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- EMDB-10339: Respiratory mucin MUC5B; C-terminal dimerization domain structure -

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Basic information

Entry
Database: EMDB / ID: EMD-10339
TitleRespiratory mucin MUC5B; C-terminal dimerization domain structure
Map dataBase of the C2 symmetric MUC5B models
Sample
  • Complex: Dimeric structure of C-terminal MUC5B base region.
    • Protein or peptide: MUC5B
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 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-multiangle 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-
UpdateApr 22, 2020-
Current statusApr 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10339.png

Downloads & links

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Map

FileDownload / File: emd_10339.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBase of the C2 symmetric MUC5B models
Voxel sizeX=Y=Z: 2.0371 Å
Density
Contour LevelBy AUTHOR: 0.128 / Movie #1: 0.128
Minimum - Maximum-0.24390915 - 1.2561007
Average (Standard dev.)-0.0004407064 (±0.022067558)
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-0.2441.256-0.000

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Supplemental data

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Sample components

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Entire : Dimeric structure of C-terminal MUC5B base region.

EntireName: Dimeric structure of C-terminal MUC5B base region.
Components
  • Complex: Dimeric structure of C-terminal MUC5B base region.
    • Protein or peptide: MUC5B

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Supramolecule #1: Dimeric structure of C-terminal MUC5B base region.

SupramoleculeName: Dimeric structure of C-terminal MUC5B base region. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293-EBNA / Recombinant plasmid: pCEP-His
Molecular weightExperimental: 260 kDa/nm

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Macromolecule #1: MUC5B

MacromoleculeName: MUC5B / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVIYNKTDRA GCHFYAVCNQ HCDID RFQG ACPTSPPPVS SAPLSSPSPA PGCDNAIPLR QVNETWTLEN CTVARCVGDN RVVLLD PKP VANVTCVNKH LPIKVSDPSQ PCDFHYECEC ICSMWGGSHY STFDGTSYTF RGNCTYV LM REIHARFGNL SLYLDNHYCT ...String:
EVIYNKTDRA GCHFYAVCNQ HCDID RFQG ACPTSPPPVS SAPLSSPSPA PGCDNAIPLR QVNETWTLEN CTVARCVGDN RVVLLD PKP VANVTCVNKH LPIKVSDPSQ PCDFHYECEC ICSMWGGSHY STFDGTSYTF RGNCTYV LM REIHARFGNL SLYLDNHYCT ASATAAAARC PRALSIHYKS MDIVLTVTMV HGKEEGLI L FDQIPVSSGF SKNGVLVSVL GTTTMRVDIP ALGVSVTFNG QVFQARLPYS LFHNNTEGQ CGTCTNNQRD DCLQRDGTTA ASCKDMAKTW LVPDSRKDGC WAPTGTPPTA SPAAPVSSTP TPTPCPPQP LCDLMLSQVF AECHNLVPPG PFFNACISDH CRGRLEVPCQ SLEAYAELCR A RGVCSDWR GATGGLCDLT CPPTKVYKPC GPIQPATCNS RNQSPQLEGM AEGCFCPEDQ IL FNAHMGI CVQACPCVGP DGFPKFPGER WVSNCQSCVC DEGSVSVQCK PLPCDAQGQP PPC NRPGFV TVTRPRAENP CCPETVCVCN TTTCPQSLPV CPPGQESICT QEEGDCCPTF RCRP QLCSY NGTFYGVGAT FPGALPCHMC TCLSGDTQDP TVQCQEDACN NTTCPQGFEY KRVAG QCCG ECVQTACLTP DGQPVQLNET WVNSHVDNCT VYLCEAEGGV HLLTPQPASC PDVSSC RGS LRKTGCCYSC EEDSCQVRIN TTILWHQGCE TEVNITFCEG SCPGASKYSA EAQAMQH QC TCCQERRVHE ETVPLHCPNG SAILHTYTHV DECGCTPFCV PAPMAPPHTR GFPAQEAT A V

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
0.025 MC8H18N2O4SHepes
0.15 MNaClSodium chlorideSodium Chloride

Details: Buffers were filtered and degassed before use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 231589
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Software - details: Ru nthrough cryoSPARC in exhaustive mode
Startup modelType of model: INSILICO MODEL
In silico model: Ab initio model was generated using particles from rounds of 2D classification in cryoSPARC. A mask was generated around the stalk of EMD-10264 and used to subtract it from the ...In silico model: Ab initio model was generated using particles from rounds of 2D classification in cryoSPARC. A mask was generated around the stalk of EMD-10264 and used to subtract it from the structure. The subtracted base of the protein was then refined with a local refinement.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.8)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.8) / Number images used: 16590

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