Structures suited for PROSS
This page answers the following questions:
- What should I do if my protein does not have a solved X-ray structure but has a Cryo-EM or an NMR structure?
- What should I do if my protein does not have a solved X-ray structure nor satisfying NMR or Cryo-EM structures?
- My protein has a solved structure but I want to stabilize a variant with few point mutations?
- How can I check if my protein has a solved X-ray structure or whether there is a homologous structure?
- Which structure should I use if there is more than one option?
What should I do if my protein does not have a solved X-ray structure but has a Cryo-EM or an NMR structure?
- An NMR structure is available - download the PDB file, select a single conformation that is most likely to best represent the structure and delete all atom lines of the other conformations from the file. Editing can also be done in PyMOL. Then use our upload option to upload your edited file.
**If an X-ray structure that has only a few point mutations is available, prefer it over an NMR based structure.
- A CryoEM structure is available - use only if it has atomic-level accuracy (i.e., the backbone and sidechain atoms of each protein are observed)
What should I do if my protein does not have a solved X-ray structure nor satisfying NMR or Cryo-EM structures?
In such a case follow these 3 steps:
- Search for an X-ray solved structure of a close homologue (close = above 40% sequence identity with your target). For this purpose you can use the BLAST and change the Database option to pdb. If you found a homologous structure, continue to the next step.
- Use SWISS-MODEL, Phyre2, or other softwares to generate a model of your input sequence. Then download the model file in PDB format. At this point you may use the generated model as input for PROSS. However, we recommend to make the check offered in clause 3.
- Open the 2 structures on PyMOL (the homologous structure that you found and the generated model), align them and examine whether the alignment makes sense - secondary structure elements should have a good overlay, loops may diverge, especially if their length is different. Free PyMOL version for students
- Selection of PROSS designs for experimental testing: carefully examine the mutations on PyMOL, we recommend to omit mutations predicted in loops longer than 5 amino acids, particularly if the loop conformations between the model and the homologue available in RCSB are highly different. Free PyMOL version for students
If you haven't found a structure of a close homologue, PROSS can't help you.
Furthermore, models are not reliable at loop regions. If your protein of interest has a low secondary structure content, avoid PROSS.
My protein has a solved structure but I want to stabilize a variant with few point mutations?
- One option is to submit the original structure. Before ordering the genes make the necessary changes in the amino acid sequences. Also, consider removing mutations offered by PROSS that are close in 3D to your point mutations.
- The other option is to follow the same steps as explained in question 2 (steps 2 & 3).
- You can manually edit the PDB file of the available structure at the mutated positions: for each mutated residue delete first the sidechain atom lines, leaving only the four backbone atom lines (N, CA, C, O). Then edit the backbone atom lines - change the amino acid name to the mutated identity.
How can I check if my protein has a solved X-ray structure or whether there is a homologous structure?
Use the BLAST and change the Database option to pdb.
If you find an X-ray hit that is 100% identical - congratulations, you have a structure!
If you find an X-ray hit with above 40% sequence identity to your input sequence - congratulations, you have a homologous structure. To proceed follow the instructions in question 1.
Which structure should I use if there is more than one option?
If the same sequence was crystallized more than once, use the structure that optimizes the following properties.
- Prefer the presence of ligands (see the Small-molecule ligands and Interacting chains submission options).
- Prefer higher X-ray resolution and less outliers (outliers summary is described in the main RCSB page of any given protein)
- Prefer higher residue content - in some structures there is quite some lack of density. use structures with less missing density.
If you have a structure with ligands that has a significantly lower resolution than another structure that has no ligand, prefer the higher resolution structure and use the Specific residues to fix option to add active-site constraints that you may derive from the structure with the ligands (details here).