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- EMDB-24321: Cryo-EM structure of the ADP state actin filament -

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Basic information

Entry
Database: EMDB / ID: EMD-24321
TitleCryo-EM structure of the ADP state actin filament
Map dataCryo-EM structure of F actin in the ADP state
Sample
  • Complex: filamentous chicken skeletal muscle actin
    • Protein or peptide: Actin, alpha skeletal muscle, intermediate form
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
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
Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesGallus gallus (chicken) / Chicken (chicken)
Methodhelical reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsGong R / Espinosa de los Reyes S / Reynolds MJ / Gurel P / Alushin GM
Funding support United States, 3 items
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 ZG / Reynolds MJ / Gurel PS / Shams A / Bowl MR / Bird JE / Alushin GM
History
DepositionJun 27, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7r8v
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7r8v
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24321.png

Downloads & links

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Map

FileDownload / File: emd_24321.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of F actin in the ADP state
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.06
Minimum - Maximum-0.12675016 - 0.30203798
Average (Standard dev.)1.5197869e-05 (±0.0033614864)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 527.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z527.360527.360527.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.1270.3020.000

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Supplemental data

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Mask #1

Fileemd_24321_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryo-EM structure of F actin in the ADP state

Fileemd_24321_half_map_1.map
Annotationcryo-EM structure of F actin in the ADP state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryo-EM structure of F actin in the ADP state

Fileemd_24321_half_map_2.map
Annotationcryo-EM structure of F actin in the ADP state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : filamentous chicken skeletal muscle actin

EntireName: filamentous chicken skeletal muscle actin
Components
  • Complex: filamentous chicken skeletal muscle actin
    • Protein or peptide: Actin, alpha skeletal muscle, intermediate form
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: filamentous chicken skeletal muscle actin

SupramoleculeName: filamentous chicken skeletal muscle actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: chicken skeletal muscle actin purified from fresh chicken breast
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 5.4 kDa/nm

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

MacromoleculeName: Actin, alpha skeletal muscle, intermediate form / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Chicken (chicken)
Molecular weightTheoretical: 41.387227 KDa
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSY E LPDGQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITAL APSTMKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKCF

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
Details: 10 mM imidazole pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.01% NaN3
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K
Detailschicken cytoskeletal actin purified from chicken breast muscle

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Average exposure time: 10.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 229833 / Software - Name: RELION (ver. 3.0)
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6bno

Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
Final reconstructionApplied symmetry - Helical parameters - Δz: 27.19 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.85 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 228334
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6bno

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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