+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-9199 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Mouse Protocadherin gamma B6 on membranes (energy filtered) | |||||||||
Map data | mouse protocadherin gamma B6 forms ordered assemblies on membranes (energy filtered) | |||||||||
Sample |
| |||||||||
Biological species | Mus (mice) / Mus musculus (house mouse) | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Brasch J / Noble AJ / Shapiro L / Carragher B / Potter CS | |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_9199.map.gz | 12.3 GB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-9199-v30.xml emd-9199.xml | 14.4 KB 14.4 KB | Display Display | EMDB header |
Images | emd_9199.png | 275.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-9199 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-9199 | HTTPS FTP |
-Validation report
Summary document | emd_9199_validation.pdf.gz | 78.9 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_9199_full_validation.pdf.gz | 78 KB | Display | |
Data in XML | emd_9199_validation.xml.gz | 499 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9199 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-9199 | HTTPS FTP |
-Related structure data
Related structure data | 9197C 9198C 9200C 6e6bC C: citing same article (ref.) |
---|---|
EM raw data | EMPIAR-10234 (Title: Single particle cryoEM of mouse protocadherin gamma B6 Data size: 438.8 / Data #1: Micrograph frames [micrographs - multiframe] Data #2: Micrographs along with all other magnification images from the collection [micrographs - single frame]) EMPIAR-10238 (Title: CryoET of mouse protocadherin gamma B6 on membranes (with energy filter) Data size: 411.3 / Data #1: K2 tilt-series frames [micrographs - multiframe] Data #2: Whole-frame aligned tilt images along with all other magnification images from the collection [micrographs - single frame] Data #3: Appion-Protomo tilt-series alignments [tilt series]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_9199.map.gz / Format: CCP4 / Size: 14.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | mouse protocadherin gamma B6 forms ordered assemblies on membranes (energy filtered) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.68 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : mouse protocadherin Gamma B6 on liposomes
Entire | Name: mouse protocadherin Gamma B6 on liposomes |
---|---|
Components |
|
-Supramolecule #1: mouse protocadherin Gamma B6 on liposomes
Supramolecule | Name: mouse protocadherin Gamma B6 on liposomes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Protocadherin gamma B6 are tethered to membranes by binding of C-terminal octa-histidine tags to Ni-NTA lipid head groups presented on the liposome surface. |
---|---|
Source (natural) | Organism: Mus (mice) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: human embryonic kidney 293 freestyle / Recombinant plasmid: pi alpha |
-Supramolecule #2: mouse protocadherin gamma B6 EC1-6
Supramolecule | Name: mouse protocadherin gamma B6 EC1-6 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all / Details: C-terminal octa-hisitidine tag |
---|---|
Source (natural) | Organism: Mus (mice) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: human embryonic kidney 293 freestyle / Recombinant plasmid: pi alpha |
-Supramolecule #3: liposomes
Supramolecule | Name: liposomes / type: complex / ID: 3 / Parent: 1 Details: liposomes composed of DOPC and DOGS-NTA (8:2 ratio) |
---|---|
Source (natural) | Organism: Mus (mice) |
-Macromolecule #1: mouse protocadherin gamma B6
Macromolecule | Name: mouse protocadherin gamma B6 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | electron tomography |
Aggregation state | 3D array |
-Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.4 Component:
| |||||||||||||||
Grid | Model: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 298.15 K / Instrument: GATAN CRYOPLUNGE 3 / Details: 2.5 second blot before plunging.. | |||||||||||||||
Details | liposomes at 0.5mM | |||||||||||||||
Cryo protectant | 5% glycerol | |||||||||||||||
Sectioning | Other: NO SECTIONING |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 1.6 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D / Number images used: 52 |
---|