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- EMDB-24932: Cryo-EM Structure of dolphin Prestin: Sensor Down II (Expanded II... -

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Basic information

Entry
Database: EMDB / ID: EMD-24932
TitleCryo-EM Structure of dolphin Prestin: Sensor Down II (Expanded II) state
Map data
Sample
  • Complex: Dolphin Prestin: Sensor Down II (Expanded II) state
    • Protein or peptide: Prestin
Function / homology
Function and homology information


cochlear outer hair cell electromotile response / secondary active sulfate transmembrane transporter activity / sensory perception of sound / regulation of cell shape / plasma membrane
Similarity search - Function
Sulphate anion transporter, conserved site / SLC26A transporters signature. / SLC26A/SulP transporter / SLC26A/SulP transporter domain / Sulfate permease family / STAS domain / STAS domain profile. / STAS domain / STAS domain superfamily
Similarity search - Domain/homology
Biological speciesTursiops truncatus (common bottlenose dolphin)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsBavi N / Clark MD / Contreras GF / Shen R / Reddy BG / Milewski W / Perozo E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01 DC019833 United States
CitationJournal: Nature / Year: 2021
Title: The conformational cycle of prestin underlies outer-hair cell electromotility.
Authors: Navid Bavi / Michael David Clark / Gustavo F Contreras / Rong Shen / Bharat G Reddy / Wieslawa Milewski / Eduardo Perozo /
Abstract: The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier. Knockout or impairment of ...The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier. Knockout or impairment of prestin causes severe hearing loss. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl or SO). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein-membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.
History
DepositionSep 20, 2021-
Header (metadata) releaseNov 3, 2021-
Map releaseNov 3, 2021-
UpdateDec 29, 2021-
Current statusDec 29, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7s9c
  • Surface level: 0.0124
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24932.png

Downloads & links

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Map

FileDownload / File: emd_24932.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.063 Å
Density
Contour LevelBy AUTHOR: 0.0124 / Movie #1: 0.0124
Minimum - Maximum-0.01576215 - 0.035583735
Average (Standard dev.)5.655637e-05 (±0.0019734881)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.128 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z272.128272.128272.128
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ364364364
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0160.0360.000

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Supplemental data

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Sample components

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Entire : Dolphin Prestin: Sensor Down II (Expanded II) state

EntireName: Dolphin Prestin: Sensor Down II (Expanded II) state
Components
  • Complex: Dolphin Prestin: Sensor Down II (Expanded II) state
    • Protein or peptide: Prestin

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Supramolecule #1: Dolphin Prestin: Sensor Down II (Expanded II) state

SupramoleculeName: Dolphin Prestin: Sensor Down II (Expanded II) state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Tursiops truncatus (common bottlenose dolphin)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Hek 293

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Macromolecule #1: Prestin

MacromoleculeName: Prestin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Tursiops truncatus (common bottlenose dolphin)
Molecular weightTheoretical: 80.97375 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDHVEETEIL AATQRYYVER PIFSHPVLQE RLHKKDKISE SIGDKLKQAF TCTPKKIRNI IYMFLPITKW LPAYRFKEYV LGDIVSGIS TGVLQLPQGL AFAMLAAVPP VFGLYSSFYP VIMYCFFGTS RHISIGPFAV ISLMIGGVAV RLVPDDIVIP G GVNATNST ...String:
MDHVEETEIL AATQRYYVER PIFSHPVLQE RLHKKDKISE SIGDKLKQAF TCTPKKIRNI IYMFLPITKW LPAYRFKEYV LGDIVSGIS TGVLQLPQGL AFAMLAAVPP VFGLYSSFYP VIMYCFFGTS RHISIGPFAV ISLMIGGVAV RLVPDDIVIP G GVNATNST EARDALRVKV AMSVTLLTGI IQFCLGVCRF GFVAIYLTEP LVRGFTTAAA VHVFTSMLKY LFGVKTKRYS GI FSVVYST VAVLQNVKNL NVCSLGVGLM VFGLLLGGKE FNERFKEKLP APIPLEFFAV VMGTGISAGF SLHESYNVDV VGT LPLGLL PPANPDTSLF HLVYVDAIAI AIVGFSVTIS MAKTLANKHG YQVDGNQELI ALGLCNSTGS LFQTFAISCS LSRS LVQEG TGGKTQLAGC LASLMILLVI LATGFLFESL PQAVLSAIVI VNLKGMFMQF SDLPFFWRTS KIELTIWLTT FVSSL FLGL DYGLITAVII ALMTVIYRTQ SPSYIVLGQL PDTDVYIDID AYEEVKEVPG IKIFQINAPI YYANSDLYSS ALKRKT GVN PAFILGARRK AMKKYAKEVG NANMANATVV KVDAEVDAED GTKPEEEEDE IKYPPIVTKS TLPEELQRFM PPGDNVH TI ILDFTQVNFM DSVGVKTLAG IVKEYGDVGI YVYLAGCSAQ VVSDLTQNQF FENPALLDLL FHSIHDAVLG SQVREALA E QEATAAPPQE DSEPNATPEA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.4 / Component - Concentration: 125.0 mM / Component - Formula: Na2SO4 / Component - Name: sodium sulfate
Details: 125 mM Na2SO4, 5mM Mg(OH)2, 20 Tris-OH, 10-15 mM methanesulfonic acid + 0.02 % GDN
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: 4.5 s blot times, blot force 3, and double filter papers on each side of the vitrobot..
DetailsMonodisperse peak at around 14 ml using SEC (Superose 6 column)

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 65000
FSC plot (resolution estimation)

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