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- EMDB-9197: Mouse Protocadherin gamma B6 dimer-of-dimers -

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Basic information

Entry
Database: EMDB / ID: EMD-9197
TitleMouse Protocadherin gamma B6 dimer-of-dimers
Map datasubtomogram averaged map of Protocadherin gamma B6 EC1-6 dimer-of-dimers in solution
Sample
  • Complex: mouse protocadherin gamma b6
    • Protein or peptide: mouse protocadherin gamma b6
Biological speciesMus (mice) / Mus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 35.0 Å
AuthorsBrasch J / Noble AJ / Shapiro L / Carragher B / Potter CS
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01MH114817 United States
Other privateSimons Foundation / SF349247 United States
National Institutes of Health/National Institute of General Medical SciencesGM103310 United States
Other privateAgouron Institute / F00316 United States
Other governmentNYSTAR United States
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-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.54
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.54
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9197.png

Downloads & links

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Map

FileDownload / File: emd_9197.map.gz / Format: CCP4 / Size: 5.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsubtomogram averaged map of Protocadherin gamma B6 EC1-6 dimer-of-dimers in solution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.04 Å/pix.
x 112 pix.
= 788.48 Å		7.04 Å/pix.
x 112 pix.
= 788.48 Å		7.04 Å/pix.
x 112 pix.
= 788.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.54 / Movie #1: 3.54
Minimum - Maximum-5.6383686 - 14.336349
Average (Standard dev.)-0.00000000002 (±0.46186543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions112112112
Spacing112112112
CellA=B=C: 788.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.047.047.04
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z788.480788.480788.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS112112112
D min/max/mean-5.63814.336-0.000

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Supplemental data

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Sample components

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Entire : mouse protocadherin gamma b6

EntireName: mouse protocadherin gamma b6
Components
  • Complex: mouse protocadherin gamma b6
    • Protein or peptide: mouse protocadherin gamma b6

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Supramolecule #1: mouse protocadherin gamma b6

SupramoleculeName: mouse protocadherin gamma b6 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus (mice)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: human embryonic kidney 293 freestyle / Recombinant plasmid: pi alpha
Molecular weightExperimental: 300 KDa

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Macromolecule #1: mouse protocadherin gamma b6

MacromoleculeName: mouse protocadherin gamma b6 / 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 VVAVDADSGH NAWLSYHVLQ ASDPGLFSLG LRTGEVRTA RALGEKDAAR QRLLVGVRDG GQPPLSATAT LLLVFADSLQ EHHHHHHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.075 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
10.0 mMTris-CLtris(hydroxymethyl)aminomethane chloride
3.0 mMCaCl2Calcium chloride
250.0 mMImidazoleImidazole
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298.15 K / Instrument: SPOTITON / Details: homemade nanowire grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: OTHER / Software - Name: Dynamo (ver. 109) / Details: FSC 0.143 map to model / Number subtomograms used: 506
ExtractionNumber tomograms: 7 / Number images used: 506 / Reference model: random subset / Method: volumes picked manually / Software - Name: Dynamo (ver. 1.1.109) / Software - details: Dipole picking
Details: Volumes were picked using Dipole set models in Dynamo. Ends of the particles were marked using 'North' and 'South' options.
Final angle assignmentType: OTHER / Software - Name: Dynamo (ver. 109) / Details: sub-volume correlation based alignment

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