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Yorodumi- PDB-7ndg: Cryo-EM structure of the ternary complex between Netrin-1, Neogen... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7ndg | |||||||||||||||||||||||||||||||||
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Title | Cryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B | |||||||||||||||||||||||||||||||||
Components |
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Keywords | SIGNALING PROTEIN / Signal transduction / cell surface receptors / neuron regeneration / cell migration / Netrin / Neogenin / Repulsive Guidance Molecule / complex structure / protein-protein interactions | |||||||||||||||||||||||||||||||||
Function / homology | Function and homology information regulation of glial cell migration / chemorepulsion of axon / DSCAM interactions / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / motor neuron migration / Regulation of commissural axon pathfinding by SLIT and ROBO / negative regulation of axon extension ...regulation of glial cell migration / chemorepulsion of axon / DSCAM interactions / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / Netrin-1 signaling / motor neuron migration / Regulation of commissural axon pathfinding by SLIT and ROBO / negative regulation of axon extension / Netrin mediated repulsion signals / substrate-dependent cell migration, cell extension / mammary gland duct morphogenesis / nuclear migration / positive regulation of cell motility / DCC mediated attractive signaling / inner ear morphogenesis / regulation of synapse assembly / BMP signaling pathway / positive regulation of glial cell proliferation / endoplasmic reticulum-Golgi intermediate compartment / basement membrane / glial cell proliferation / positive regulation of axon extension / coreceptor activity / side of membrane / cell-cell adhesion / actin cytoskeleton / Ras protein signal transduction / DNA-binding transcription factor activity, RNA polymerase II-specific / cell adhesion / membrane raft / RNA polymerase II cis-regulatory region sequence-specific DNA binding / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / apoptotic process / 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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.98 Å | |||||||||||||||||||||||||||||||||
Authors | Robinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. ...Robinson, R.A. / Griffiths, S.C. / van de Haar, L.L. / Malinauskas, T. / van Battum, E.Y. / Zelina, P. / Schwab, R.A. / Karia, D. / Malinauskaite, L. / Brignani, S. / van den Munkhof, M. / Dudukcu, O. / De Ruiter, A.A. / Van den Heuvel, D.M.A. / Bishop, B. / Elegheert, J. / Aricescu, A.R. / Pasterkamp, R.J. / Siebold, C. | |||||||||||||||||||||||||||||||||
Funding support | United Kingdom, Netherlands, 10items
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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 | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ndg.cif.gz | 537.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ndg.ent.gz | 437 KB | Display | PDB format |
PDBx/mmJSON format | 7ndg.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ndg_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 7ndg_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 7ndg_validation.xml.gz | 80.5 KB | Display | |
Data in CIF | 7ndg_validation.cif.gz | 122.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nd/7ndg ftp://data.pdbj.org/pub/pdb/validation_reports/nd/7ndg | HTTPS FTP |
-Related structure data
Related structure data | 12286MC 7ne0C 7ne1C M: map data used to model this data 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
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 4 types, 12 molecules AGDBHEFCINMO
#1: Protein | Mass: 49227.402 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: NTN1, NTN1L / Plasmid: pHLsec Details (production host): Aricescu et al., Acta Crystallogr D Biol Crystallogr, 2006, PMID 17001101 Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: O95631 #2: Protein | Mass: 39268.199 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Neo1 / Plasmid: pHLsec Details (production host): Aricescu et al., Acta Crystallogr D Biol Crystallogr, 2006, PMID 17001101 Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: Q7TQG5 #3: Protein | Mass: 28104.494 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Details: Repulsive Guidance Molecule B / Source: (gene. exp.) Homo sapiens (human) / Gene: RGMB / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q6NW40 #4: Protein | Mass: 13022.377 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Details: Repulsive Guidance Molecule B / Source: (gene. exp.) Homo sapiens (human) / Gene: RGMB / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q6NW40 |
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-Non-polymers / Sugars , 2 types, 15 molecules
#5: Chemical | #6: Sugar | ChemComp-NAG / |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
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Molecular weight |
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3 | ||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.07 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES 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% ...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. | ||||||||||||||||||||||||||||||||||||
Specimen support | 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 Grid material: COPPER / Grid type: PELCO Ultrathin Carbon with Lacey Carbon | ||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: -700 nm / Nominal defocus min: -500 nm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K |
Image recording | Average exposure time: 37.26 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1635 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. |
EM imaging optics | Phase plate: VOLTA PHASE PLATE |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 280158 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 5.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68541 Details: In total 1635 movies were collected and drift correction, beam-induced motion and dose-weighting were performed with MotionCor2 RELION 3.1 (Zivanov et al., 2018) for 1635 movies. Contrast ...Details: In total 1635 movies were collected and drift correction, beam-induced motion and dose-weighting were performed with MotionCor2 RELION 3.1 (Zivanov et al., 2018) for 1635 movies. Contrast transfer function (CTF) was estimated using CTFFIND 4.1 (Rohou and Grigorieff, 2015) implemented in RELION. 280158 particles were picked using Warp (Tegunov and Cramer, 2019). 2D classification in cryoSPARC v2 (Punjani et al., 2017) were performed for these particles and best 2D class averaged with 100674 particles were used to generate ab-initio 3D model with C3 symmetry. C1 symmetry did not generate reasonable 3D model. All Warp picked particles were used for 3D classification in cryoSPARC v2 and the best class with 177056 particles was used for refinement in RELION 3.1 with initial model generated in cryoSPARC v2. Byesian particle polishing improved the resolution to 5.44 angstrom, however the map for RGMB was very weak. Last step of 3D classification without alignment with T regularisation parameter set to 16 was performed and gave one class with more continuous map for RGMB, which was refined to 5.98 angstrom resolution, as estimated using the Fourier shell correlation (FSC) = 0.143 criterion. Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient 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. |