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- PDB-9qe2: Mouse otoferlin (216-1931) in complex with an MSP2N2 lipid nanodi... -

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Basic information

Entry
Database: PDB / ID: 9qe2
TitleMouse otoferlin (216-1931) in complex with an MSP2N2 lipid nanodisc (30 mol% DOPS, 10 mol% PI(4,5)P2)
ComponentsOtoferlin
KeywordsMEMBRANE PROTEIN / Ferlins / otoferlin / lipid nanodisc / synaptic vesicle / exocytosis / auditory nerve / deafness
Function / homology
Function and homology information


cell projection / sensory perception of sound / synaptic vesicle membrane / presynaptic membrane / basolateral plasma membrane / Golgi membrane / endoplasmic reticulum membrane / metal ion binding
Similarity search - Function
Ferlin B-domain / FerIin domain / Ferlin, C-terminal domain / Ferlin, second C2 domain / Ferlin family / Ferlin, third C2 domain / Ferlin, fourth C2 domain / Ferlin, fifth C2 domain / Ferlin, sixth C2 domain / Ferlin, first C2 domain ...Ferlin B-domain / FerIin domain / Ferlin, C-terminal domain / Ferlin, second C2 domain / Ferlin family / Ferlin, third C2 domain / Ferlin, fourth C2 domain / Ferlin, fifth C2 domain / Ferlin, sixth C2 domain / Ferlin, first C2 domain / : / FerB (NUC096) domain / FerI (NUC094) domain / Ferlin C-terminus / Ferlin dsRNA-binding domain-like domain / FerB / FerI / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily
Similarity search - Domain/homology
1,2-DICAPROYL-SN-PHOSPHATIDYL-L-SERINE / Otoferlin
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsCretu, C. / Moser, T.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)CR 937/2-1, EXC 2067/1-390729940 and MO896/5 Germany
CitationJournal: Sci Adv / Year: 2025
Title: Structure and function of otoferlin, a synaptic protein of sensory hair cells essential for hearing.
Authors: Han Chen / Constantin Cretu / Abigail Trebilcock / Natalia Evdokimova / Norbert Babai / Laura Feldmann / Florian Leidner / Fritz Benseler / Sophia Mutschall / Klara Esch / Csaba Zoltan ...Authors: Han Chen / Constantin Cretu / Abigail Trebilcock / Natalia Evdokimova / Norbert Babai / Laura Feldmann / Florian Leidner / Fritz Benseler / Sophia Mutschall / Klara Esch / Csaba Zoltan Kibedi Szabo / Vladimir Pena / Constantin Pape / Helmut Grubmüller / Nicola Strenzke / Nils Brose / Carolin Wichmann / Julia Preobraschenski / Tobias Moser /
Abstract: Hearing relies upon speedy synaptic transmission of sound information from inner hair cells (IHCs) to spiral ganglion neurons. To accomplish this, IHCs use a sophisticated presynaptic machinery ...Hearing relies upon speedy synaptic transmission of sound information from inner hair cells (IHCs) to spiral ganglion neurons. To accomplish this, IHCs use a sophisticated presynaptic machinery including the multi-C domain protein otoferlin that is affected by human deafness mutations. Otoferlin is essential for IHC exocytosis, but how it binds Ca and the target membrane to serve synaptic vesicle (SV) tethering, docking, and fusion remained unclear. Here, we obtained cryo-electron microscopy structures of otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding by otoferlin involves CB-CG domains and repositions CF and CG domains. Disruption of Ca-binding sites of the CD domain in mice altered synaptic sound encoding and eliminated the Ca cooperativity of IHC exocytosis, indicating that it requires the binding of several Ca-ions by otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV docking and support the role of otoferlin as Ca sensor of SV fusion in IHCs.
History
DepositionMar 7, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Otoferlin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,68912
Polymers200,4181
Non-polymers1,27211
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Otoferlin


Mass: 200417.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cytosolic domain of mouse otoferlin (residues 216-1931) expressed in Sf9 insect cells in frame with an N-terminal twin-StrepII tag.
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Otof / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0R4J1K2
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-PSF / 1,2-DICAPROYL-SN-PHOSPHATIDYL-L-SERINE / PHOSPHATIDYLSERINE


Mass: 455.437 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H34NO10P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Mouse otoferlin (residues 216-1931) in complex with an MSP2N2 lipid nanodisc (comprising 30 mol% DOPS and 10 mol% PI(4,5)P2)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acidHEPES1
2150 mMpotassium chlorideKCl1
31.25 % (v/v)glycerolC3H8O31
40.5 mMTris(2-carboxyethyl)phosphine hydrochlorideTCEP1
50.5 mMcalcium chlorideCaCl21
SpecimenConc.: 0.92 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Prior to cryo-EM grid preparation, the otoferlin (216-1931)-nanodisc complex was crosslinked in batch on ice with 0.07% (v/v) glutaraldehyde (GA) for 12 min. The reaction was quenched with ...Details: Prior to cryo-EM grid preparation, the otoferlin (216-1931)-nanodisc complex was crosslinked in batch on ice with 0.07% (v/v) glutaraldehyde (GA) for 12 min. The reaction was quenched with 50 mM L-Lysine and L-Arginine and the sample was purified on a Superose 6 10/300 Increase column.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.49 sec. / Electron dose: 37.85 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12458 / Details: 12448 movies were accepted after curation
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3390520
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 751004 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 102.02 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 103.83 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004212767
ELECTRON MICROSCOPYf_angle_d0.761617283
ELECTRON MICROSCOPYf_chiral_restr0.04821847
ELECTRON MICROSCOPYf_plane_restr0.00662266
ELECTRON MICROSCOPYf_dihedral_angle_d5.57551729

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