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- PDB-6zs5: 3.5 A cryo-EM structure of human uromodulin filament core -

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Basic information

Entry
Database: PDB / ID: 6zs5
Title3.5 A cryo-EM structure of human uromodulin filament core
ComponentsUromodulin
KeywordsANTIMICROBIAL PROTEIN / Uromodulin / Umod / THP / immunoglobulin-like fold / Tamm-Horsfall protein / glycoprotein / ZP module / Zona Pellucida / fold complementation / beta-strand complementation / cryoSPARC / filament / soluble adhesion antagonist
Function / homology
Function and homology information


citric acid secretion / metanephric thick ascending limb development / metanephric distal convoluted tubule development / connective tissue replacement / protein transport into plasma membrane raft / Asparagine N-linked glycosylation / organ or tissue specific immune response / collecting duct development / metanephric ascending thin limb development / micturition ...citric acid secretion / metanephric thick ascending limb development / metanephric distal convoluted tubule development / connective tissue replacement / protein transport into plasma membrane raft / Asparagine N-linked glycosylation / organ or tissue specific immune response / collecting duct development / metanephric ascending thin limb development / micturition / urea transmembrane transport / protein localization to vacuole / regulation of protein transport / juxtaglomerular apparatus development / antibacterial innate immune response / intracellular chloride ion homeostasis / renal urate salt excretion / urate transport / glomerular filtration / renal sodium ion absorption / neutrophil migration / intracellular phosphate ion homeostasis / response to water deprivation / potassium ion homeostasis / regulation of urine volume / intracellular sodium ion homeostasis / endoplasmic reticulum organization / IgG binding / heterophilic cell-cell adhesion / extrinsic component of membrane / ciliary membrane / leukocyte cell-cell adhesion / cellular response to unfolded protein / cellular defense response / multicellular organismal response to stress / renal water homeostasis / side of membrane / : / ERAD pathway / RNA splicing / tumor necrosis factor-mediated signaling pathway / apoptotic signaling pathway / lipid metabolic process / autophagy / Golgi lumen / regulation of blood pressure / intracellular calcium ion homeostasis / spindle pole / response to lipopolysaccharide / defense response to Gram-negative bacterium / basolateral plasma membrane / cilium / apical plasma membrane / response to xenobiotic stimulus / inflammatory response / negative regulation of cell population proliferation / calcium ion binding / cell surface / endoplasmic reticulum / extracellular space / extracellular exosome / membrane
Similarity search - Function
Uromodulin-like, D8C domain / EGF domain / EGF domain / : / Zona pellucida domain, conserved site / ZP domain signature. / : / : / ZP-N domain / Zona pellucida, ZP-C domain ...Uromodulin-like, D8C domain / EGF domain / EGF domain / : / Zona pellucida domain, conserved site / ZP domain signature. / : / : / ZP-N domain / Zona pellucida, ZP-C domain / ZP-C domain / Zona pellucida (ZP) domain / ZP domain profile. / Zona pellucida domain / : / Calcium-binding EGF domain / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsStanisich, J.J. / Zyla, D. / Afanasyev, P. / Xu, J. / Pilhofer, M. / Boeringer, D. / Glockshuber, R.
Funding support Switzerland, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_176403/1 Switzerland
Swiss National Science Foundation31003A_156304 Switzerland
Swiss National Science Foundation31003A_179255 Switzerland
European Research Council (ERC)679209 Switzerland
CitationJournal: Elife / Year: 2020
Title: The cryo-EM structure of the human uromodulin filament core reveals a unique assembly mechanism.
Authors: Jessica J Stanisich / Dawid S Zyla / Pavel Afanasyev / Jingwei Xu / Anne Kipp / Eric Olinger / Olivier Devuyst / Martin Pilhofer / Daniel Boehringer / Rudi Glockshuber /
Abstract: The glycoprotein uromodulin (UMOD) is the most abundant protein in human urine and forms filamentous homopolymers that encapsulate and aggregate uropathogens, promoting pathogen clearance by urine ...The glycoprotein uromodulin (UMOD) is the most abundant protein in human urine and forms filamentous homopolymers that encapsulate and aggregate uropathogens, promoting pathogen clearance by urine excretion. Despite its critical role in the innate immune response against urinary tract infections, the structural basis and mechanism of UMOD polymerization remained unknown. Here, we present the cryo-EM structure of the UMOD filament core at 3.5 Å resolution, comprised of the bipartite zona pellucida (ZP) module in a helical arrangement with a rise of ~65 Å and a twist of ~180°. The immunoglobulin-like ZPN and ZPC subdomains of each monomer are separated by a long linker that interacts with the preceding ZPC and following ZPN subdomains by β-sheet complementation. The unique filament architecture suggests an assembly mechanism in which subunit incorporation could be synchronized with proteolytic cleavage of the C-terminal pro-peptide that anchors assembly-incompetent UMOD precursors to the membrane.
History
DepositionJul 15, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Uromodulin
D: Uromodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,9794
Polymers139,6432
Non-polymers1,3352
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5360 Å2
ΔGint-8 kcal/mol
Surface area15840 Å2
MethodPISA

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Components

#1: Protein Uromodulin / Tamm-Horsfall urinary glycoprotein / THP


Mass: 69821.680 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07911
#2: 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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#3: 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.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}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 human uromodulin filament core / Type: COMPLEX
Details: Native uromodulin was purified from healthy human urine.
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 1.28 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.2
Buffer componentConc.: 0.5 mM / Name: Ethylenediaminetetraacetic acid / Formula: EDTA
SpecimenConc.: 1.58 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: individual, isolated fibers
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 282 K
Details: 3.5 ul sample, 30 s wait time, 0.5 s drain time, 13.5 s blotting from the back

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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: 130000 X / Nominal defocus max: 3300 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9543 / Details: Data was joined from two sessions
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.18.2model refinement
10Rosettamodel refinement
11cryoSPARC2.15initial Euler assignment
12cryoSPARC2.15final Euler assignment
13cryoSPARC2.15classification
14cryoSPARC2.153D reconstruction
Image processingDetails: Motion correction with dose-weighting
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1310000
Details: Autopicking based on the 2D classes from manually chosen filaments
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145000 / Symmetry type: POINT
Atomic model buildingB value: 49.9714 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 4WRN

4wrn


Pdb chain-ID: A / Accession code: 4WRN / Pdb chain residue range: 400-710 / Source name: PDB / Type: experimental model

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