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- PDB-7r8v: Cryo-EM structure of the ADP state actin filament -

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Basic information

Entry
Database: PDB / ID: 7r8v
TitleCryo-EM structure of the ADP state actin filament
ComponentsActin, alpha skeletal muscle, intermediate form
KeywordsCELL ADHESION / filamentous actin / cytoskeleton / adhesion / contractility
Function / homology
Function and homology information


Striated Muscle Contraction / skeletal muscle thin filament assembly / striated muscle thin filament / skeletal muscle fiber development / stress fiber / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / hydrolase activity / ATP binding
Similarity search - Function
Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsGong, R. / Espinosa de los Reyes, S. / Reynolds, M.J. / Gurel, P. / Alushin, G.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institute of Dental and Craniofacial ResearchDP5OD017885 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
DepositionJun 27, 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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Assembly

Deposited unit
B: Actin, alpha skeletal muscle, intermediate form
A: Actin, alpha skeletal muscle, intermediate form
C: Actin, alpha skeletal muscle, intermediate form
D: Actin, alpha skeletal muscle, intermediate form
E: Actin, alpha skeletal muscle, intermediate form
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,19415
Polymers206,9365
Non-polymers2,25810
Water0
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: 41387.227 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P68139
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / 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

ComponentName: filamentous chicken skeletal muscle actin / Type: COMPLEX
Details: chicken skeletal muscle actin purified from fresh chicken breast
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 5.4 kDa/nm / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
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
Details: chicken cytoskeletal actin purified from chicken breast muscle
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen 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 FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
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)
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
9RELION3initial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.85 ° / Axial rise/subunit: 27.19 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 229833
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 228334 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6BNO

6bno

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