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- PDB-28yj: Molecular basis of ZPD homopolymerization: cryo-EM structure of a... -

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Basic information

Entry
Database: PDB / ID: 28yj
TitleMolecular basis of ZPD homopolymerization: cryo-EM structure of a native vertebrate egg coat filament
ComponentsUromodulin
KeywordsSTRUCTURAL PROTEIN / Epidermal 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 / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsBanjara, S. / Okumura, H. / Jovine, L.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Swedish Research Council2020-04936 Sweden
Swedish Research Council2024-05336 Sweden
Knut and Alice Wallenberg Foundation2018.0042 Sweden
Citation
Journal: To Be Published
Title: AlphaFold as a Prior: Experimental Structure Determination Conditioned on a Pretrained Neural Network
Authors: Fadini, A. / Li, M. / McCoy, A.J. / Banjara, S. / Okumura, H. / Napier, E. / Fontana, P. / Khan, A.R. / Jovine, L. / Terwilliger, T.C. / Read, R.J. / Hekstra, D.R. / AlQuraishi, M.
#1: Journal: Biochem J / Year: 2004
Title: A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.
Authors: Hiroki Okumura / Yoshinori Kohno / Yuki Iwata / Hitoshi Mori / Naohito Aoki / Chihiro Sato / Ken Kitajima / Daita Nadano / Tsukasa Matsuda /
Abstract: Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We ...Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.
#2: Journal: Curr Top Dev Biol / Year: 2018
Title: Egg-Coat and Zona Pellucida Proteins of Chicken as a Typical Species of Aves.
Authors: Shunsuke Nishio / Hiroki Okumura / Tsukasa Matsuda /
Abstract: Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. ...Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.
#3: Journal: EMBO J / Year: 2020
Title: Cryo-EM structure of native human uromodulin, a zona pellucida module polymer.
Authors: Alena Stsiapanava / Chenrui Xu / Martina Brunati / Sara Zamora-Caballero / Céline Schaeffer / Marcel Bokhove / Ling Han / Hans Hebert / Marta Carroni / Shigeki Yasumasu / Luca Rampoldi / ...Authors: Alena Stsiapanava / Chenrui Xu / Martina Brunati / Sara Zamora-Caballero / Céline Schaeffer / Marcel Bokhove / Ling Han / Hans Hebert / Marta Carroni / Shigeki Yasumasu / Luca Rampoldi / Bin Wu / Luca Jovine /
Abstract: Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous ...Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo-electron microscopy study of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of heteromeric vertebrate egg coat filaments identify a common sperm-binding region at the interface between subunits.
#4: Journal: 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, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 18, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 18, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Uromodulin
B: Uromodulin
C: Uromodulin
D: Uromodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,48012
Polymers147,3014
Non-polymers5,1798
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
Uromodulin / Zona pellucida protein D


Mass: 36825.234 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / Organ: Ovary
Plasmid details: Zona pellucida (specialized extracellular matrix surrounding the oocyte)
Tissue: Oocyte / References: UniProt: Q766V2
#2: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken)
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-6[DManpa1-3]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-e1_e3-f1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Native chicken ZPD homopolymeric filament / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Cellular location: Zona pellucida (specialized extracellular matrix surrounding the oocyte)
Organ: Ovary / Tissue: Oocyte
Buffer solutionpH: 7
Buffer componentConc.: 10 mM / Name: HEPES / Formula: C8H18N2O4S
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 700 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: 2.75 sec. / Electron dose: 53 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 19953
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
7UCSF Chimeramodel fitting
8ISOLDEmodel fitting
9Cootmodel fitting
14cryoSPARC3D reconstruction
15EMReady3D reconstruction
16REFMACmodel refinement
17Servalcatmodel refinement
18PHENIX2.0_5936model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1165059
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 498339 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Model 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 ...Details: Model 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.
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 4.6 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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