Oct 30, 2023

Public workspaceModel building and refinement of RCKW and FL-LRRK2 bound to inhibitors

DOI
dx.doi.org/10.17504/protocols.io.81wgbx5m1lpk/v1
  • 1University of California, San Diego
Marta Sanz Murillo
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgbx5m1lpk/v1
Protocol Citation: Marta Sanz Murillo 2023. Model building and refinement of RCKW and FL-LRRK2 bound to inhibitors . protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx5m1lpk/v1
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 24, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 89833
Keywords: ASAPCRN, cryo-EM, LRRK2, inhibitors
Funders Acknowledgement:
Aligning Science Across Parkinson's: ASAP
Grant ID: ASAP-000519
Abstract
Protocol to model and refine a PDB against a cryo-EM map.
Materials
ChimeraX1
COOT2,3
Phenix4
Rosetta5
Before start
Install the needed software (Chimera, COOT) and Phenix or Rosetta.
Split into domains protein models using Chimera that will be used as a starting point (PDBs 6VP7 and 7LHW for RCKW and FL LRRK2, respectively). Save each domain in a PDB file separately.
Open maps with the best resolution in Chimera and fit every domain PDB into the map. Save the new position for each PDB domain file.
Open PDB files in COOT and merge all files. Save a new PDB file with all domains.
Check all amino acids manually and refine/regularize them against the cryo-EM map. You can find a basic COOT tutorial for cryo-EM here:
https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/tutorial/Coot-Cryo-EM-basics.html
You might want to model amino acids not present in your starting point PDBs if you have empty density. To do that:
1. Add residue (Right side, eighth icon from the bottom)
2. Click on the alpha carbon of the last amino acid.
3. Mutate residue (right side, tenth icon from the bottom). It pops up a list with all amino acids. Click on the amino acid you want to add).
Once you manually checked your model, save it. Then, add your ligand. To do that:
Save your ligand in a PDB file. Open Phenix and use elBOW application to generate restrains for your ligand.
1. On the right side, click Ligands > elBOW
2. On elBOW setup, click PDB file with hydrogens and/or CONECT records > OK
3. Click Use simple optimization >OK
4. In geometry file, browse your PDB_ligand.pdb
5. Browse your output directory
6. Type Output file prefix
7. Run the job.
Note
If you don't have a PDB and you have instead a SMILES notation for your ligand, you can generate a PDB and restrains for it by clicking on the step 2 SMILES string option instead

Open the ligand PDB_Ligand.pdb in Chimera, fit it into the map, and save a new file. Open it in COOT together with your model protein and merge them. Save a new PDB with ligan plus protein.
Refine/Regularize your ligand against the map in COOT. Save your new PDB.
Refine your model against your map in Phenix or Rosetta:

In Phenix:
1. Click Cryo-EM > Refinement > Real-Space Refinement
2. Input/Output Tab:
2.a: Type your Job Title
2.b: Add file > PDB_protein+ligand.pdb
2.c: Add file > map.mrc
2.d: Add file > Ligand_restrains.cif
3. Add map resolution
4. In refinement settings Tab: Keep it as default
5. Run the job
6. Once the job is done, open your new PDB_protein+ligand_new.pdb in COOT and check amino acids and restrains.
7. Check Ramachandran plot. Manually correct amino acid outliers and allowed


In Rosetta:
1. Remove inhibitors and nucleotides from the model.
2. Rosseta/1.3 for refinement.
3. Move map and pdb into your directory
4. Load rosetta using module load rosetta/3.13
5. Once is finished go to COOT, add your ligand and remove all the hydrogens from your structure
5.a: To remove hydrogens in COOT, go to Calculate > Scripting
5.b: Type delete_hydrogens(#) (# is the model)
Check validation parameters and fix outliers you may have in your PDB. Run a refinement in Phenix or Rosetta (it is an iterative process, so you might have to correct/refine it several times).
Protocol references
1. E. F. Pettersen, T. D. Goddard, C. C. Huang, E. C. Meng, G. S. Couch, T. I. Croll, J. H. Morris, T. E. Ferrin, UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021)
2. P. Emsley, K. Cowtan, ıt Coot: model-building tools for molecular graphics. Acta Crystallographica Section D. 60, 2126–2132 (2004).
3. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of ıt Coot. Acta Crystallographica Section D. 66, 486–501 (2010).
4. D. Liebschner, P. V. Afonine, M. L. Baker, G. Bunkóczi, V. B. Chen, T. I. Croll, B. Hintze, L.-W. Hung, S. Jain, A. J. McCoy, N. W. Moriarty, R. D. Oeffner, B. K. Poon, M. G. Prisant, R. J. Read, J. S. Richardson, D. C. Richardson, M. D. Sammito, O. V. Sobolev, D. H. Stockwell, T. C. Terwilliger, A. G. Urzhumtsev, L. L. Videau, C. J. Williams, P. D. Adams, Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in ıt Phenix. Acta Crystallographica Section D. 75, 861–877 (2019).
5. Wang, R. Y.-R. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).