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- EMDB-12286: Cryo-EM structure of the ternary complex between Netrin-1, Neogen... -

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Basic information

Entry
Database: EMDB / ID: EMD-12286
TitleCryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B
Map data
Sample
  • Complex: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex
    • Complex: Netrin 1
      • Protein or peptide: Netrin-1
    • Complex: NeogeninNEO1
      • Protein or peptide: NeogeninNEO1
    • Complex: Repulsive Guidance Molecule B
      • Protein or peptide: Repulsive Guidance Molecule B (C-terminal region)
    • Complex: Repulsive Guidance Molecule B C-terminal region (chain D)
      • Ligand: CALCIUM IONCalcium
  • Protein or peptide: RGM domain family member B
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


regulation of glial cell migration / chemorepulsion of axon / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / 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 ...regulation of glial cell migration / chemorepulsion of axon / anterior/posterior axon guidance / Cdc42 protein signal transduction / Role of second messengers in netrin-1 signaling / 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 / regulation of synapse assembly / inner ear morphogenesis / DSCAM interactions / basement membrane / endoplasmic reticulum-Golgi intermediate compartment / positive regulation of axon extension / glial cell proliferation / BMP signaling pathway / coreceptor activity / side of membrane / positive regulation of glial cell proliferation / 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 / apoptotic process / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / signal transduction / extracellular region / nucleoplasm / membrane / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Repulsive guidance molecule, C-terminal / Repulsive guidance molecule, N-terminal / Repulsive guidance molecule / Repulsive guidance molecule (RGM) C-terminus / Repulsive guidance molecule (RGM) N-terminus / Neogenin, C-terminal / Neogenin C-terminus / Laminin, N-terminal / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. ...Repulsive guidance molecule, C-terminal / Repulsive guidance molecule, N-terminal / Repulsive guidance molecule / Repulsive guidance molecule (RGM) C-terminus / Repulsive guidance molecule (RGM) N-terminus / Neogenin, C-terminal / Neogenin C-terminus / Laminin, N-terminal / Laminin N-terminal (Domain VI) / Laminin N-terminal domain profile. / Laminin N-terminal domain (domain VI) / Laminin-type EGF-like (LE) domain profile. / Laminin-type EGF-like (LE) domain signature. / Laminin-type epidermal growth factor-like domai / Laminin EGF domain / Laminin-type EGF domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / Netrin domain / NTR domain profile. / Tissue inhibitor of metalloproteinases-like, OB-fold / Immunoglobulin domain / Immunoglobulin domain / Immunoglobulin I-set / Immunoglobulin I-set domain / EGF-like domain signature 1. / Fibronectin type III domain / Fibronectin type 3 domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Fibronectin type-III domain profile. / Galactose-binding-like domain superfamily / Fibronectin type III / Fibronectin type III superfamily / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Netrin-1 / Repulsive guidance molecule B / Neogenin
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.98 Å
AuthorsRobinson 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
OrganizationGrant numberCountry
Cancer Research UKC20724/A14414 United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
European Research Council (ERC)647278 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L017776/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
Wellcome Trust099675/Z/12/Z United Kingdom
Wellcome Trust090532/Z/09/Z United Kingdom
Netherlands Organisation for Scientific Research (NWO)ALW-VICI Netherlands
H2020 Marie Curie Actions of the European Commission289581 Netherlands
CitationJournal: 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
DepositionFeb 1, 2021-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ndg
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12286.png

Downloads & links

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Map

FileDownload / File: emd_12286.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.0066733146 - 0.017858697
Average (Standard dev.)0.00019691909 (±0.0013854764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 299.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z299.200299.200299.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0070.0180.000

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Supplemental data

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Mask #1

Fileemd_12286_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_12286_additional_1.map
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Half map: #2

Fileemd_12286_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_12286_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Gu...

EntireName: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex
Components
  • Complex: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex
    • Complex: Netrin 1
      • Protein or peptide: Netrin-1
    • Complex: NeogeninNEO1
      • Protein or peptide: NeogeninNEO1
    • Complex: Repulsive Guidance Molecule B
      • Protein or peptide: Repulsive Guidance Molecule B (C-terminal region)
    • Complex: Repulsive Guidance Molecule B C-terminal region (chain D)
      • Ligand: CALCIUM IONCalcium
  • Protein or peptide: RGM domain family member B
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Gu...

SupramoleculeName: Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 500 KDa

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Supramolecule #2: Netrin 1

SupramoleculeName: Netrin 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 390 KDa

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Supramolecule #3: Neogenin

SupramoleculeName: Neogenin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 39 KDa

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Supramolecule #4: Repulsive Guidance Molecule B

SupramoleculeName: Repulsive Guidance Molecule B / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 180 KDa

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Supramolecule #5: Repulsive Guidance Molecule B C-terminal region (chain D)

SupramoleculeName: Repulsive Guidance Molecule B C-terminal region (chain D)
type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5

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Macromolecule #1: Netrin-1

MacromoleculeName: Netrin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.227402 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGLSMFAGQ AAQPDPCSDE NGHPRRCIPD FVNAAFGKDV RVSSTCGRPP ARYCVVSERG EERLRSCHLC NASDPKKAHP PAFLTDLNN PHNLTCWQSE NYLQFPHNVT LTLSLGKKFE VTYVSLQFCS PRPESMAIYK SMDYGRTWVP FQFYSTQCRK M YNRPHRAP ...String:
GPGLSMFAGQ AAQPDPCSDE NGHPRRCIPD FVNAAFGKDV RVSSTCGRPP ARYCVVSERG EERLRSCHLC NASDPKKAHP PAFLTDLNN PHNLTCWQSE NYLQFPHNVT LTLSLGKKFE VTYVSLQFCS PRPESMAIYK SMDYGRTWVP FQFYSTQCRK M YNRPHRAP ITKQNEQEAV CTDSHTDMRP LSGGLIAFST LDGRPSAHDF DNSPVLQDWV TATDIRVAFS RLHTFGDENE DD SELARDS YFYAVSDLQV GGRCKCNGHA ARCVRDRDDS LVCDCRHNTA GPECDRCKPF HYDRPWQRAT AREANECVAC NCN LHARRC RFNMELYKLS GRKSGGVCLN CRHNTAGRHC HYCKEGYYRD MGKPITHRKA CKACDCHPVG AAGKTCNQTT GQCP CKDGV TGITCNRCAK GYQQSRSPIA PCIKGSGTET SQVAPA

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Macromolecule #2: Neogenin

MacromoleculeName: Neogenin / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 39.268199 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ETGETRVPEV PSSLHVRPLV TSIVVSWTPP ENQNIVVRGY AIGYGIGSPH AQTIKVDYKQ RYYTIENLDP SSHYVITLKA FNNVGEGIP LYESAVTRPH TVPDPTPMMP PVGVQASILS HDTIRITWAD NSLPKHQKIT DSRYYTVRWK TNIPANTKYK N ANATTLSY ...String:
ETGETRVPEV PSSLHVRPLV TSIVVSWTPP ENQNIVVRGY AIGYGIGSPH AQTIKVDYKQ RYYTIENLDP SSHYVITLKA FNNVGEGIP LYESAVTRPH TVPDPTPMMP PVGVQASILS HDTIRITWAD NSLPKHQKIT DSRYYTVRWK TNIPANTKYK N ANATTLSY LVTGLKPNTL YEFSVMVTKG RRSSTWSMTA HGATFELVPT SPPKDVTVVS KEGKPRTIIV NWQPPSEANG KI TGYIIYY STDVNAEIHD WVIEPVVGNR LTHQIQELTL DTPYYFKIQA RNSKGMGPMS EAVQFRTPKA LGSAGKGSRL PDL GSDYKP PMSGSNSPHG SPTSPLDSNG TKHHHHHH

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Macromolecule #3: Repulsive Guidance Molecule B (C-terminal region)

MacromoleculeName: Repulsive Guidance Molecule B (C-terminal region) / type: protein_or_peptide / ID: 3 / Details: Repulsive Guidance Molecule B / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.104494 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: PHLRTFKDNF QTCKVEGAWP LIDNNYLSVQ VTNVPVVPGS SATATNKITI IFKAHHECTD QKVYQAVTDD LPAAFVDGTT SGGDSDAKS LRIVERESGH YVEMHARYIG TTVFVRQVGR YLTLAIRMPE DLAMSYEESQ DLQLCVNGCP LSERIDDGQG Q VSAILGHS ...String:
PHLRTFKDNF QTCKVEGAWP LIDNNYLSVQ VTNVPVVPGS SATATNKITI IFKAHHECTD QKVYQAVTDD LPAAFVDGTT SGGDSDAKS LRIVERESGH YVEMHARYIG TTVFVRQVGR YLTLAIRMPE DLAMSYEESQ DLQLCVNGCP LSERIDDGQG Q VSAILGHS LPRTSLVQAW PGYTLETANT QCHEKMPVKD IYFQSCVFDL LTTGDANFTA AAHSALEDVE ALHPRKERWH IF PSSGTKH HHHHH

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Macromolecule #4: RGM domain family member B

MacromoleculeName: RGM domain family member B / type: protein_or_peptide / ID: 4 / Details: Repulsive Guidance Molecule B / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.022377 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
ETGQCRIQKC TTDFVSLTSH LNSAVDGFDS EFCKALRAYA GCTQRTSKAC RGNLVYHSAV LGISDLMSQR NCSKDGPTSS TNPEVTHDP CNYHSHAGAR EHRRGDQNPP SYLFCGLFGD

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Details: Repulsive Guidance Molecule B / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.07 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium chloride
2.0 mMCaCl2Calcium chloride
1.0 mMC12H14Na8O27S8Sucrose octasulfate, sodium salt
0.01 % (w/v)NaN3Sodium azide

Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.01% NaN3
GridModel: 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
VitrificationCryogen 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..
DetailsThe 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.7000000000000001 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 96000
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm 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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 280158
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 3 / Avg.num./class: 59019 / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3)
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.
Number images used: 68541
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsCrystal 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.
RefinementProtocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-7ndg:
Cryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B

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