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- EMDB-25050: Tomogram of mouse stereocilia containing PCDH15 molecules labeled... -

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Entry
Database: EMDB / ID: EMD-25050
TitleTomogram of mouse stereocilia containing PCDH15 molecules labeled with 39G7-AuNPs
Map dataTomogram of mouse stereocilia containing PCDH15 molecules labeled with 39G7-AuNPs
Sample
  • Organelle or cellular component: Stereocilia labeled with 39G7-AuNP
    • Organelle or cellular component: Mouse stereocilia
    • Complex: 39G7-AuNP conjugate
Biological speciesMus musculus (house mouse) / Oryctolagus cuniculus (rabbit)
Methodelectron tomography / cryo EM
AuthorsElferich J / Clark S / Ge J / Goehring A / Matsui A / Gouaux E
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Elife / Year: 2021
Title: Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography.
Authors: Johannes Elferich / Sarah Clark / Jingpeng Ge / April Goehring / Aya Matsui / Eric Gouaux /
Abstract: Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor ...Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the 'tip link.' Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location, and conformational states of intact tip links, on stereocilia, remains unknown. Here, we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.
#1: Journal: Biorxiv / Year: 2021
Title: Molecular structure and conformation of stereocilia tip-links elucidated by cryo-electron tomography
Authors: Elferich J / Clark S / Ge J / Goehring A / Matsui A / Gouaux E
History
DepositionSep 29, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateJan 12, 2022-
Current statusJan 12, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_25050.png

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Map

FileDownload / File: emd_25050.map.gz / Format: CCP4 / Size: 746.9 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationTomogram of mouse stereocilia containing PCDH15 molecules labeled with 39G7-AuNPs
Voxel sizeX=Y=Z: 6.611 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)25.829845 (±35.03582)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-512
Dimensions14681042512
Spacing10421468512
CellA: 6888.662 Å / B: 9704.948 Å / C: 3384.832 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z6.6116.6116.611
M x/y/z10421468512
origin x/y/z0.0000.0000.000
length x/y/z6888.6629704.9483384.832
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS00-512
NC/NR/NS10421468512
D min/max/mean-128.000127.00025.830

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Supplemental data

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Sample components

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Entire : Stereocilia labeled with 39G7-AuNP

EntireName: Stereocilia labeled with 39G7-AuNP
Components
  • Organelle or cellular component: Stereocilia labeled with 39G7-AuNP
    • Organelle or cellular component: Mouse stereocilia
    • Complex: 39G7-AuNP conjugate

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Supramolecule #1: Stereocilia labeled with 39G7-AuNP

SupramoleculeName: Stereocilia labeled with 39G7-AuNP / type: organelle_or_cellular_component / ID: 1 / Parent: 0

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Supramolecule #2: Mouse stereocilia

SupramoleculeName: Mouse stereocilia / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: 39G7-AuNP conjugate

SupramoleculeName: 39G7-AuNP conjugate / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.4 / Details: DMEM/F12
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: Coated with polylysine
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: PELCO / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 41 / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: TomoAlign (ver. Jan2019) / Number images used: 41
CTF correctionSoftware - Name: IMOD

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