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Yorodumi- EMDB-12286: Cryo-EM structure of the ternary complex between Netrin-1, Neogen... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12286 | |||||||||||||||||||||||||||||||||
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Title | Cryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B | |||||||||||||||||||||||||||||||||
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Sample |
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Function / homology | Function and homology information regulation of glial cell migration / DSCAM interactions / chemorepulsion of axon / anterior/posterior axon guidance / Role of second messengers in netrin-1 signaling / Cdc42 protein signal transduction / Netrin-1 signaling / Regulation of commissural axon pathfinding by SLIT and ROBO / motor neuron migration / negative regulation of axon extension ...regulation of glial cell migration / DSCAM interactions / chemorepulsion of axon / anterior/posterior axon guidance / Role of second messengers in netrin-1 signaling / Cdc42 protein signal transduction / Netrin-1 signaling / Regulation of commissural axon pathfinding by SLIT and ROBO / motor neuron migration / negative regulation of axon extension / substrate-dependent cell migration, cell extension / Netrin mediated repulsion signals / mammary gland duct morphogenesis / positive regulation of cell motility / nuclear migration / DCC mediated attractive signaling / inner ear morphogenesis / regulation of synapse assembly / endoplasmic reticulum-Golgi intermediate compartment / basement membrane / glial cell proliferation / positive regulation of axon extension / BMP signaling pathway / coreceptor activity / side of membrane / positive regulation of glial cell proliferation / cell-cell adhesion / actin cytoskeleton / Ras protein signal transduction / cell adhesion / DNA-binding transcription factor activity, RNA polymerase II-specific / membrane raft / RNA polymerase II cis-regulatory region sequence-specific DNA binding / regulation of transcription by RNA polymerase II / apoptotic process / positive regulation of DNA-templated transcription / signal transduction / extracellular region / nucleoplasm / identical protein binding / membrane / plasma membrane / cytosol Similarity search - Function | |||||||||||||||||||||||||||||||||
Biological species | Homo sapiens (human) / Mus musculus (house mouse) | |||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.98 Å | |||||||||||||||||||||||||||||||||
Authors | Robinson RA / Griffiths SC / van de Haar LL / Malinauskas T / van Battum EY / Zelina P / Schwab RA / Karia D / Malinauskaite L / Brignani S ...Robinson RA / Griffiths SC / van de Haar LL / Malinauskas T / van Battum EY / Zelina P / Schwab RA / Karia D / Malinauskaite L / Brignani S / van den Munkhof M / Dudukcu O / De Ruiter AA / Van den Heuvel DMA / Bishop B / Elegheert J / Aricescu AR / Pasterkamp RJ / Siebold C | |||||||||||||||||||||||||||||||||
Funding support | United Kingdom, Netherlands, 10 items
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Citation | Journal: Cell / Year: 2021 Title: Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling. Authors: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / ...Authors: Ross A Robinson / Samuel C Griffiths / Lieke L van de Haar / Tomas Malinauskas / Eljo Y van Battum / Pavol Zelina / Rebekka A Schwab / Dimple Karia / Lina Malinauskaite / Sara Brignani / Marleen H van den Munkhof / Özge Düdükcü / Anna A De Ruiter / Dianne M A Van den Heuvel / Benjamin Bishop / Jonathan Elegheert / A Radu Aricescu / R Jeroen Pasterkamp / Christian Siebold / Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin ...During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs. | |||||||||||||||||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12286.map.gz | 24.9 MB | EMDB map data format | |
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Header (meta data) | emd-12286-v30.xml emd-12286.xml | 34.9 KB 34.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12286_fsc.xml | 16.3 KB | Display | FSC data file |
Images | emd_12286.png | 461 KB | ||
Masks | emd_12286_msk_1.map | 166.4 MB | Mask map | |
Others | emd_12286_additional_1.map.gz emd_12286_half_map_1.map.gz emd_12286_half_map_2.map.gz | 155.4 MB 131.8 MB 131.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12286 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12286 | HTTPS FTP |
-Validation report
Summary document | emd_12286_validation.pdf.gz | 362.8 KB | Display | EMDB validaton report |
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Full document | emd_12286_full_validation.pdf.gz | 361.9 KB | Display | |
Data in XML | emd_12286_validation.xml.gz | 18.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12286 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-12286 | HTTPS FTP |
-Related structure data
Related structure data | 7ndgMC 7ne0C 7ne1C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10637 (Title: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex Data size: 1.2 TB Data #1: Unaligned multi-frame micrographs of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_12286.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.85 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_12286_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: #1
File | emd_12286_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_12286_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_12286_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Gu...
+Supramolecule #1: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Gu...
+Supramolecule #2: Netrin 1
+Supramolecule #3: Neogenin
+Supramolecule #4: Repulsive Guidance Molecule B
+Supramolecule #5: Repulsive Guidance Molecule B C-terminal region (chain D)
+Macromolecule #1: Netrin-1
+Macromolecule #2: Neogenin
+Macromolecule #3: Repulsive Guidance Molecule B (C-terminal region)
+Macromolecule #4: RGM domain family member B
+Macromolecule #5: CALCIUM ION
+Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.07 mg/mL | ||||||||||||||||||
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Buffer | pH: 7.5 Component:
Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3 | ||||||||||||||||||
Grid | Model: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa Details: Agar Scientific Ultra-thin carbon support film, 3 nm - on lacey carbon. https://www.agarscientific.com/ultra-thin-carbon-support-film-3-nm-on-holey-carbon | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: Lacey carbon grids with 3 nm ultrathin carbon support film were glow discharged for 30 seconds at high RF level using Harrick Plasma Cleaner, model PDC-002-CE, and then 3.5 microl of the ...Details: Lacey carbon grids with 3 nm ultrathin carbon support film were glow discharged for 30 seconds at high RF level using Harrick Plasma Cleaner, model PDC-002-CE, and then 3.5 microl of the sample was pipetted per grid. Excess protein was blotted away for 3 seconds using filter paper (round filter paper for Vitrobot from Agar Scientific, catalogue number 47000-100) and Vitrobot Mark IV (Thermo Fisher Scientific) (relative force -15) at 95-100% humidity. Grids were plunge frozen in liquid ethane.. | ||||||||||||||||||
Details | The ternary NEO1-NET1-RGMB complex was purified by SEC on a S200 10/300 Increase column with a running buffer of 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3 at 4 degrees C . The peak fraction containing the ternary complex was diluted to 0.07 mg per ml in SEC buffer. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 77.0 K |
Specialist optics | Phase plate: VOLTA PHASE PLATE |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1635 / Average exposure time: 37.26 sec. / Average electron dose: 40.0 e/Å2 Details: Cryo-EM data were collected on a Titan Krios G3i microscope (Thermo Fisher Scientific) operating at 300 kV with a 50 microm C2 aperture and Volta phase plate (Thermo Fisher Scientific), at ...Details: Cryo-EM data were collected on a Titan Krios G3i microscope (Thermo Fisher Scientific) operating at 300 kV with a 50 microm C2 aperture and Volta phase plate (Thermo Fisher Scientific), at the Division of Structural Biology, University of Oxford. Movies were recorded using a FEI Falcon III direct electron detector in electron counting mode using EPU software at a nominal magnification of 96000x, corresponding to a physical pixel size of 0.85 angstrom/pixel. A total dose of 40 electrons per square angstrom was used at a dose rate of 0.77 electrons/pix/sec. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -0.7000000000000001 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 96000 |
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
-Atomic model buiding 1
Details | Crystal structure used as a model for fitting and rigid body refinement will be described by Robinson, Griffiths, van de Haar, Malinauskas et al., Cell, 2021. |
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Refinement | Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient |
Output model | PDB-7ndg: |