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- EMDB-56971: Molecular basis of ZPD homopolymerization: cryo-EM structure of a... -

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Basic information

Entry
Database: EMDB / ID: EMD-56971
TitleMolecular basis of ZPD homopolymerization: cryo-EM structure of a native vertebrate egg coat filament
Map datacryoSPARC map
Sample
  • Complex: Native chicken ZPD homopolymeric filament
    • Protein or peptide: Uromodulin
KeywordsEpidermal growth factor domain / EGF domain / zona pellucida module / zona pellucida domain / ZP module / ZP domain / ZP-N domain / ZP-C domain / interdomain linker / extracellular matrix / glycoprotein / N-glycan / structural protein / protein filament / protein polymerization / fertilization / egg coat
Function / homology
Function and homology information


: / : / ZP-N domain / Zona pellucida, ZP-C domain / ZP-C domain / Zona pellucida (ZP) domain / ZP domain profile. / Zona pellucida domain / EGF-like domain / Epidermal growth factor-like domain. ...: / : / ZP-N domain / Zona pellucida, ZP-C domain / ZP-C domain / Zona pellucida (ZP) domain / ZP domain profile. / Zona pellucida domain / EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsBanjara S / Okumura H / Jovine L
Funding support Sweden, 3 items
OrganizationGrant numberCountry
Swedish Research Council2020-04936 Sweden
Swedish Research Council2024-05336 Sweden
Knut and Alice Wallenberg Foundation2018.0042 Sweden
CitationJournal: Cell / Year: 2024
Title: ZP2 cleavage blocks polyspermy by modulating the architecture of the egg coat.
Authors: Shunsuke Nishio / Chihiro Emori / Benjamin Wiseman / Dirk Fahrenkamp / Elisa Dioguardi / Sara Zamora-Caballero / Marcel Bokhove / Ling Han / Alena Stsiapanava / Blanca Algarra / Yonggang Lu ...Authors: Shunsuke Nishio / Chihiro Emori / Benjamin Wiseman / Dirk Fahrenkamp / Elisa Dioguardi / Sara Zamora-Caballero / Marcel Bokhove / Ling Han / Alena Stsiapanava / Blanca Algarra / Yonggang Lu / Mayo Kodani / Rachel E Bainbridge / Kayla M Komondor / Anne E Carlson / Michael Landreh / Daniele de Sanctis / Shigeki Yasumasu / Masahito Ikawa / Luca Jovine /
Abstract: Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N- ...Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm.
History
DepositionMar 2, 2026-
Header (metadata) releaseMar 18, 2026-
Map releaseMar 18, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56971.png

Downloads & links

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Map

FileDownload / File: emd_56971.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoSPARC map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 512 pix.
= 375.603 Å		0.73 Å/pix.
x 512 pix.
= 375.603 Å		0.73 Å/pix.
x 512 pix.
= 375.603 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7336 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.10123206 - 0.33351332
Average (Standard dev.)-0.0004094335 (±0.0048670517)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 375.6032 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: LocScale-2.0 feature-enhanced map, boxed around model

Fileemd_56971_additional_1.map
AnnotationLocScale-2.0 feature-enhanced map, boxed around model
Projections & Slices
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Additional map: EMReady2 postprocessed map, boxed around model

Fileemd_56971_additional_2.map
AnnotationEMReady2 postprocessed map, boxed around model
Projections & Slices
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Half map: cryoSPARC half-map 1

Fileemd_56971_half_map_1.map
AnnotationcryoSPARC half-map 1
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Half map: cryoSPARC half-map 2

Fileemd_56971_half_map_2.map
AnnotationcryoSPARC half-map 2
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AxesZYX

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Sample components

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Entire : Native chicken ZPD homopolymeric filament

EntireName: Native chicken ZPD homopolymeric filament
Components
  • Complex: Native chicken ZPD homopolymeric filament
    • Protein or peptide: Uromodulin

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Supramolecule #1: Native chicken ZPD homopolymeric filament

SupramoleculeName: Native chicken ZPD homopolymeric filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gallus gallus (chicken) / Organ: Ovary / Tissue: Oocyte
Location in cell: Zona pellucida (specialized extracellular matrix surrounding the oocyte)

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Macromolecule #1: Uromodulin

MacromoleculeName: Uromodulin / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken) / Organ: Ovary / Tissue: Oocyte
Molecular weightTheoretical: 36.825234 KDa
SequenceString: NKSELVSPHN SRGRFALRAK RSSDACVPNP CQHHGGCQVI EDRPICSCKP GFTGAFCQDV VLKLACEEEH MKMMVRKEVF ELLKIPREL VHLKNQACKV SEREEEGEMF FAATLTGENH TACGSVIQQN SSHVSYSNII ETGREAHRGV ISRSFQLEVH F SCVYAYEQ ...String:
NKSELVSPHN SRGRFALRAK RSSDACVPNP CQHHGGCQVI EDRPICSCKP GFTGAFCQDV VLKLACEEEH MKMMVRKEVF ELLKIPREL VHLKNQACKV SEREEEGEMF FAATLTGENH TACGSVIQQN SSHVSYSNII ETGREAHRGV ISRSFQLEVH F SCVYAYEQ VVKMPFALTP VDKLVQFMVR EGHFNVSMRL YKTASYLEPY DLLTAAVPIT DTLYVMLKIE GQHQLRYFLL SV EDCWATP SADPYQDVLH ELIEQGCPHD ETVTYLNAIG ESTTAKFSFQ MFQFVGYPKV FLHCRVRLCL PDGPEPCAKQ CPT LWRS

UniProtKB: Uromodulin

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7 / Component - Concentration: 10.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 19953 / Average exposure time: 2.75 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.7000000000000001 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: 1165059
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: cryoSPARC, EMReady) / Number images used: 498339
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsModel building was initiated using a local installation of AlphaFold 3 to predict a minimal filament fragment comprising one full-length subunit (chain A) and two partial subunits (chains B and C). The top-ranked prediction was rigid-body fitted into an initial 8.6 A-resolution map (postprocessed with EMReady2) using UCSF Chimera, followed by flexible fitting with Namdinator. Non-resolved terminal regions were trimmed, and well-defined N-glycan densities were manually built in Coot. The model was refined by real-space refinement in Phenix using NCS constraints and increased non-bonded interaction weights, followed by ADP refinement against the unsharpened map. This model served as a starting point for extension with an additional EGF and ZP-N domain from a fourth subunit (chain D). The extended model was docked into the present 4.6 A-resolution map, manually adjusted, and subjected to flexible fitting using the cryo-EM minimizer from cg2all; subsequently, it was refined using Refmac Servalcat task of CCP-EM Doppio, applying global NCS restraints, ProSMART-derived self-restraints, and increased non-bonded interaction weights. Following additional rounds of manual model rebuilding in Coot and real-space refinement in PHENIX (as described above), with positional refinement performed against a LocScale2-postprocessed map and ADP refinement against the unsharpened map, the model was validated using MolProbity and PHENIX. Note that the EGF domain of chain A (and, to a lesser extent, portions of its ZP-N domain near the postprocessed map boundary and the distal regions of the EGF domains in chains C and D) are weakly defined in the density, consistent with their elevated B-factors. These regions were retained in the model to preserve biological completeness, with their conformations constrained by NCS during refinement.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-28yj:
Molecular basis of ZPD homopolymerization: cryo-EM structure of a native vertebrate egg coat filament

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