
Similarity search - Function
Class XV myosin, motor domain / Myosin-XV, FERM domain C-lobe / : / MyTH4 domain / MyTH4 domain superfamily / MyTH4 domain / MyTH4 domain profile. / Domain in Myosin and Kinesin Tails / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #530 / IQ calmodulin-binding motif ...Class XV myosin, motor domain / Myosin-XV, FERM domain C-lobe / : / MyTH4 domain / MyTH4 domain superfamily / MyTH4 domain / MyTH4 domain profile. / Domain in Myosin and Kinesin Tails / Methane Monooxygenase Hydroxylase; Chain G, domain 1 - #530 / IQ calmodulin-binding motif / Variant SH3 domain / FERM central domain / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / FERM central domain / FERM superfamily, second domain / FERM domain / FERM domain profile. / Kinesin motor domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / Src homology 3 domains / ATPase, nucleotide binding domain / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / PH-like domain superfamily / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / Mainly Alpha
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle / Unconventional myosin-XV
Similarity search - Component
Biological speciesMus musculus (house mouse)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsGong, R. / Reynolds, M.J. / Gurel, P. / Alushin, G.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DP5OD017885 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM141044 United States
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01DC018827 United States
Citation
Journal: Sci Adv / Year: 2022
Title: Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia.
Authors: Rui Gong / Fangfang Jiang / Zane G Moreland / Matthew J Reynolds / Santiago Espinosa de Los Reyes / Pinar Gurel / Arik Shams / James B Heidings / Michael R Bowl / Jonathan E Bird / Gregory M Alushin /
Abstract: The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin ...The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament ("F-actin") assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, ""). Here, we present cryo-electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin's D-loop, which mediates inter-subunit contacts, while the mutant locks the D-loop in a single conformation. Adenosine diphosphate-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin's structural plasticity in a myosin nucleotide state-dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.
#1: Journal: Biorxiv / Year: 2021
Title: Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia
Authors: Gong, R. / Jiang, F. / Moreland, Z.G. / Reynolds, M.J. / Gurel, P.S. / Shams, A. / Bowl, M.R. / Bird, J.E. / Alushin, G.M.
History
DepositionJul 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Author supporting evidence / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle, intermediate form
D: Isoform 3 of Unconventional myosin-XV
B: Actin, alpha skeletal muscle, intermediate form
C: Actin, alpha skeletal muscle, intermediate form
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,88312
Polymers203,0774
Non-polymers1,8068
Water00
1


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

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Components

#1: Protein Actin, alpha skeletal muscle, intermediate form


Mass: 41631.430 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P68139
#2: Protein Isoform 3 of Unconventional myosin-XV / Unconventional myosin-15


Mass: 78182.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Myo15a, Myo15
Production host: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
References: UniProt: Q9QZZ4
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
1The ADP state wild type myosin15 in complex with filamentous actinCOMPLEX#1-#20MULTIPLE SOURCES
2ActinCOMPLEX#11NATURAL
3Isoform 3 of Unconventional myosin-XVCOMPLEX#21RECOMBINANT
Molecular weightValue: 5.4 kDa/nm / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Gallus gallus (chicken)9031
23Mus musculus (house mouse)10090
Buffer solutionpH: 7
Details: 10 mM imidazole pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.01% NaN3
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.52 ° / Axial rise/subunit: 27.06 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 139547
3D reconstructionResolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125232 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6BNO

6bno

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