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- EMDB-31759: Thermostabilized human prestin in complex with salicylate -

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Basic information

Entry
Database: EMDB / ID: EMD-31759
TitleThermostabilized human prestin in complex with salicylate
Map datapostprocess_masked.mrc from RELION
Sample
  • Complex: prestin in complex with chloride ion
    • Protein or peptide: prestin
  • Ligand: 2-HYDROXYBENZOIC ACIDSalicylic acid
  • Ligand: CHOLESTEROL
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsFutamata H / Fukuda M / Yamashita K / Nishizawa T / Nureki O
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of thermostabilized prestin provide mechanistic insights underlying outer hair cell electromotility.
Authors: Haon Futamata / Masahiro Fukuda / Rie Umeda / Keitaro Yamashita / Atsuhiro Tomita / Satoe Takahashi / Takafumi Shikakura / Shigehiko Hayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / ...Authors: Haon Futamata / Masahiro Fukuda / Rie Umeda / Keitaro Yamashita / Atsuhiro Tomita / Satoe Takahashi / Takafumi Shikakura / Shigehiko Hayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Kazuaki Homma / Osamu Nureki /
Abstract: Outer hair cell elecromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized prestin (Pres), complexed with ...Outer hair cell elecromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized prestin (Pres), complexed with chloride, sulfate, or salicylate at 3.52-3.63 Å resolutions. The central positively-charged cavity allows flexible binding of various anion species, which likely accounts for the known distinct modulations of nonlinear capacitance (NLC) by different anions. Comparisons of these Pres structures with recent prestin structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insights into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.
History
DepositionAug 21, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31759.png

Downloads & links

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Map

FileDownload / File: emd_31759.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocess_masked.mrc from RELION
Voxel sizeX=Y=Z: 1.10667 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.18893655 - 0.28762716
Average (Standard dev.)0.0012412918 (±0.009328953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 188.13391 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_31759_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Half map: run half1 class001 unfil.mrc from RELION

Fileemd_31759_half_map_1.map
Annotationrun_half1_class001_unfil.mrc from RELION
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: run half2 class001 unfil.mrc from RELION

Fileemd_31759_half_map_2.map
Annotationrun_half2_class001_unfil.mrc from RELION
Projections & Slices
AxesZYX

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Slices (1/2)
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Sample components

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Entire : prestin in complex with chloride ion

EntireName: prestin in complex with chloride ion
Components
  • Complex: prestin in complex with chloride ion
    • Protein or peptide: prestin
  • Ligand: 2-HYDROXYBENZOIC ACIDSalicylic acid
  • Ligand: CHOLESTEROL
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

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Supramolecule #1: prestin in complex with chloride ion

SupramoleculeName: prestin in complex with chloride ion / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: prestin

MacromoleculeName: prestin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 82.503375 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDHAEENEIL AATQRYVVER PVYSQELLEE ELEKKDRVPK TLGDKLKKSF RCSPKKAKNL LLSFFPILEW LPKYNLKEWL LGDLIAGLT VGSLQIPQGI AFALLAGLPP IYGLYSSFFP PLIYAFFGTS RHISVGPFAV VSLLVGSVVE RLVPDDIVLP G GVNATNGT ...String:
MDHAEENEIL AATQRYVVER PVYSQELLEE ELEKKDRVPK TLGDKLKKSF RCSPKKAKNL LLSFFPILEW LPKYNLKEWL LGDLIAGLT VGSLQIPQGI AFALLAGLPP IYGLYSSFFP PLIYAFFGTS RHISVGPFAV VSLLVGSVVE RLVPDDIVLP G GVNATNGT EARDALRVQV AFTLTFLAGI IQLALGLLRL GFLVDFLSEP LISGFTTGAA IHILLSQLKY LLGLKIPRHS GP FSVIYSV ISVFHNIPNT NIATLGVSLL SFVLLLVVKE LNKRFKKKLP VPIPAELIVV ILATLISYYF NLAEKYGVSI VGH IPKGLP PPSVPDLSLF PLVIGDAIAI AIVGLAVSIS VGKTFAKKHG YQIDGNQELI ALGLMNIVGS FFSCYPATGS FSRS AVNES AGGKTQVAGI VAALVVLLVL LFLGPLFYYL PKAVLAAIII VNLKGLLMQF KDAPKLWKVD KLDFLIWLVT FLGVV FLSV EIGLLVGVGF SLLTVLLRTQ RPRTSVLGRI PGTDIYRDVK QYPEAEEVPG VKIFRIDSPI YFANSEYFKE RLKRKT GVD PVKVLAARKK ALKKIEKEIK KANLANKTVV KADAEVDGED ATKPEEEDGE VKYPPIVIQS DWPSELPRFV PPKVDFH TL ILDFSAVSFV DTVGVKTLKE IVKEYREIGV QVYLAGCNAS VVEKLERGGF FDDGITKEHL FLSVHDAVLF AQARKALA E QEASAPPSQE DLEPNATPAT PEAGTENLYF QG

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Macromolecule #2: 2-HYDROXYBENZOIC ACID

MacromoleculeName: 2-HYDROXYBENZOIC ACID / type: ligand / ID: 2 / Number of copies: 1 / Formula: SAL
Molecular weightTheoretical: 138.121 Da
Chemical component information

ChemComp-SAL:
2-HYDROXYBENZOIC ACID / Salicylic acid

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 2 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #4: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 4 / Number of copies: 8 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM / POPC

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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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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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 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 113410
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5da0

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-7v75:
Thermostabilized human prestin in complex with salicylate

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