Oct 01, 2023
Model Building of PI3KC3-C1/RAB1A
- 1University of California, Berkeley;
- 2ASAP CRN
Protocol Citation: Annan SI Cook 2023. Model Building of PI3KC3-C1/RAB1A. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xn84g1b/v1
Manuscript citation:
Pettersen, E. F. et al. UCSF ChimeraX: Structure visualization for researchers,
educators, and developers. Protein Sci30, 70-82 (2021). https://doi.org:10.1002/pro.3943
Croll, T. I. ISOLDE: a physically realistic
environment for model building into low-resolution electron-density maps. Acta Crystallogr D Struct Biol 74, 519-530 (2018). https://doi.org:10.1107/S2059798318002425
Pavel V Afonine, et al. Real-space refinement in PHENIX for cryo-EM and crystallography, ,Acta Crystallogr D Struct Biol. 2018 Jun 1; 74(Pt 6): 531–544.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 88629
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Details model building of the PI3KC3-C1/RAB!A complex using chimeraX1.5-1.6 and ISOLDE. Refinement was done using Phenix real space refinement.
Generate Starting model
Generate Starting model
Use Alphafold2 to generate a starting model containing the PI3KC3-C1 components (VPS34, VPS15, ATG14, BECN1) and RAB1A respectively.
Docking and model building/refinement
Docking and model building/refinement
Dock each starting model into the experimental map
Use ISOLDE to relax the entire PI3KC3-C1 complex and RAB1A into the experimental data in ChimeraX, selectively using restraints and ignoring clashes until the model simulates correctly. Trim obvious disordered regions by deleting them in Chimera.
Build ligands into the map, use the ignore command to ignore clashes selectively until the clashes have been eliminated.
Iteratively refine the model by building sidechains into density features, and improving the Ramachandran angles with selective restraints.
Assess the model to map fit using the built in Q-score function and Ramachandran plot
When model fit to density and Ramachandran angles start to look good, export the model and refine using Phenix real space refinement.
Repeat steps 5-7 until you are satisfied with the final model and the metrics cease improvement.