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- EMDB-21192: human non-clustered delta protocadherin 10 on membranes tomogram 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-21192
Titlehuman non-clustered delta protocadherin 10 on membranes tomogram 2
Map datahuman delta protocadherin 10 full ectodomains on membranes tomogram 2
Sample
  • Complex: trans dimers of protocadherin 10 extracellular domains formed between membranes of liposomes
    • Complex: human delta protocadherin 10
      • Protein or peptide: human protocadherin 10
    • Complex: liposomes
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsBrasch J / Harrison OJ / Noble AJ / Carragher B / Potter CS
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01GM118584 United States
National Institutes of Health/National Institute of General Medical SciencesGM103310 United States
National Institutes of Health/National Institute of Mental HealthR01MH114817 United States
Other governmentNYSTAR United States
Other privatethe Simons Foundation (SF349247) United States
National Institutes of Health/National Institute of General Medical SciencesF32GM128303 United States
CitationJournal: Cell Rep / Year: 2020
Title: Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins.
Authors: Oliver J Harrison / Julia Brasch / Phinikoula S Katsamba / Goran Ahlsen / Alex J Noble / Hanbin Dan / Rosemary V Sampogna / Clinton S Potter / Bridget Carragher / Barry Honig / Lawrence Shapiro /
Abstract: Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the ...Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of δ1- and δ2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all δ-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, δ1- and δ2-protocadherin trans dimers formed through antiparallel EC1-EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins. Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning.
History
DepositionJan 6, 2020-
Header (metadata) releaseFeb 5, 2020-
Map releaseMar 11, 2020-
UpdateMar 11, 2020-
Current statusMar 11, 2020Processing site: RCSB / Status: Released

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_21192.png

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Map

FileDownload / File: emd_21192.map.gz / Format: CCP4 / Size: 2.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman delta protocadherin 10 full ectodomains on membranes tomogram 2
Voxel sizeX=Y=Z: 7.34344 Å
Density
Minimum - Maximum-7.278857 - 6.3346405
Average (Standard dev.)-0.00000000441 (±0.99999964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-410
Dimensions12811152410
Spacing11521281410
CellA: 8459.643 Å / B: 9406.946 Å / C: 3010.8103 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.34344010416677.34343950039037.3434390243902
M x/y/z11521281410
origin x/y/z0.0000.0000.000
length x/y/z8459.6439406.9463010.810
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS00-410
NC/NR/NS11521281410
D min/max/mean-7.2796.335-0.000

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Supplemental data

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Sample components

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Entire : trans dimers of protocadherin 10 extracellular domains formed bet...

EntireName: trans dimers of protocadherin 10 extracellular domains formed between membranes of liposomes
Components
  • Complex: trans dimers of protocadherin 10 extracellular domains formed between membranes of liposomes
    • Complex: human delta protocadherin 10
      • Protein or peptide: human protocadherin 10
    • Complex: liposomes

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Supramolecule #1: trans dimers of protocadherin 10 extracellular domains formed bet...

SupramoleculeName: trans dimers of protocadherin 10 extracellular domains formed between membranes of liposomes
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: purified full ectodomains of protocadherin 10 are tethered to Ni-NTA lipid head groups presented on the liposome surface through binding of C-terminal hexa-histidine tags
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: human delta protocadherin 10

SupramoleculeName: human delta protocadherin 10 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Details: full ectodomain of human delta protocadherin 10 with a C-termin hexa histidine tag

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Supramolecule #3: liposomes

SupramoleculeName: liposomes / type: complex / ID: 3 / Parent: 1
Details: liposomes composed of DOPC and DOGS-NTA (8:2 ratio) with 100nm diameter

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Macromolecule #1: human protocadherin 10

MacromoleculeName: human protocadherin 10 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SQLHYTVQEE QEHGTFVGNI AEDLGLDITK LSARGFQTVP NSR TPYLDL NLETGVLYVN EKIDREQICK QSPSCVLHLE VFLENPLELF QVEIEVLDIN DNPP SFPEP DLTVEISESA TPGTRFPLES AFDPDVGTNS LRDYEITPNS YFSLDVQTQG DGNRF AELV ...String:
SQLHYTVQEE QEHGTFVGNI AEDLGLDITK LSARGFQTVP NSR TPYLDL NLETGVLYVN EKIDREQICK QSPSCVLHLE VFLENPLELF QVEIEVLDIN DNPP SFPEP DLTVEISESA TPGTRFPLES AFDPDVGTNS LRDYEITPNS YFSLDVQTQG DGNRF AELV LEKPLDREQQ AVHRYVLTAV DGGGGGGVGE GGGGGGGAGL PPQQQRTGTA LLTIRV LDS NDNVPAFDQP VYTVSLPENS PPGTLVIQLN ATDPDEGQNG EVVYSFSSHI SPRAREL FG LSPRTGRLEV SGELDYEESP VYQVYVQAKD LGPNAVPAHC KVLVRVLDAN DNAPEISF S TVKEAVSEGA APGTVVALFS VTDRDSEENG QVQCELLGDV PFRLKSSFKN YYTIVTEAP LDREAGDSYT LTVVARDRGE PALSTSKSIQ VQVSDVNDNA PRFSQPVYDV YVTENNVPGA YIYAVSATD RDEGANAQLA YSILECQIQG MSVFTYVSIN SENGYLYALR SFDYEQLKDF S FQVEARDA GSPQALAGNA TVNILIVDQN DNAPAIVAPL PGRNGTPARE VLPRSAEPGY LL TRVAAVD ADDGENARLT YSIVRGNEMN LFRMDWRTGE LRTARRVPAK RDPQRPYELV IEV RDHGQP PLSSTATLVV QLVDGAVEPQ GGGGSGGGGS GEHQRPSRSG GGETSLDLTH HHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation state3D array

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
3.0 mMCaCl2Calcium chloride
5.0 v/v %glycerolglycerol
GridSupport film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3 / Details: 2.5 second blot before plunging..
Detailsaggregated liposomes were deposited onto EM grids
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Spherical aberration corrector: Cs corrector (CEOS GmbH) / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 60 / Average exposure time: 1.1 sec. / Average electron dose: 3.15 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm
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

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D / Software - details: part of Appion-protomo 2.4.1 / Number images used: 53

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