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- PDB-7v74: Thermostabilized human prestin in complex with sulfate -

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Basic information

Entry
Database: PDB / ID: 7v74
TitleThermostabilized human prestin in complex with sulfate
Componentsprestin
KeywordsMEMBRANE PROTEIN / motor protein / prestin / SLC26A5 / electromotility
Function / homologyCHOLESTEROL / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsFutamata, H. / Fukuda, M. / Yamashita, K. / Nishizawa, T. / Nureki, O.
Funding support Japan, 1items
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, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / refine
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: prestin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,45312
Polymers82,5031
Non-polymers6,95011
Water00
1
A: prestin
hetero molecules

A: prestin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)178,90724
Polymers165,0072
Non-polymers13,90022
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (-1), (1)188.133336, 188.133336

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Components

#1: Protein prestin


Mass: 82503.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293SGNTI- / Production host: Homo sapiens (human)
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate


Mass: 760.076 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: prestin in complex with chloride ion / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0337 / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9REFMACmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 249144 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
Atomic model buildingPDB-ID: 5DA0

5da0


Accession code: 5DA0 / Source name: PDB / Type: experimental model
RefinementResolution: 3.63→3.63 Å / Cor.coef. Fo:Fc: 0.921 / SU B: 8.208 / SU ML: 0.133 / ESU R: 0.221
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34144 --
obs0.34144 124524 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 177.349 Å2
Refinement stepCycle: 1 / Total: 4874
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.0124976
ELECTRON MICROSCOPYr_bond_other_d00.0165243
ELECTRON MICROSCOPYr_angle_refined_deg2.021.6236720
ELECTRON MICROSCOPYr_angle_other_deg0.6151.54912088
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.0675594
ELECTRON MICROSCOPYr_dihedral_angle_2_deg22.46916.66742
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.72310.087806
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0880.2799
ELECTRON MICROSCOPYr_gen_planes_refined0.010.025322
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021042
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it28.20215.8672385
ELECTRON MICROSCOPYr_mcbond_other28.14815.8692385
ELECTRON MICROSCOPYr_mcangle_it37.08223.9182976
ELECTRON MICROSCOPYr_mcangle_other37.07623.9252977
ELECTRON MICROSCOPYr_scbond_it33.51420.1022591
ELECTRON MICROSCOPYr_scbond_other33.50820.1012592
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other48.84428.7413745
ELECTRON MICROSCOPYr_long_range_B_refined59.26321.0219829
ELECTRON MICROSCOPYr_long_range_B_other59.261321.0219830
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork2.741 9118 -
obs--100 %

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