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

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Basic information

Entry
Database: EMDB / ID: EMD-4296
TitleRecombinant human MUC5B mucin N-terminal
Map dataNone
Sample
  • Complex: MUC5B N-terminal oligomeric state
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.5 Å
AuthorsTrillo-Muoy S / Nilsson HE / Hebert H / Hansson GC
Funding support Sweden, United States, 16 items
OrganizationGrant numberCountry
The Swedish Heart-Lung Foundation Sweden
Magnus Bergvalls Foundation Sweden
Lederhausens Center for CF Research at University Gothenburg Sweden
Center for Innovative Medicine (KI) Sweden
Cystic Fibrosis Foundation14X0 United States
Swedish CF Foundation Sweden
Cystic Fibrosis Foundation077X0 United States
Swedish Research Council Sweden
The Knut and Alice Wallenberg Foundation Sweden
The Swedish Cancer Foundation Sweden
Sahlgrenska University Hospital (ALF) Sweden
IngaBritt and Arne Lundberg Foundation Sweden
National Institute of Allergy and Infectious DiseasesU01AI095473 Sweden
Wilhelm and Martina Lundgrens Foundation Sweden
Erica Lederhausens Foundation Sweden
The Swedish Foundation for Strategic Research Sweden
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.
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 ...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.
History
DepositionFeb 13, 2018-
Header (metadata) releaseFeb 21, 2018-
Map releaseMar 20, 2019-
UpdateOct 2, 2019-
Current statusOct 2, 2019Processing site: PDBe / Status: Released

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Structure visualization

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

emd_4296.png

Downloads & links

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Map

FileDownload / File: emd_4296.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 2.0785 Å
Density
Contour LevelBy AUTHOR: 1.14 / Movie #1: 1.14
Minimum - Maximum-0.92020476 - 3.0594723
Average (Standard dev.)0.009275456 (±0.13923985)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 399.07202 Å
α=β=γ: 90.0 °

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/NZ000
NX/NY/NZ320320320
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
Components
  • Complex: MUC5B N-terminal oligomeric state

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Supramolecule #1: MUC5B N-terminal oligomeric state

SupramoleculeName: MUC5B N-terminal oligomeric state / type: complex / ID: 1 / Parent: 0
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
Source (natural)Organism: Homo sapiens (human) / Strain: Human / Organ: submucosal gland / Tissue: gland
Recombinant expressionOrganism: Cricetinae gen. sp. (mammal) / Recombinant strain: ovary / Recombinant cell: CHO-K1 cells
Recombinant plasmid: mammalian episomal expression vector pCEP-His
Molecular weightExperimental: 570 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 6.2 / Component - Concentration: 20.0 mM / Component - Formula: C6H13NO4S / Component - Name: MES
StainingType: NEGATIVE / Material: Uranyl acetate / Details: 2% (w/v) uranyl acetate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.005 kPa
DetailsSample was incubated at room temperature overnight and then fixed in 0.6% glutaraldehyde for 4 min Before the negtive stain procedure

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 72165 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL
Image recordingFilm or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.0 µm / Number grids imaged: 3 / Number real images: 95 / Average exposure time: 0.5 sec. / Average electron dose: 15.0 e/Å2

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Image processing

Particle selectionNumber selected: 4456
Details: Negative monitor contrast fascilated particle picking
CTF correctionSoftware - Name: EMAN2 (ver. 2.12)
Software - details: EMAN2 e2boxer.py was used to automatically select particle images
Details: For low resolution data EMAN2 uses a 1D structure factor during the CTF procedure
Startup modelType of model: OTHER
Details: The initial model was based on a subset of class averages representing different views.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2 (ver. 2.12)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: EMAN2 (ver. 2.12)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 25.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.12) / Number images used: 4056
FSC plot (resolution estimation)

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