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- EMDB-4296: Recombinant human MUC5B mucin N-terminal -

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Basic information

Entry
Database: EMDB / Id: 4296
TitleRecombinant human MUC5B mucin N-terminal
Map data
SampleMUC5B N-terminal oligomeric state:
SourceHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.5 Å
AuthorsTrillo-Muoy S / Nilsson HE / Hebert H / Hansson GC
CitationJournal: J. Biol. Chem. / Year: 2018
Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.
P-authors: Sergio Trillo-Muyo / Harriet E Nilsson / Christian V Recktenwald / Anna Ermund / Caroline Ridley / Lauren N Meiss / Andrea Bähr / Nikolai Klymiuk / Jeffrey J Wine / Philip J B Koeck / David J Thornton / Hans Hebert / Gunnar C Hansson /
Abstract: Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of ...Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.
DateDeposition: Feb 13, 2018 / Header (metadata) release: Feb 21, 2018 / Map release: Mar 20, 2019 / Last update: Mar 20, 2019

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Strvis

Movie
  • Surface view with section colored by density value
  • Surface level: 1.14
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.14
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downlink

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Map

Fileemd_4296.map.gz (map file in CCP4 format, 28312 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
2.08 Å/pix.
= 399.072 Å
192 pix
2.08 Å/pix.
= 399.072 Å
192 pix
2.08 Å/pix.
= 399.072 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.0785 Å
Density
Contour Level:1.14 (by author), 1.14 (movie #1):
Minimum - Maximum-0.92020476 - 3.0594723
Average (Standard dev.)0.009275456 (0.13923985)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin-96.0-96.0-96.0
Limit95.095.095.0
Spacing192192192
CellA=B=C: 399.07202 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.07852.07852.0785
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z399.072399.072399.072
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.9203.0590.009

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Supplemental data

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Sample components

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Entire MUC5B N-terminal oligomeric state

EntireName: MUC5B N-terminal oligomeric state
Details: The MUC5B N-terminal covalent dimers form non-covalent tetramers at low pH, high Ca2+ in the secretory vesicles of the mucus secreting cells
Number of components: 1

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Component #1: protein, MUC5B N-terminal oligomeric state

ProteinName: MUC5B N-terminal oligomeric state
Details: The MUC5B N-terminal covalent dimers form non-covalent tetramers at low pH, high Ca2+ in the secretory vesicles of the mucus secreting cells
Recombinant expression: No
MassExperimental: 570 kDa
SourceSpecies: Homo sapiens (human) / Strain: Human
Source (engineered)Expression System: Cricetinae gen. sp. (mammal) / Vector: mammalian episomal expression vector pCEP-His / Strain: ovary / Cell of expression system: CHO-K1 cells
Source (natural)Organ or tissue: submucosal gland

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.015 mg/ml / Ph: 6.2
Staining2% (w/v) uranyl acetate
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2100F
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 15 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000.0 X (nominal), 72165.0 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: JEOL
CameraDetector: TVIPS TEMCAM-F415 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 95 / Sampling size: 15 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D2 (2*2 fold dihedral) / Number of projections: 4056
3D reconstructionAlgorithm: FOURIER SPACE / Software: EMAN2
CTF correction: For low resolution data EMAN2 uses a 1D structure factor during the CTF procedure
Resolution: 25.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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