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- EMDB-40675: Cryogenic electron microscopy map of human plakophilin-3 -

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Basic information

Entry
Database: EMDB / ID: EMD-40675
TitleCryogenic electron microscopy map of human plakophilin-3
Map data
Sample
  • Organelle or cellular component: plakophilin-3
    • Protein or peptide: plakophilin-3
Keywordsactin cytoskeleton / armadillo / desmosomes / phosphatidylinositol 4 / 5-bisphosphate / plasma membrane / plakophilin / CELL ADHESION
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.03 Å
AuthorsGupta J / Izard T / Rangarajan ES
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Int J Mol Sci / Year: 2023
Title: Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin.
Authors: Jyoti Gupta / Erumbi S Rangarajan / Regina B Troyanovsky / Indrajyoti Indra / Sergey M Troyanovsky / Tina Izard /
Abstract: Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat ...Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.
History
DepositionMay 2, 2023-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJun 21, 2023-
Current statusJun 21, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40675.png

Downloads & links

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Map

FileDownload / File: emd_40675.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.44 Å
Density
Contour LevelBy AUTHOR: 0.202
Minimum - Maximum-0.98487544 - 1.696459
Average (Standard dev.)0.0006192126 (±0.04583657)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 184.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_40675_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_40675_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : plakophilin-3

EntireName: plakophilin-3
Components
  • Organelle or cellular component: plakophilin-3
    • Protein or peptide: plakophilin-3

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Supramolecule #1: plakophilin-3

SupramoleculeName: plakophilin-3 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.2 KDa

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Macromolecule #1: plakophilin-3

MacromoleculeName: plakophilin-3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHHHSS GLEVLFQGPG SHRTLQ RLS SGFDDID LP SAVKYLMA S DPNLQVLGA AYIQHKCYSD AAAKKQARS L QAVPRLVK LF NHANQEV QRH ATGAMR NLIY DNADN KLALV EENG IFELLR TLR EQDDELR KN VTGILWNL S SSDHLKDRL ...String:
HHHHHHHHSS GLEVLFQGPG SHRTLQ RLS SGFDDID LP SAVKYLMA S DPNLQVLGA AYIQHKCYSD AAAKKQARS L QAVPRLVK LF NHANQEV QRH ATGAMR NLIY DNADN KLALV EENG IFELLR TLR EQDDELR KN VTGILWNL S SSDHLKDRL ARDTLEQLTD LVLSPLSGA G GPPLIQQN AS EAEIFYN ATG FLRNLS SASQ ATRQK MRECH GLVD ALVTSI NHA LDAGKCE DK SVENAVCV L RNLSYRLYD EMPPSALQRL EGRGRRDLA G APPGEVVG CF TPQSRRL REL PLAADA LTFA EVSKD PKGLE WLWS PQIVGL YNR LLQRCEL NR HTTEAAAG A LQNITAGDR RWAGVLSRLA LEQERILNP L LDRVRTAD HH QLRSLTG LIR NLSRNA RNKD EMSTK VVSHL IEKL PGSVGE KSP PAEVLVN II AVLNNLVV A SPIAARDLL YFDGLRKLIF IKKKRDSPD S EKSSRAAS SL LANLWQY NKL HRDFRA KGYR KEDFL GP

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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

Concentration0.2 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris, 150 mM NaCl, 1 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 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 / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 294650
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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