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- EMDB-54540: Cryo-EM structure of Gephyrin E domain in complex with Darpin 27F3 -

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Basic information

Entry
Database: EMDB / ID: EMD-54540
TitleCryo-EM structure of Gephyrin E domain in complex with Darpin 27F3
Map data
Sample
  • Complex: Gephyrin in complex with Darpin 27F3 partner
    • Protein or peptide: Isoform 5 of Gephyrin
    • Protein or peptide: Darpin
Keywordsscaffolding protein / postsynaptic density / protein-protein interaction / linker-mediated regulation / STRUCTURAL PROTEIN. / STRUCTURAL PROTEIN
Function / homology
Function and homology information


Molybdenum cofactor biosynthesis / glycine receptor clustering / molybdopterin cofactor biosynthetic process / establishment of synaptic specificity at neuromuscular junction / molybdopterin adenylyltransferase activity / nitrate reductase activity / molybdopterin molybdotransferase activity / molybdopterin adenylyltransferase / molybdopterin molybdotransferase / gamma-aminobutyric acid receptor clustering ...Molybdenum cofactor biosynthesis / glycine receptor clustering / molybdopterin cofactor biosynthetic process / establishment of synaptic specificity at neuromuscular junction / molybdopterin adenylyltransferase activity / nitrate reductase activity / molybdopterin molybdotransferase activity / molybdopterin adenylyltransferase / molybdopterin molybdotransferase / gamma-aminobutyric acid receptor clustering / postsynaptic specialization / inhibitory synapse / Mo-molybdopterin cofactor biosynthetic process / glycinergic synapse / molybdopterin cofactor binding / postsynaptic specialization, intracellular component / response to metal ion / neurotransmitter receptor localization to postsynaptic specialization membrane / synaptic transmission, GABAergic / protein targeting / synapse assembly / synaptic membrane / establishment of protein localization / tubulin binding / GABA-ergic synapse / cytoplasmic side of plasma membrane / molecular adaptor activity / dendritic spine / cytoskeleton / protein-macromolecule adaptor activity / postsynaptic membrane / postsynaptic density / postsynapse / signaling receptor binding / neuronal cell body / dendrite / ATP binding / metal ion binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Molybdenum cofactor biosynthesis proteins signature 2. / Molybdenum cofactor biosynthesis proteins signature 1. / MoeA, N-terminal and linker domain / MoeA, C-terminal, domain IV / MoeA, N-terminal and linker domain superfamily / MoeA, C-terminal, domain IV superfamily / Molybdopterin biosynthesis protein MoeA-like / MoeA N-terminal region (domain I and II) / MoeA C-terminal region (domain IV) / Molybdenum cofactor biosynthesis, conserved site ...Molybdenum cofactor biosynthesis proteins signature 2. / Molybdenum cofactor biosynthesis proteins signature 1. / MoeA, N-terminal and linker domain / MoeA, C-terminal, domain IV / MoeA, N-terminal and linker domain superfamily / MoeA, C-terminal, domain IV superfamily / Molybdopterin biosynthesis protein MoeA-like / MoeA N-terminal region (domain I and II) / MoeA C-terminal region (domain IV) / Molybdenum cofactor biosynthesis, conserved site / MoaB/Mog domain / MoaB/Mog-like domain superfamily / Probable molybdopterin binding domain / Probable molybdopterin binding domain
Similarity search - Domain/homology
Biological speciesRattus norvegicus (Norway rat) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsOrtiz-Lopez D / Hove T / Boettcher B / Schindelin H
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research Foundation (DFG)232550447 Germany
German Research Foundation (DFG)456578072 Germany
German Research Foundation (DFG)359471283 Germany
German Research Foundation (DFG)525040890 Germany
CitationJournal: Nat Commun / Year: 2026
Title: Cryo-EM structures of higher order Gephyrin oligomers reveal principles of inhibitory postsynaptic scaffold organization.
Authors: Diego Ortiz-López / Tamsanqa T Hove / Christiane Huhn / Serena Camuso / Pia M van Gen Hassend / Bodo Sander / Benjamin F N Campbell / Shiva K Tyagarajan / Andreas Plückthun / Christian G ...Authors: Diego Ortiz-López / Tamsanqa T Hove / Christiane Huhn / Serena Camuso / Pia M van Gen Hassend / Bodo Sander / Benjamin F N Campbell / Shiva K Tyagarajan / Andreas Plückthun / Christian G Specht / Hans M Maric / Bettina Böttcher / Hermann Schindelin /
Abstract: Gephyrin, the principal scaffolding protein of inhibitory postsynaptic densities, clusters glycine and GABA receptors via multivalent interactions. It features structured N and C terminal domains ...Gephyrin, the principal scaffolding protein of inhibitory postsynaptic densities, clusters glycine and GABA receptors via multivalent interactions. It features structured N and C terminal domains connected by an intrinsically disordered linker. Although the structural and functional properties of its terminal domains are well characterized, the mechanism by which full-length gephyrin organizes into higher-order complexes remains unresolved. Here, we combine biochemical reconstitution, cryo-electron microscopy, and mutational analyses to elucidate the structural logic of gephyrin oligomerization. We demonstrate that gephyrin adopts a stable dimeric assembly which constitutes the basic unit for both linear and oblique tetramers as well as linear hexameric arrangements. High resolution structures reveal a critical segment of the flexible linker that adopts two distinct conformations, one of which occludes the receptor-binding site. This segment harbors key phosphorylation sites, suggesting a regulatory control mechanism. Our findings redefine the architecture of inhibitory postsynaptic sites and reconcile gephyrin oligomerization models with published in-situ postsynaptic densities characterized by cryo-electron tomography.
History
DepositionJul 24, 2025-
Header (metadata) releaseJun 24, 2026-
Map releaseJun 24, 2026-
UpdateJun 24, 2026-
Current statusJun 24, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54540.png

Downloads & links

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Map

FileDownload / File: emd_54540.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 288 pix.
= 262.8 Å		0.91 Å/pix.
x 288 pix.
= 262.8 Å		0.91 Å/pix.
x 288 pix.
= 262.8 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9125 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.078
Minimum - Maximum-0.3017489 - 0.6162725
Average (Standard dev.)-0.00010850684 (±0.011952791)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 262.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_54540_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_54540_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Gephyrin in complex with Darpin 27F3 partner

EntireName: Gephyrin in complex with Darpin 27F3 partner
Components
  • Complex: Gephyrin in complex with Darpin 27F3 partner
    • Protein or peptide: Isoform 5 of Gephyrin
    • Protein or peptide: Darpin

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Supramolecule #1: Gephyrin in complex with Darpin 27F3 partner

SupramoleculeName: Gephyrin in complex with Darpin 27F3 partner / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Isoform 5 of Gephyrin

MacromoleculeName: Isoform 5 of Gephyrin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: molybdopterin adenylyltransferase
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 46.775766 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ITKVARRHRM SPFPLTSMDK AFITVLEMTP VLGTEIINYR DGMGRVLAQD VYAKDNLPPF PASVKDGYAV RAADGPGDRF IIGESQAGE QPTQTVMPGQ VMRVTTGAPI PCGADAVVQV EDTELIRESD DGTEELEVRI LVQARPGQDI RPIGHDIKRG E CVLAKGTH ...String:
ITKVARRHRM SPFPLTSMDK AFITVLEMTP VLGTEIINYR DGMGRVLAQD VYAKDNLPPF PASVKDGYAV RAADGPGDRF IIGESQAGE QPTQTVMPGQ VMRVTTGAPI PCGADAVVQV EDTELIRESD DGTEELEVRI LVQARPGQDI RPIGHDIKRG E CVLAKGTH MGPSEIGLLA TVGVTEVEVN KFPVVAVMST GNELLNPEDD LLPGKIRDSN RSTLLATIQE HGYPTINLGI VG DNPDDLL NALNEGISRA DVIITSGGVS MGEKDYLKQV LDIDLHAQIH FGRVFMKPGL PTTFATLDID GVRKIIFALP GNP VSAVVT CNLFVVPALR KMQGILDPRP TIIKARLSCD VKLDPRPEYH RCILTWHHQE PLPWAQSTGN QMSSRLMSMR SANG LLMLP PKTEQYVELH KGEVVDVMVI GRL

UniProtKB: Gephyrin

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Macromolecule #2: Darpin

MacromoleculeName: Darpin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.777062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRGSHHHHHH HHGSDLGKKL LEAARAGQDD EVRILMANGA DVNASDVVGQ TPLHLAAWSG HLEIVEVLLK TGADVNAEDM VGNTPLHLA AYVGHLEIVE VLLKAGADVN ADDFRGRTPL HLAAYYGHLE IVEVLLKHGA DVNAQDKFGK TPFDLAIDNG N EDIAEVLQ KAAKLNDYKD DDDK

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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

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
5.0 mMC2H6OS2-mercaptoethanol
1.0 mMC10H16N2O8EDTA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 3.5 sec. / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.5) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab initio reconstruction using CryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5) / Number images used: 780576
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.5)

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