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- EMDB-42112: Cryo-EM structure of dolphin Prestin in low Cl buffer -

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Basic information

Entry
Database: EMDB / ID: EMD-42112
TitleCryo-EM structure of dolphin Prestin in low Cl buffer
Map data
Sample
  • Complex: Dolphin Prestin solubilized in GDN in low Cl buffer
    • Protein or peptide: Prestin
  • Ligand: CHLORIDE IONChloride
KeywordsSolute Carrier / Electromotility / Voltage Sensitive / Mechanosensitive / MEMBRANE PROTEIN
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: 3.4 Å
AuthorsHaller P / Bavi N / Perozo E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)5R01DC019833-03 United States
CitationJournal: Elife / Year: 2023
Title: Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.
Authors: Xiaoxuan Lin / Patrick R Haller / Navid Bavi / Nabil Faruk / Eduardo Perozo / Tobin R Sosnick /
Abstract: Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs ...Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.
History
DepositionSep 25, 2023-
Header (metadata) releaseDec 13, 2023-
Map releaseDec 13, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42112.png

Downloads & links

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Map

FileDownload / File: emd_42112.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.068 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.7817509 - 3.142457
Average (Standard dev.)0.013492993 (±0.05920199)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.408 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_42112_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42112_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42112_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : Dolphin Prestin solubilized in GDN in low Cl buffer

EntireName: Dolphin Prestin solubilized in GDN in low Cl buffer
Components
  • Complex: Dolphin Prestin solubilized in GDN in low Cl buffer
    • Protein or peptide: Prestin
  • Ligand: CHLORIDE IONChloride

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Supramolecule #1: Dolphin Prestin solubilized in GDN in low Cl buffer

SupramoleculeName: Dolphin Prestin solubilized in GDN in low Cl buffer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Tursiops truncatus (common bottlenose dolphin)

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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

UniProtKB: Prestin

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Macromolecule #2: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
190.0 mMC8H18N2O4SHEPES
1.0 mMNaClSodium chloridesodium chloride

Details: 190 mM HEPES, 95 mM Tris-base, 1mM NaCl, 3mM DTT, 1mM EDTA, 0.02 % GDN
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: 3.5s Blot time, Blot force 1.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 170000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7s8x


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8uc1:
Cryo-EM structure of dolphin Prestin in low Cl buffer

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