The maximum resolution obtained by crystallography? - Can we determine the primary structure of a protein by that? (Mar/02/2009 )
Hi
Can we characterize a protein sequence as well as its post-translational map by crystallography. I know that, to some extent, we can obtain this information by MS/MS but I assume if we have enough protein then crystallography would provide more information. Does any body know if I'm right in this regard or not; obviously, my knowledge about crystallography is very limited, I only have some experience with mass-spectrometry. And my problem is that I cannot have a PTM map of the protein with 100% certainty and a full sequence coverage.
Opal
Opal on Mar 3 2009, 05:56 AM said:
Can we characterize a protein sequence as well as its post-translational map by crystallography. I know that, to some extent, we can obtain this information by MS/MS but I assume if we have enough protein then crystallography would provide more information. Does any body know if I'm right in this regard or not; obviously, my knowledge about crystallography is very limited, I only have some experience with mass-spectrometry. And my problem is that I cannot have a PTM map of the protein with 100% certainty and a full sequence coverage.
Opal
As far as I'm aware, no, you can't calculate a protein sequence by crystallography (if that's what you meant by "characterise"). Crystallography requires the sequence in order to generate the structure. You might be able to do it by NMR, but only if you don't have too many prolines or modified residues (unless you know the modification, and have a spectrum of it). You could do some Edman sequencing, but it has its own set of caveats, limitations and problems.
Why in particular do you need these data?
Well, at this point I'm not going to do any more sequencing analysis on my protein, other than what I have from LC/MS/MS. But for building my discussion I want to mention the most commonly ( and reliably) techniques used to map Post-translational modifications on a protein.
Yes, you can see the side chains easily if you have resolution in the 2.5 angstrom range, preferably 2.0A. Some parts of the protein may be disordered and not visible in the electron density.
However, you will need phases to solve your structure, preferably a close homology model if you don't want to use heavy atom methods.
Of course, your protein may never crystallize!
Opal on Mar 2 2009, 11:56 AM said:
Can we characterize a protein sequence as well as its post-translational map by crystallography. I know that, to some extent, we can obtain this information by MS/MS but I assume if we have enough protein then crystallography would provide more information. Does any body know if I'm right in this regard or not; obviously, my knowledge about crystallography is very limited, I only have some experience with mass-spectrometry. And my problem is that I cannot have a PTM map of the protein with 100% certainty and a full sequence coverage.
Opal