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- EMDB-11471: Extended cryo-EM map of native human uromodulin filament core at ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11471
TitleExtended cryo-EM map of native human uromodulin filament core at 4.7 A resolution
Map datasharpened map
Sample
  • Complex: Native human uromodulin filament core
    • Protein or peptide: Native human uromodulin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsStanisich JJ / Zyla D / Afanasyev P / Xu J / Pilhofer M / Boehringer 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 27, 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: 4.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11471.png

Downloads & links

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Map

FileDownload / File: emd_11471.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.084 Å
Density
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-5.4416604 - 15.860672
Average (Standard dev.)0.021716958 (±0.52223665)
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-5.44215.8610.022

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Supplemental data

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Mask #1

Fileemd_11471_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_11471_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_11471_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_11471_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: Native human 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: Native human uromodulin

MacromoleculeName: Native human uromodulin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: LLEHRLECGA NDMKVSLGKC QLKSLGFDKV FMY LSDSRC SGFNDRDNRD WVSVVTPARD GPCGTVLTRN ETHATYSNTL YLADEIIIRD LNIK INFAC SYPLDMKVSL KTALQPMVSA LNIRVGGTGM FTVRMALFQT PSYTQPYQGS SVTLS TEAF LYVGTMLDGG ...String:
LLEHRLECGA NDMKVSLGKC QLKSLGFDKV FMY LSDSRC SGFNDRDNRD WVSVVTPARD GPCGTVLTRN ETHATYSNTL YLADEIIIRD LNIK INFAC SYPLDMKVSL KTALQPMVSA LNIRVGGTGM FTVRMALFQT PSYTQPYQGS SVTLS TEAF LYVGTMLDGG DLSRFALLMT NCYATPSSNA TDPLKYFIIQ DRCPHTRDST IQVVEN GES SQGRFSVQMF RFAGNYDLVY LHCEVYLCDT MNEKCKPTCS G

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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 / 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: 485000 / Details: Manual picking of straight filaments
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER
Details: The 3D class from cryoSPARC was used as a starting model
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cisTEM (ver. 1)
Final 3D classificationNumber classes: 10 / Software - Name: cisTEM (ver. 1) / Details: only the best resolved 3D class was selected
Final angle assignmentType: NOT APPLICABLE / Software - Name: cisTEM (ver. 1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1) / Number images used: 330000
DetailsMotion correction with dose-weighting

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