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- EMDB-54809: Mouse otoferlin (216-1931) in the lipid-free Ca2+-bound state, "o... -

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Basic information

Entry
Database: EMDB / ID: EMD-54809
TitleMouse otoferlin (216-1931) in the lipid-free Ca2+-bound state, "open" conformation (class 1)
Map dataCalcium-bound lipid-free otoferlin, overall map (class 1)
Sample
  • Complex: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "open" conformation (class 1)
    • Protein or peptide: Otoferlin
  • Ligand: CALCIUM ION
KeywordsFerlin / otoferlin / multi-C2 domain / Ca2+-binding / membrane fusion / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsCretu C / Moser T
Funding support Germany, 1 items
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 18, 2025-
Header (metadata) releaseNov 5, 2025-
Map releaseNov 5, 2025-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54809.png

Downloads & links

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Map

FileDownload / File: emd_54809.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCalcium-bound lipid-free otoferlin, overall map (class 1)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.72 Å/pix.
x 360 pix.
= 259.2 Å		0.72 Å/pix.
x 360 pix.
= 259.2 Å		0.72 Å/pix.
x 360 pix.
= 259.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.102543294 - 0.16002807
Average (Standard dev.)-0.0001457397 (±0.0036650875)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_54809_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Calcium-bound lipid-free otoferlin, half map B (class 1)

Fileemd_54809_half_map_1.map
AnnotationCalcium-bound lipid-free otoferlin, half map B (class 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Calcium-bound lipid-free otoferlin, half map A (class 1)

Fileemd_54809_half_map_2.map
AnnotationCalcium-bound lipid-free otoferlin, half map A (class 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "o...

EntireName: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "open" conformation (class 1)
Components
  • Complex: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "open" conformation (class 1)
    • Protein or peptide: Otoferlin
  • Ligand: CALCIUM ION

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Supramolecule #1: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "o...

SupramoleculeName: Lipid-free mouse otoferlin (216-1931) in the Ca2+-bound state, "open" conformation (class 1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 188 KDa

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Macromolecule #1: Otoferlin

MacromoleculeName: Otoferlin / type: protein_or_peptide / ID: 1
Details: Mouse otoferlin (residues 216-1931) cloned in-frame with a twin-StrepII affinity tag and an HRV3C protease cleavage site
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 200.417609 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MASWSHPQFE KGGGSGGGSG GGSWSHPQFE KLEVLFQGPG SGDKDCEQSN AMEDLDHLAI QLGDGLDPDS VSLASVTALT SNVSNKRSK PDIKMEPSAG RPMDYQVSIT VIEARQLVGL NMDPVVCVEV GDDKKYTSMK ESTNCPYYNE YFVFDFHVSP D VMFDKIIK ...String:
MASWSHPQFE KGGGSGGGSG GGSWSHPQFE KLEVLFQGPG SGDKDCEQSN AMEDLDHLAI QLGDGLDPDS VSLASVTALT SNVSNKRSK PDIKMEPSAG RPMDYQVSIT VIEARQLVGL NMDPVVCVEV GDDKKYTSMK ESTNCPYYNE YFVFDFHVSP D VMFDKIIK ISVIHSKNLL RSGTLVGSFK MDVGTVYSQP EHQFHHKWAI LSDPDDISAG LKGYVKCDVA VVGKGDNIKT PH KANETDE DDIEGNLLLP EGVPPERQWA RFYVKIYRAE GLPRMNTSLM ANVKKAFIGE NKDLVDPYVQ VFFAGQKGKT SVQ KSSYEP LWNEQVVFTD LFPPLCKRMK VQIRDSDKVN DVAIGTHFID LRKISNDGDK GFLPTLGPAW VNMYGSTRNY TLLD EHQDL NEGLGEGVSF RARLMLGLAV EILDTSNPEL TSSTEVQVEQ ATPVSESCTG RMEEFFLFGA FLEASMIDRK NGDKP ITFE VTIGNYGNEV DGMSRPLRPR PRKEPGDEEE VDLIQNSSDD EGDEAGDLAS VSSTPPMRPQ ITDRNYFHLP YLERKP CIY IKSWWPDQRR RLYNANIMDH IADKLEEGLN DVQEMIKTEK SYPERRLRGV LEELSCGCHR FLSLSDKDQG RSSRTRL DR ERLKSCMREL ESMGQQAKSL RAQVKRHTVR DKLRSCQNFL QKLRFLADEP QHSIPDVFIW MMSNNKRIAY ARVPSKDL L FSIVEEELGK DCAKVKTLFL KLPGKRGFGS AGWTVQAKLE LYLWLGLSKQ RKDFLCGLPC GFEEVKAAQG LGLHSFPPI SLVYTKKQAF QLRAHMYQAR SLFAADSSGL SDPFARVFFI NQSQCTEVLN ETLCPTWDQM LVFDNLELYG EAHELRDDPP IIVIEIYDQ DSMGKADFMG RTFAKPLVKM ADEAYCPPRF PPQLEYYQIY RGSATAGDLL AAFELLQIGP SGKADLPPIN G PVDMDRGP IMPVPVGIRP VLSKYRVEVL FWGLRDLKRV NLAQVDRPRV DIECAGKGVQ SSLIHNYKKN PNFNTLVKWF EV DLPENEL LHPPLNIRVV DCRAFGRYTL VGSHAVSSLR RFIYRPPDRS APNWNTTGEV VVSMEPEEPV KKLETMVKLD ATS DAVVKV DVAEDEKERK KKKKKGPSEE PEEEEPDESM LDWWSKYFAS IDTMKEQLRQ HETSGTDLEE KEEMESAEGL KGPM KSKEK SRAAKEEKKK KNQSPGPGQG SEAPEKKKAK IDELKVYPKE LESEFDSFED WLHTFNLLRG KTGDDEDGST EEERI VGRF KGSLCVYKVP LPEDVSREAG YDPTYGMFQG IPSNDPINVL VRIYVVRATD LHPADINGKA DPYIAIKLGK TDIRDK ENY ISKQLNPVFG KSFDIEASFP MESMLTVAVY DWDLVGTDDL IGETKIDLEN RFYSKHRATC GIAQTYSIHG YNIWRDP MK PSQILTRLCK EGKVDGPHFG PHGRVRVANR VFTGPSEIED ENGQRKPTDE HVALSALRHW EDIPRVGCRL VPEHVETR P LLNPDKPGIE QGRLELWVDM FPMDMPAPGT PLDISPRKPK KYELRVIVWN TDEVVLEDDD FFTGEKSSDI FVRGWLKGQ QEDKQDTDVH YHSLTGEGNF NWRYLFPFDY LAAEEKIVMS KKESMFSWDE TEYKIPARLT LQIWDADHFS ADDFLGAIEL DLNRFPRGA KTAKQCTMEM ATGEVDVPLV SIFKQKRVKG WWPLLARNEN DEFELTGKVE AELHLLTAEE AEKNPVGLAR N EPDPLEK

UniProtKB: Otoferlin

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 7 / Formula: CA
Molecular weightTheoretical: 40.078 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

Concentration0.42 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMKClpotassium chloride
1.25 %C3H8O3glycerol
0.5 mMTCEPtris(2-carboxyethyl)phosphine
0.5 mMCaCl2calcium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: Harrick Plasma cleaner, medium settings, in air
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsPrior to vitrification, the Ca2+-bound lipid-free otoferlin (216-1931) was crosslinked with glutaraldehyde in batch and purified by size-exclusion chromatography

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
SoftwareName: EPU (ver. v.3.6)
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 12269 / Average exposure time: 2.46 sec. / Average electron dose: 38.13 e/Å2 / Details: 11981 exposures were selected after curation
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5201261
CTF correctionSoftware - Name: cryoSPARC (ver. v.4.7) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.4.7) / Number images used: 189916
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.4.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.4.7)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. v.4.7)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9qe2


Chain - Source name: Other / Chain - Initial model type: experimental model / Details: lipid-bound otoferlin (216-1931)
SoftwareName: UCSF ChimeraX (ver. v.1.8)
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-9seg:
Mouse otoferlin (216-1931) in the lipid-free Ca2+-bound state, "open" conformation (class 1)

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