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- PDB-8jj5: Porcine uroplakin complex -

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Basic information

Entry
Database: PDB / ID: 8jj5
TitlePorcine uroplakin complex
Components
  • Tetraspanin
  • UPK1B
  • Uroplakin 2
  • Uroplakin 3A
KeywordsMEMBRANE PROTEIN / Urothelium / asymmetric unit membrane / urinary bladder / urinary tract infection / lipid raft
Function / homology
Function and homology information


apical plasma membrane urothelial plaque / urea transport / urinary bladder development / water transport / sodium ion homeostasis / potassium ion homeostasis / epithelial cell differentiation / kidney development / cell morphogenesis / membrane / plasma membrane
Similarity search - Function
Uroplakin-3a / Uroplakin-3 / Tetraspanin, animals / Tetraspanin, EC2 domain superfamily / Tetraspanin/Peripherin / Tetraspanin family
Similarity search - Domain/homology
Uroplakin 3A / UPK1B / Tetraspanin
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsOda, T. / Yanagisawa, H. / Kikkawa, M.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP21H02654 Japan
Japan Society for the Promotion of Science (JSPS)JP22H05538 Japan
Japan Society for the Promotion of Science (JSPS)JP21H05248 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121002 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121002 Japan
CitationJournal: Commun Biol / Year: 2023
Title: Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.
Authors: Haruaki Yanagisawa / Yoshihiro Kita / Toshiyuki Oda / Masahide Kikkawa /
Abstract: The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane ...The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.
History
DepositionMay 29, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 8, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tetraspanin
B: Uroplakin 2
C: Uroplakin 3A
D: UPK1B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,03310
Polymers108,2444
Non-polymers2,7896
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules ABCD

#1: Protein Tetraspanin


Mass: 28747.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Urinary bladder / Tissue: Epithelium / References: UniProt: Q06AT5
#2: Protein Uroplakin 2


Mass: 19299.869 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: NCBI Reference Sequence: NP_999177.1 / Source: (natural) Sus scrofa (pig) / Organ: Urinary bladder / Tissue: Epithelium
#3: Protein Uroplakin 3A


Mass: 30339.234 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Urinary bladder / Tissue: Epithelium / References: UniProt: A0A287AEW0
#4: Protein UPK1B / Uroplakin-1b


Mass: 29857.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Urinary bladder / Tissue: Epithelium / References: UniProt: Q06AT4

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Sugars , 4 types, 6 molecules

#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: 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.: 1
Source method: isolated from a genetically manipulated source
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
#7: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 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]{}}}}LINUCSPDB-CARE
#8: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Uroplakin complex / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Asymmetric unit membrane isolated by sarkosyl extraction
Entity ID: #1-#4 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig) / Organ: Urinary bladder / Tissue: Urothelium
Buffer solutionpH: 7.4
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 609567 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421012
ELECTRON MICROSCOPYf_angle_d0.67528638
ELECTRON MICROSCOPYf_dihedral_angle_d5.6862865
ELECTRON MICROSCOPYf_chiral_restr0.0433273
ELECTRON MICROSCOPYf_plane_restr0.0053600

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