Hello, I try to solve a small protein structure ( 132 aa). I diffracted some crystal and the best resolution which I obtained was 1,42A with a I422 space group. I have other dataset at 1,55A and 1,7A in the same space group and some dataset at 1,98A in a C2 space group. The first strategy was try to solve it through Molecular Replacement with a other protein (18% sequence identity). But Both PHASER and Molrep didn't give good results ( bad contrast and low TfZ).
The secundary prediction of my protein is all beta. Other group is trying with ARCIMBOLDO and SHREDDER but at this moment they don´t obtain any results.
So the question is, If anyone know some programs/methods to solver these datasets.
Thank you.
With such low homology models, molecular replacement is unlikely to work. But given your high resolution, high symmetry tetragonal crystals, you might have luck phasing on the protein's endogenous sulfurs (does it have any Cys or Met?). Search for recent papers and methods of single-wavelength anomalous dispersion using S atoms (S-SAD). Can you freeze your crystals for a synchrotron trip? This would allow you to obtain the best sulfur absorption at 1.77A.
Here are some starter references:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118829/
Nature Methods12,131–133(2015)doi:10.1038/nmeth.3211
http://www.diamond.ac.uk/Home/Corporate-Literature/Annual-Review/Review2015/Villages/Macromolecular-Crystallography-Village/Macromolecular-Crystallography-Village-Developments/Inverse-beam-and-the-use-of-the-mini-Kappa-for-sulphur-SAD-phasing-on-I04.html
Might be good to once try Auto-Rickshaw server to do molecular replacement (no need to provide a search model). It's quick and easy, but at such very low identity nothing is guaranteed.
See link below:
http://www.embl-hamburg.de/Auto-Rickshaw/
Hi David,
Several questions to:
1. Did you check if your molecule fits in the asymmetric unit cell?
2. If there are more than two molecules, do you see Non-Crystallographic peaks on your self-rotation function?
3. Did you search PDB for the structures with similar unit cell parameters?
And did you check if they could be a search models for your structure?
If you still have not get your structure solved, then I would recommend a server SIMBAD.
https://www.ccp4.ac.uk/testserv/index.jsp
This a beta version. Just create an account and try. You need to upload only your data. The program will check structures with the similar unit cell parameters, then structures, which are common contaminants, and finally will check all possible domains from Protein Data Bank (blind search).
If you will not get any result, then you have to get Se-Met derivative of your protein. In the case if you have no Met in your sequence, you may mutate some of your Leu, or Ile to Met, and get Se-Met. If you have Cys or His, you may try to soak in Hg or in Pt, and get heavy atom derivative.
It is long way, but if you are working on the interesting structure it is worth to solve it.
Good luck!
George.
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