Cryo-EM structure of the ternary complex between Netrin-1, Neogenin and Repulsive Guidance Molecule B
ELECTRON MICROSCOPY
Sample
Cryo-EM structure of the ternary Netrin 1-Neogenin 1-Repulsive Guidance Molecule B complex
Sample Components
Netrin 1
Neogenin
Repulsive Guidance Molecule B
Repulsive Guidance Molecule B C-terminal region (chain D)
Specimen Preparation
Sample Aggregation State
PARTICLE
Vitrification Instrument
FEI VITROBOT MARK IV
Cryogen Name
ETHANE
Sample Vitrification 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 PD ...
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.
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
68541
Reported Resolution (Å)
5.98
Resolution Method
FSC 0.143 CUT-OFF
Other 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 ...
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.
Refinement Type
Symmetry Type
POINT
Point Symmetry
C3
Map-Model Fitting and Refinement
Id
1
Refinement Space
Refinement Protocol
RIGID BODY FIT
Refinement Target
Correlation coefficient
Overall B Value
Fitting Procedure
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., Cel ...
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.
