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- EMDB-10336: Respiratory mucin MUC5B: C-terminal dimerization domain structure -

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Basic information

Entry
Database: EMDB / ID: EMD-10336
TitleRespiratory mucin MUC5B: C-terminal dimerization domain structure
Map data
SampleDimeric structure of C-terminal MUC5B with C2 symmetry:
MUC5B
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsLockhart-Lockhart MP / Ridley C / Collins RF / Baldock C / Thornton DJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/R002800/1 United Kingdom
CitationJournal: J. Biol. Chem. / Year: 2019
Title: The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.
Authors: Caroline Ridley / Michael P Lockhart-Cairns / Richard F Collins / Thomas A Jowitt / Durai B Subramani / Mehmet Kesimer / Clair Baldock / David J Thornton /
Abstract: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is ...Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multi-angle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.
History
DepositionSep 25, 2019-
Header (metadata) releaseOct 9, 2019-
Map releaseOct 9, 2019-
UpdateOct 9, 2019-
Current statusOct 9, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0728
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0728
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10336.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.04 Å/pix.
x 256 pix.
= 521.498 Å
2.04 Å/pix.
x 256 pix.
= 521.498 Å
2.04 Å/pix.
x 256 pix.
= 521.498 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.0371 Å
Density
Contour LevelBy AUTHOR: 0.0728 / Movie #1: 0.0728
Minimum - Maximum-0.21519406 - 0.88516355
Average (Standard dev.)-0.00033244176 (±0.018416813)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 521.4976 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.03710156252.03710156252.0371015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z521.498521.498521.498
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2150.885-0.000

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Supplemental data

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Sample components

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Entire Dimeric structure of C-terminal MUC5B with C2 symmetry

EntireName: Dimeric structure of C-terminal MUC5B with C2 symmetry
Number of components: 2

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Component #1: protein, Dimeric structure of C-terminal MUC5B with C2 symmetry

ProteinName: Dimeric structure of C-terminal MUC5B with C2 symmetry
Recombinant expression: No
MassExperimental: 260 MDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Vector: pCEP-His / Cell of expression system: HEK293-EBNA

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Component #2: protein, MUC5B

ProteinName: MUC5B / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.25 mg/mL
Buffer solution: Buffers were filtered and degassed before use.
pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 16590
3D reconstructionSoftware: cryoSPARC / Resolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF

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