Our stabilization algorithm must receive an X-ray structure as input. PROSS is not compatible with NMR structures. Use pdb format only.
This page answers the following questions:
- What should I do if my protein does not have a solved X-ray structure?
- 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?
1. What should I do if my protein does not have a solved X-ray structure?
In such a case follow these 3 steps:
If you haven't found a structure of a close homologue, PROSS can't help you.
- 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 HHpred server. If you found a homologous structure, you may continue to clause 2.
- Use SWISS-MODEL to generate a model for 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 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.
2. 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 1 (clauses 2 & 3).
3. How can I check if my protein has a solved X-ray structure or whether there is a homologous structure?
Use the HHpred server.
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.
4. Which structure should I use if there is more than one option?
If the same sequence was crystallized more than once, choose the structure that contains more information.
Information may include:
- Presence of ligands (see small-molecule ligands and interacting chains).
- More residues - in some structures there is a lot of missing density, use structures with less missing density.
- Structure resolution and outliers - higher resolution and less outliers are preferred.