Late-stage 48S Initiation Complex (LS48S IC) guided by the trans-RNA of 15 nt length, purification method of biotinylated DNA-oligo pull-down
Specimen Preparation
Sample Aggregation State
PARTICLE
Vitrification Instrument
FEI VITROBOT MARK IV
Cryogen Name
ETHANE
Sample Vitrification Details
The sample was incubated on the grid for 30 s and then blotted with filter paper from both sides for 1.5 s with the blot force 5
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
1000
Reported Resolution (Å)
2.9
Resolution Method
OTHER
Other Details
Refinement Type
Symmetry Type
POINT
Map-Model Fitting and Refinement
Id
1 (8P09, 6YAM, 6FYX)
Refinement Space
Refinement Protocol
RIGID BODY FIT
Refinement Target
Overall B Value
Fitting Procedure
Details
The atomic model of LS48S IC complex (PDB: 8P09) was initially rigid body fitted in density using ChimeraX and further model building was done for mRN ...
The atomic model of LS48S IC complex (PDB: 8P09) was initially rigid body fitted in density using ChimeraX and further model building was done for mRNA, eIF5 N-terminal domain, eIF2gamma, eIF3d and eIF3 octamer in ChimeraX. Densities for eIF5 N-terminal domain, eIF2gamma, eIF3d and eIF3 octamer were observed in the map and therefore were included in the model. The 3D structure of the eIF5-N terminal domain was generated by Alphafold2 (PDB: G1TGW1). It was then accommodated into the model by aligning structurally to its counterparts in the yeast atomic model of the py48S-eIF5N pre-initiation complex (PDB: 6FYX). IF3d and eIF3 octamer were taken from the atomic model of LS48S IC complex with eIF3 proteins (PDB: 6YAM) and placed into density by rigid-body fitting. The atomic models with and without eIF3 proteins were inspected visually and performed stereochemical, torsional, angle refinement and optimization by using ISOLDE v1.6. Figures featuring cryo-EM densities and atomic models were visualized by using ChimeraX.
