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- EMDB-26460: cryo-EM structure of the ADP state wild type myosin-15-F-actin co... -

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Basic information

Entry
Database: EMDB / ID: EMD-26460
Titlecryo-EM structure of the ADP state wild type myosin-15-F-actin complex (symmetry expansion and re-centering)
Map data
Sample
  • Complex: the rigor state wild type myosin-15-F-actin complex
    • Complex: Actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Isoform 3 of Unconventional myosin-XV
      • Protein or peptide: Unconventional myosin-XV
    • Complex: chicken muscle regulatory light chain
      • Protein or peptide: regulatory light chain
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


Striated Muscle Contraction / stereocilium bundle / stereocilium / myosin complex / inner ear morphogenesis / response to light stimulus / cytoskeletal motor activity / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development ...Striated Muscle Contraction / stereocilium bundle / stereocilium / myosin complex / inner ear morphogenesis / response to light stimulus / cytoskeletal motor activity / striated muscle thin filament / skeletal muscle thin filament assembly / skeletal muscle fiber development / stress fiber / actin filament / locomotory behavior / sensory perception of sound / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / actin cytoskeleton / 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 / IQ calmodulin-binding motif / Variant SH3 domain ...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 / 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 / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / ATPase, nucleotide binding domain / PH-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Actin, alpha skeletal muscle / Unconventional myosin-XV
Similarity search - Component
Biological speciesGallus gallus (chicken) / Mus musculus (house mouse) / chicken (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsGong R / Reynolds MJ / Alushin GM
Funding support United States, 3 items
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
CitationJournal: 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.
History
DepositionMar 20, 2022-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26460.png

Downloads & links

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Map

FileDownload / File: emd_26460.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 512 pix.
= 527.36 Å		1.03 Å/pix.
x 512 pix.
= 527.36 Å		1.03 Å/pix.
x 512 pix.
= 527.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.074856766 - 0.10682238
Average (Standard dev.)3.066733e-05 (±0.0020277533)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_26460_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26460_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26460_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : the rigor state wild type myosin-15-F-actin complex

EntireName: the rigor state wild type myosin-15-F-actin complex
Components
  • Complex: the rigor state wild type myosin-15-F-actin complex
    • Complex: Actin
      • Protein or peptide: Actin, alpha skeletal muscle
    • Complex: Isoform 3 of Unconventional myosin-XV
      • Protein or peptide: Unconventional myosin-XV
    • Complex: chicken muscle regulatory light chain
      • Protein or peptide: regulatory light chain
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: the rigor state wild type myosin-15-F-actin complex

SupramoleculeName: the rigor state wild type myosin-15-F-actin complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1, #3
Molecular weightExperimental: 5.4 kDa/nm

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Supramolecule #2: Actin

SupramoleculeName: Actin / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Supramolecule #3: Isoform 3 of Unconventional myosin-XV

SupramoleculeName: Isoform 3 of Unconventional myosin-XV / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)

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Supramolecule #4: chicken muscle regulatory light chain

SupramoleculeName: chicken muscle regulatory light chain / type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: chicken (chicken)
Molecular weightTheoretical: 41.63143 KDa
SequenceString: DETTALVCDN GSGLVKAGFA GDDAPRAVFP SIVGRPRHQG VMVGMGQKDS YVGDEAQSKR GILTLKYPIE (HIC)GIITN WDD MEKIWHHTFY NELRVAPEEH PTLLTEAPLN PKANREKMTQ IMFETFNVPA MYVAIQAVLS LYASGRTTGI VLDSGDG VT HNVPIYEGYA ...String:
DETTALVCDN GSGLVKAGFA GDDAPRAVFP SIVGRPRHQG VMVGMGQKDS YVGDEAQSKR GILTLKYPIE (HIC)GIITN WDD MEKIWHHTFY NELRVAPEEH PTLLTEAPLN PKANREKMTQ IMFETFNVPA MYVAIQAVLS LYASGRTTGI VLDSGDG VT HNVPIYEGYA LPHAIMRLDL AGRDLTDYLM KILTERGYSF VTTAEREIVR DIKEKLCYVA LDFENEMATA ASSSSLEK S YELPDGQVIT IGNERFRCPE TLFQPSFIGM ESAGIHETTY NSIMKCDIDI RKDLYANNVM SGGTTMYPGI ADRMQKEIT ALAPSTMKIK IIAPPERKYS VWIGGSILAS LSTFQQMWIT KQEYDEAGPS IVHRKCF

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Macromolecule #2: Unconventional myosin-XV

MacromoleculeName: Unconventional myosin-XV / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 82.393773 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: EDGVEDMTQL EDLQETTVLA NLKTRFERNL IYTYIGSILV SVNPYRMFAI YGPEQVQQYS GRALGENPPH LFAIANLAFA KMLDAKQNQ CVIISGESGS GKTEATKLIL RCLAAMNQRR DVMQQIKILE ATPLLEAFGN AKTVRNDNSS RFGKFVEIFL E GGVICGAI ...String:
EDGVEDMTQL EDLQETTVLA NLKTRFERNL IYTYIGSILV SVNPYRMFAI YGPEQVQQYS GRALGENPPH LFAIANLAFA KMLDAKQNQ CVIISGESGS GKTEATKLIL RCLAAMNQRR DVMQQIKILE ATPLLEAFGN AKTVRNDNSS RFGKFVEIFL E GGVICGAI TSQYLLEKSR IVFQAKNERN YHIFYELLAG LPAQLRQAFS LQEAETYYYL NQGGNCEIAG KSDADDFRRL LA AMEVLGF TSEDQDSIFR ILASILHLGN VYFEKHETDA QEVASVVSAR EIQAVAELLQ VSPEGLQKAI TFKVTETIRE KIF TPLTVE SAVDARDAIA KVLYALLFGW LITRVNALVS PKQDTLSIAI LDIYGFEDLS FNSFEQLCIN YANENLQYLF NKIV FQEEQ EEYIREQMDW REIAFADNQP CINLISLKPY GILRILDDQC CFPQATDHTF LQKCHYHHGA NPLYSKPKMP LPEFT IKHY AGKVTYQVHK FLDKNHDQVR QDVLDLFVHS RTRVVAHLFS SHAAQTAPPR LGKSSSITRL YKAHTVAAKF QQSLLD LVE KMERCNPLFV RCLKPNHKKE PGLFEPDVMM AQLRYSGVLE TVRIRKEGFP VRLPFQVFID RYRCLVALKL NVPADGD MC VSLLSRLCTV TPDMYRVGIS KLFLKEHLHQ LLESMRERVQ NRAALTLQRY LRGFFIQRHF RSLRRKIILL QSRARGF

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Macromolecule #3: regulatory light chain

MacromoleculeName: regulatory light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 16.936826 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString:
VFAMFDQSQI QEFKEAFNMI DQNRDGFIDK EDLHDMLASL GKNPTDEYLD AMMNEAPGPI NFTMFLTMFG EKLNGTDPED VIRNAFACF DEEATGFIQE DYLRELLTTM GDRFTDEEVD ELYREAPIDK KGNFNYIEFT RILKHGA

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle 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 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: LEICA EM GP
Detailsthe ADP state wild type myosin-15 bound to F-actin

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 375696
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6bno

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 189104
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
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-7udu:
cryo-EM structure of the ADP state wild type myosin-15-F-actin complex (symmetry expansion and re-centering)

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