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- EMDB-9200: Mouse Protocadherin gamma B6 cis mutant V563D on membranes (energ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9200
TitleMouse Protocadherin gamma B6 cis mutant V563D on membranes (energy filtered)
Map dataProtocadherin gamma B6 cis mutant on liposomes
Sample
  • Complex: mouse protocadherin Gamma B6 cis mutant V563D on liposomes
    • Complex: mouse protocadherin gamma B6 cis mutant V563D EC1-6
      • Protein or peptide: mouse protocadherin gamma B6 cis mutant V563D
    • Complex: liposomes
Biological speciesMus (mice) / Mus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsBrasch J / Noble AJ / Shapiro L / Carragher B / Potter CS
CitationJournal: Nature / Year: 2019
Title: Visualization of clustered protocadherin neuronal self-recognition complexes.
Authors: Julia Brasch / Kerry M Goodman / Alex J Noble / Micah Rapp / Seetha Mannepalli / Fabiana Bahna / Venkata P Dandey / Tristan Bepler / Bonnie Berger / Tom Maniatis / Clinton S Potter / Bridget ...Authors: Julia Brasch / Kerry M Goodman / Alex J Noble / Micah Rapp / Seetha Mannepalli / Fabiana Bahna / Venkata P Dandey / Tristan Bepler / Bonnie Berger / Tom Maniatis / Clinton S Potter / Bridget Carragher / Barry Honig / Lawrence Shapiro /
Abstract: Neurite self-recognition and avoidance are fundamental properties of all nervous systems. These processes facilitate dendritic arborization, prevent formation of autapses and allow free interaction ...Neurite self-recognition and avoidance are fundamental properties of all nervous systems. These processes facilitate dendritic arborization, prevent formation of autapses and allow free interaction among non-self neurons. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, β- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities. Avoidance is observed between neurons that express identical protocadherin repertoires, and single-isoform differences are sufficient to prevent self-recognition. Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers. Although these interactions have previously been characterized in isolation, structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains.
History
DepositionOct 12, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseApr 17, 2019-
UpdateMay 22, 2019-
Current statusMay 22, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • 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_9200.png

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Map

FileDownload / File: emd_9200.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProtocadherin gamma B6 cis mutant on liposomes
Voxel sizeX=Y=Z: 7.04 Å
Density
Minimum - Maximum-0.14761552 - 0.4655733
Average (Standard dev.)0.11592891 (±0.03637942)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-53
Dimensions13141224374
Spacing12241314374
CellA: 8616.96 Å / B: 9250.56 Å / C: 2632.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.047.047.04
M x/y/z12241314374
origin x/y/z0.0000.0000.000
length x/y/z8616.9609250.5602632.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS00-53
NC/NR/NS12241314374
D min/max/mean-0.1480.4660.116

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Supplemental data

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Sample components

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Entire : mouse protocadherin Gamma B6 cis mutant V563D on liposomes

EntireName: mouse protocadherin Gamma B6 cis mutant V563D on liposomes
Components
  • Complex: mouse protocadherin Gamma B6 cis mutant V563D on liposomes
    • Complex: mouse protocadherin gamma B6 cis mutant V563D EC1-6
      • Protein or peptide: mouse protocadherin gamma B6 cis mutant V563D
    • Complex: liposomes

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Supramolecule #1: mouse protocadherin Gamma B6 cis mutant V563D on liposomes

SupramoleculeName: mouse protocadherin Gamma B6 cis mutant V563D on liposomes
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: mutant protocadherin gamma B6 ectodomains are tethered to membranes through binding of C-terminal octa-histidine tags to Ni-NTA lipid head groups presented on the liposome surface.
Source (natural)Organism: Mus (mice)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: human embryonic kidney 293 freestyle / Recombinant plasmid: pi alpha

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Supramolecule #2: mouse protocadherin gamma B6 cis mutant V563D EC1-6

SupramoleculeName: mouse protocadherin gamma B6 cis mutant V563D EC1-6 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all / Details: C-terminal octa-hisitidine tag, mutation V563D
Source (natural)Organism: Mus (mice)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: human embryonic kidney 293 freestyle / Recombinant plasmid: pi alpha

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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)
Source (natural)Organism: Mus (mice)

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Macromolecule #1: mouse protocadherin gamma B6 cis mutant V563D

MacromoleculeName: mouse protocadherin gamma B6 cis mutant V563D / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GQPVRYSIPE ELDRGSVVGK LAKDLGLSVL EVSSRKLRVS AEKLHFSVDS ESGDLLVKDR IDREQICKG RRKCELQLEA VLENPLNIFH VVVGIEDVND NAPQFEKKET RLEILETVAV G TRIPLEPA TDPDINLNSV KDYQISSNPY FSLMVRVNPD GGKTPELSLE ...String:
GQPVRYSIPE ELDRGSVVGK LAKDLGLSVL EVSSRKLRVS AEKLHFSVDS ESGDLLVKDR IDREQICKG RRKCELQLEA VLENPLNIFH VVVGIEDVND NAPQFEKKET RLEILETVAV G TRIPLEPA TDPDINLNSV KDYQISSNPY FSLMVRVNPD GGKTPELSLE KLLDREEQRS HR LILTALD GGDPPRSATT QIEISVKDNN DNPPVFSKEE YWVSVSENLS PGSSVLQVTA TDE DEGVNA EILYYFRSTA QSTRHVFSLD EKTGVIKNNQ SLDFEDIERY TMEVEAKDGG GLST RCKII IEVLDENDNS PEITITSLSD HILENSPPGV VVVLFKTRDR DFGGNGEVTC DIGKD LPFK IQASSSNYYK LVTDGALDRE QNPQYNVTIT ATDKGKPALS SSTTIVLHIT DINDNA PAF QKSSYIVHVA ENNPPGASIA QVSASDPDLG ANGHVSYSII ASDLEPKSLW SYVTVNA QS GVVFAQRAFD HEQLRSFQLT LQARDQGKPS LSANVSMRVL VGDRNDNAPR VLYPALEP D GSALFDMVPR AAEPGYLVTK VDAVDADSGH NAWLSYHVLQ ASDPGLFSLG LRTGEVRTA RALGEKDAAR QRLLVGVRDG GQPPLSATAT LLLVFADSLQ EHHHHHHHH

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
25.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
3.0 mMCaCl2Calcium chloride
10.0 % v/vglycerolglycerol
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3 / Details: 2.5 second blot before plunging..
Detailsliposomes at 0.5mM
Cryo protectant5% glycerol
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 1.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D / Number images used: 54

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