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- EMDB-25886: Full-length human E-cadherin bound to activating Fab 59D2 -

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Basic information

Entry
Database: EMDB / ID: EMD-25886
TitleFull-length human E-cadherin bound to activating Fab 59D2
Map datacisTEM generate3D full map
Sample
  • Complex: Complex of human E-cadherin monomer with activating Fab 59D2
    • Complex: Full length E-cadherin with Twin-Strep tag
      • Protein or peptide: E-cadherin (CDH1) with C-terminal Twin-Strep tag
    • Complex: Activating antibody fragment 59D2 Fab
      • Protein or peptide: Activating antibody fragment 59D2 Fab heavy chain
      • Protein or peptide: Activating antibody fragment 59D2 Fab light chain
KeywordsE-cadherin / Fab / CDH1 / functional antibody / CELL ADHESION
Function / homology
Function and homology information


response to Gram-positive bacterium / regulation of protein catabolic process at postsynapse, modulating synaptic transmission / pituitary gland development / gamma-catenin binding / negative regulation of axon extension / cell-cell adhesion mediated by cadherin / cellular response to indole-3-methanol / flotillin complex / calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules / Formation of definitive endoderm ...response to Gram-positive bacterium / regulation of protein catabolic process at postsynapse, modulating synaptic transmission / pituitary gland development / gamma-catenin binding / negative regulation of axon extension / cell-cell adhesion mediated by cadherin / cellular response to indole-3-methanol / flotillin complex / calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules / Formation of definitive endoderm / Adherens junctions interactions / catenin complex / Apoptotic cleavage of cell adhesion proteins / GTPase activating protein binding / cell-cell junction assembly / adherens junction organization / apical junction complex / ankyrin binding / negative regulation of cell-cell adhesion / cellular response to lithium ion / homophilic cell adhesion via plasma membrane adhesion molecules / lateral plasma membrane / RHO GTPases activate IQGAPs / Integrin cell surface interactions / cell adhesion molecule binding / synapse assembly / InlA-mediated entry of Listeria monocytogenes into host cells / Degradation of the extracellular matrix / negative regulation of cell migration / protein tyrosine kinase binding / protein localization to plasma membrane / adherens junction / cell morphogenesis / trans-Golgi network / cytoplasmic side of plasma membrane / response to toxic substance / beta-catenin binding / cell-cell adhesion / positive regulation of protein import into nucleus / neuron projection development / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / actin cytoskeleton / lamellipodium / cell junction / postsynapse / regulation of gene expression / endosome / response to xenobiotic stimulus / cadherin binding / glutamatergic synapse / calcium ion binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / extracellular exosome / extracellular region / identical protein binding / membrane / plasma membrane
Similarity search - Function
Cadherin prodomain like / Cadherin prodomain / Cadherin prodomain like / Cadherin, Y-type LIR-motif / Cadherin, Y-type LIR-motif / Catenin binding domain superfamily / Cadherin / Cadherin conserved site / Cadherin domain signature. / Cadherin repeats. ...Cadherin prodomain like / Cadherin prodomain / Cadherin prodomain like / Cadherin, Y-type LIR-motif / Cadherin, Y-type LIR-motif / Catenin binding domain superfamily / Cadherin / Cadherin conserved site / Cadherin domain signature. / Cadherin repeats. / Cadherin domain / Cadherins domain profile. / Cadherin-like / Cadherin-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.11 Å
AuthorsMaker A / Gumbiner BM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122467 United States
CitationJournal: Protein Expr Purif / Year: 2022
Title: Reconstitution of the full transmembrane cadherin-catenin complex.
Authors: Allison Maker / Barry M Gumbiner /
Abstract: The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α- ...The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α-catenin, β-catenin, and the E-cadherin cytoplasmic domain. However, p120-catenin and the full-length E-cadherin including the extracellular, transmembrane, and intra-cellular domains are vital to the understanding of the relationship between extracellular adhesion and intracellular signaling. Here, we reconstitute the complete and full-length cadherin-catenin complex, including full-length E-cadherin, α-catenin, β-catenin, and p120-catenin, into nanodiscs. We are able to observe the cadherin in nanodiscs by cryo-EM. We also reconstitute α-catenin, β-catenin, and p120-catenin with the E-cadherin cytoplasmic tail alone in order to analyze the affinities of their binding interactions. We find that p120-catenin does not associate strongly with α- or β-catenin and binds much more transiently to the cadherin cytoplasmic tail than does β-catenin. Overall, this work creates many new possibilities for biochemical studies understanding transmembrane signaling of cadherins and the role of p120-catenin in adhesion activation.
History
DepositionJan 8, 2022-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.65
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.65
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25886.png

Downloads & links

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Map

FileDownload / File: emd_25886.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcisTEM generate3D full map
Voxel sizeX=Y=Z: 1.68 Å
Density
Contour LevelBy AUTHOR: 5.65 / Movie #1: 5.65
Minimum - Maximum-8.632336 - 15.715873
Average (Standard dev.)-0.002000301 (±0.25059876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.681.681.68
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z537.600537.600537.600
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-8.63215.716-0.002

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Supplemental data

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Half map: cisTEM generate3D half map 2

Fileemd_25886_half_map_1.map
AnnotationcisTEM generate3D half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cisTEM generate3D half map 1

Fileemd_25886_half_map_2.map
AnnotationcisTEM generate3D half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of human E-cadherin monomer with activating Fab 59D2

EntireName: Complex of human E-cadherin monomer with activating Fab 59D2
Components
  • Complex: Complex of human E-cadherin monomer with activating Fab 59D2
    • Complex: Full length E-cadherin with Twin-Strep tag
      • Protein or peptide: E-cadherin (CDH1) with C-terminal Twin-Strep tag
    • Complex: Activating antibody fragment 59D2 Fab
      • Protein or peptide: Activating antibody fragment 59D2 Fab heavy chain
      • Protein or peptide: Activating antibody fragment 59D2 Fab light chain

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Supramolecule #1: Complex of human E-cadherin monomer with activating Fab 59D2

SupramoleculeName: Complex of human E-cadherin monomer with activating Fab 59D2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Cadherin was embedded in MSP1D1 nanodisc not visible in final map.
Molecular weightTheoretical: 130 KDa

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Supramolecule #2: Full length E-cadherin with Twin-Strep tag

SupramoleculeName: Full length E-cadherin with Twin-Strep tag / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 80 KDa

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Supramolecule #3: Activating antibody fragment 59D2 Fab

SupramoleculeName: Activating antibody fragment 59D2 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 / Details: His tagged recombinant Fab fragment
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 50 KDa

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Macromolecule #1: E-cadherin (CDH1) with C-terminal Twin-Strep tag

MacromoleculeName: E-cadherin (CDH1) with C-terminal Twin-Strep tag / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DWVIPPISCP ENEKGPFPKN LVQIKSNKDK EGKVFYSITG QGADTPPVGV FIIERETGWL KVTEPLDRER IATYTLFSHA VSSNGNAVED PMEILITVTD QNDNKPEFTQ EVFKGSVMEG ALPGTSVMEV TATDADDDVN TYNAAIAYTI LSQDPELPDK NMFTINRNTG ...String:
DWVIPPISCP ENEKGPFPKN LVQIKSNKDK EGKVFYSITG QGADTPPVGV FIIERETGWL KVTEPLDRER IATYTLFSHA VSSNGNAVED PMEILITVTD QNDNKPEFTQ EVFKGSVMEG ALPGTSVMEV TATDADDDVN TYNAAIAYTI LSQDPELPDK NMFTINRNTG VISVVTTGLD RESFPTYTLV VQAADLQGEG LSTTATAVIT VTDTNDNPPI FNPTTYKGQV PENEANVVIT TLKVTDADAP NTPAWEAVYT ILNDDGGQFV VTTNPVNNDG ILKTAKGLDF EAKQQYILHV AVTNVVPFEV SLTTSTATVT VDVLDVNEAP IFVPPEKRVE VSEDFGVGQE ITSYTAQEPD TFMEQKITYR IWRDTANWLE INPDTGAIST RAELDREDFE HVKNSTYTAL IIATDNGSPV ATGTGTLLLI LSDVNDNAPI PEPRTIFFCE RNPKPQVINI IDADLPPNTS PFTAELTHGA SANWTIQYND PTQESIILKP KMALEVGDYK INLKLMDNQN KDQVTTLEVS VCDCEGAAGV CRKAQPVEAG LQIPAILGIL GGILALLILI LLLLLFLRRR AVVKEPLLPP EDDTRDNVYY YDEEGGGEED QDFDLSQLHR GLDARPEVTR NDVAPTLMSV PRYLPRPANP DEIGNFIDEN LKAADTDPTA PPYDSLLVFD YEGSGSEAAS LSSLNSSESD KDQDYDYLNE WGNRFKKLAD MYGGGEDHSA WSHPQFEKGG GSGGGSGGSA WSHPQFEK

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Macromolecule #2: Activating antibody fragment 59D2 Fab heavy chain

MacromoleculeName: Activating antibody fragment 59D2 Fab heavy chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MGWSCIILFL VATATGVHSE VQLQQSGADL VKPGASVKLS CTASGFNIKV TYMHWMIQRP DQGLDWIGRI DPANGYSDYD PKLRGKATIT ADTSSNTVYL QLSSLTSEDT AVYYCATGFF GNYAYWGQGT TLTVSSAKTT PPSVYPLAPG SAAQTNSMVT LGCLVKGYFP ...String:
MGWSCIILFL VATATGVHSE VQLQQSGADL VKPGASVKLS CTASGFNIKV TYMHWMIQRP DQGLDWIGRI DPANGYSDYD PKLRGKATIT ADTSSNTVYL QLSSLTSEDT AVYYCATGFF GNYAYWGQGT TLTVSSAKTT PPSVYPLAPG SAAQTNSMVT LGCLVKGYFP EPVTVTWNSG SLSSGVHTFP AVLQSDLYTL SSSVTVPSSP RPSETVTCNV AHPASSTKVD KKIVPRDCHH HHHH

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Macromolecule #3: Activating antibody fragment 59D2 Fab light chain

MacromoleculeName: Activating antibody fragment 59D2 Fab light chain / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MKLPVRLLVL MFWIPASTSD VVMTQTPLSL PVSLGDQASI SCRSSQSLVH TNGNTYLHWY LQKPGQSPKL LIFKVSNRFS GVPDSFSGSG SGTDFTLKIS RVEAEDLGVY FCSQSTHVPH TFGGGTKLEI KRADAAPTVS IFPPSSEQLT SGGASVVCFL NNFYPKDINV ...String:
MKLPVRLLVL MFWIPASTSD VVMTQTPLSL PVSLGDQASI SCRSSQSLVH TNGNTYLHWY LQKPGQSPKL LIFKVSNRFS GVPDSFSGSG SGTDFTLKIS RVEAEDLGVY FCSQSTHVPH TFGGGTKLEI KRADAAPTVS IFPPSSEQLT SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC

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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

Concentration0.10 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mM(HOCH2)3CNH2Tris
150.0 mMNaClsodium chloride
1.0 mMCaCl2calcium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsNanodiscs averaged out in final reconstruction.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 47.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1077980 / Details: template picking
Startup modelType of model: INSILICO MODEL / In silico model: De novo model created in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0) / Number images used: 331400
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14)
FSC plot (resolution estimation)

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