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- EMDB-11388: 3.5 A cryo-EM structure of human uromodulin filament core -

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Basic information

Entry
Database: EMDB / ID: EMD-11388
Title3.5 A cryo-EM structure of human uromodulin filament core
Map datahalf map A
Sample
  • Complex: Native human uromodulin filament core
    • Protein or peptide: Uromodulin
Function / homology
Function and homology information


citric acid secretion / metanephric thick ascending limb development / metanephric distal convoluted tubule development / connective tissue replacement / urea transmembrane transport / Asparagine N-linked glycosylation / protein transport into plasma membrane raft / micturition / organ or tissue specific immune response / urate transport ...citric acid secretion / metanephric thick ascending limb development / metanephric distal convoluted tubule development / connective tissue replacement / urea transmembrane transport / Asparagine N-linked glycosylation / protein transport into plasma membrane raft / micturition / organ or tissue specific immune response / urate transport / metanephric ascending thin limb development / collecting duct development / protein localization to vacuole / renal urate salt excretion / regulation of protein transport / antibacterial innate immune response / intracellular phosphate ion homeostasis / intracellular chloride ion homeostasis / renal sodium ion absorption / juxtaglomerular apparatus development / glomerular filtration / neutrophil migration / response to water deprivation / intracellular sodium ion homeostasis / potassium ion homeostasis / regulation of urine volume / endoplasmic reticulum organization / renal water homeostasis / IgG binding / ciliary membrane / heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules / leukocyte cell-cell adhesion / extrinsic component of membrane / multicellular organismal response to stress / cellular response to unfolded protein / cellular defense response / side of membrane / chaperone-mediated protein folding / : / tumor necrosis factor-mediated signaling pathway / RNA splicing / apoptotic signaling pathway / cilium / lipid metabolic process / intracellular calcium ion homeostasis / autophagy / regulation of blood pressure / spindle pole / Golgi lumen / basolateral plasma membrane / defense response to Gram-negative bacterium / response to lipopolysaccharide / response to xenobiotic stimulus / inflammatory response / apical plasma membrane / negative regulation of cell population proliferation / calcium ion binding / cell surface / endoplasmic reticulum / extracellular space / extracellular exosome / membrane
Similarity search - Function
: / Zona pellucida domain, conserved site / Zona pellucida, ZP-C domain / ZP domain signature. / EGF domain / Zona pellucida-like domain / Zona pellucida (ZP) domain / EGF domain / ZP domain profile. / Zona pellucida domain ...: / Zona pellucida domain, conserved site / Zona pellucida, ZP-C domain / ZP domain signature. / EGF domain / Zona pellucida-like domain / Zona pellucida (ZP) domain / EGF 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) / Human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsStanisich JJ / Zyla D / Afanasyev P / Xu J / Pilhofer M / Boeringer D / Glockshuber R
Funding support Switzerland, 4 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_176403/1 Switzerland
Swiss National Science Foundation31003A_179255 Switzerland
Swiss National Science Foundation31003A_156304 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, 2020-
Header (metadata) releaseSep 2, 2020-
Map releaseSep 2, 2020-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zs5
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zya
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zs5
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zya
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11388.png

Downloads & links

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Map

FileDownload / File: emd_11388.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhalf map A
Voxel sizeX=Y=Z: 1.084 Å
Density
Contour LevelBy AUTHOR: 1 / Movie #1: 1
Minimum - Maximum-2.3153067 - 6.7981424
Average (Standard dev.)0.000833728 (±0.081922434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 346.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0841.0841.084
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z346.880346.880346.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-2.3156.7980.001

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Supplemental data

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Mask #1

Fileemd_11388_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: density modified main map

Fileemd_11388_additional.map
Annotationdensity modified main map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_11388_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 B

Fileemd_11388_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native human uromodulin filament core

EntireName: Native human uromodulin filament core
Components
  • Complex: Native human uromodulin filament core
    • Protein or peptide: Uromodulin

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Supramolecule #1: Native human uromodulin filament core

SupramoleculeName: Native human uromodulin filament core / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Native uromodulin was purified from healthy human urine.
Source (natural)Organism: Homo sapiens (human)
Molecular weightExperimental: 1.28 kDa/nm

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Macromolecule #1: Uromodulin

MacromoleculeName: Uromodulin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Human (human)
Molecular weightTheoretical: 69.82168 KDa
SequenceString: MGQPSLTWML MVVVASWFIT TAATDTSEAR WCSECHSNAT CTEDEAVTTC TCQEGFTGDG LTCVDLDECA IPGAHNCSAN SSCVNTPGS FSCVCPEGFR LSPGLGCTDV DECAEPGLSH CHALATCVNV VGSYLCVCPA GYRGDGWHCE CSPGSCGPGL D CVPEGDAL ...String:
MGQPSLTWML MVVVASWFIT TAATDTSEAR WCSECHSNAT CTEDEAVTTC TCQEGFTGDG LTCVDLDECA IPGAHNCSAN SSCVNTPGS FSCVCPEGFR LSPGLGCTDV DECAEPGLSH CHALATCVNV VGSYLCVCPA GYRGDGWHCE CSPGSCGPGL D CVPEGDAL VCADPCQAHR TLDEYWRSTE YGEGYACDTD LRGWYRFVGQ GGARMAETCV PVLRCNTAAP MWLNGTHPSS DE GIVSRKA CAHWSGHCCL WDASVQVKAC AGGYYVYNLT APPECHLAYC TDPSSVEGTC EECSIDEDCK SNNGRWHCQC KQD FNITDI SLLEHRLECG ANDMKVSLGK CQLKSLGFDK VFMYLSDSRC SGFNDRDNRD WVSVVTPARD GPCGTVLTRN ETHA TYSNT LYLADEIIIR DLNIKINFAC SYPLDMKVSL KTALQPMVSA LNIRVGGTGM FTVRMALFQT PSYTQPYQGS SVTLS TEAF LYVGTMLDGG DLSRFALLMT NCYATPSSNA TDPLKYFIIQ DRCPHTRDST IQVVENGESS QGRFSVQMFR FAGNYD LVY LHCEVYLCDT MNEKCKPTCS GTRFRSGSVI DQSRVLNLGP ITRKGVQATV SRAFSSLGLL KVWLPLLLSA TLTLTFQ

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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

Concentration1.58 mg/mL
BufferpH: 8.2 / Component - Concentration: 0.5 mM / Component - Formula: EDTAEthylenediaminetetraacetic acid / Component - Name: Ethylenediaminetetraacetic acid
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3.0 nm
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 282 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5 ul sample, 30 s wait time, 0.5 s drain time, 13.5 s blotting from the back.
Detailsindividual, isolated fibers

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.3 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 2 / Number real images: 9543 / Average exposure time: 6.0 sec. / Average electron dose: 45.0 e/Å2 / Details: Data was joined from two sessions
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1310000
Details: Autopicking based on the 2D classes from manually chosen filaments
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: Ab initio map generated in cryoSPARC 2
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 2.15)
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. 2.15) / Details: only the best resolved 3D class was selected
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 2.15)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 145000
DetailsMotion correction with dose-weighting

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Atomic model buiding 1

Initial modelPDB ID:

4wrn


Chain - Chain ID: A / Chain - Residue range: 400-710
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 49.9714 / Target criteria: Correlation coefficient
Output model

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

PDB-6zya:
Extended human uromodulin filament core at 3.5 A resolution

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