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- PDB-9si1: Mouse otoferlin (216-1931) in the lipid-free Ca2+-bound state, "c... -

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Basic information

Entry
Database: PDB / ID: 9si1
TitleMouse otoferlin (216-1931) in the lipid-free Ca2+-bound state, "closed-like" conformation
ComponentsOtoferlin
KeywordsMEMBRANE PROTEIN / Ferlin / otoferlin / multi-C2 domain / Ca2+-binding / membrane fusion
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
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCretu, C. / Moser, T.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)CR 937/2-1 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
DepositionAug 28, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 5, 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)200,77810
Polymers200,4181
Non-polymers3619
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: Mouse otoferlin (residues 216-1931) cloned in-frame with a twin-StrepII affinity tag and an HRV3C protease cleavage site
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
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: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "closed-like" conformation
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.188 MDa / Experimental value: YES
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-piperazineethanesulfonic acidHEPES1
2150 mMpotassium chlorideKCl1
31.25 %glycerolC3H8O31
40.5 mMtris(2-carboxyethyl)phosphineTCEP1
50.5 mMcalcium chlorideCaCl21
SpecimenConc.: 0.42 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Prior to vitrification, the Ca2+-bound lipid-free otoferlin (216-1931) was crosslinked with glutaraldehyde in batch and purified by size-exclusion chromatography
Specimen supportDetails: Harrick Plasma cleaner, medium settings, in air / Grid mesh size: 300 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: 2.46 sec. / Electron dose: 38.13 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12269 / Details: 11981 exposures were selected after curation
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv.4.7particle selection
2EPUv.3.6image acquisition
4cryoSPARCv.4.7CTF correction
7UCSF ChimeraXv.1.8model fitting
9cryoSPARCv.4.7initial Euler assignment
10cryoSPARCv.4.7final Euler assignment
11cryoSPARCv.4.7classification
12cryoSPARCv.4.73D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5201261
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66665 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 98.4 / Protocol: OTHER / Space: REAL
Atomic model buildingAccession code: 9QE2 / Details: lipid-bound otoferlin (216-1931) / Source name: Other / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.05 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004612419
ELECTRON MICROSCOPYf_angle_d0.985616814
ELECTRON MICROSCOPYf_chiral_restr0.05631800
ELECTRON MICROSCOPYf_plane_restr0.00872201
ELECTRON MICROSCOPYf_dihedral_angle_d7.23681650

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