Hi,
I had crystallised a protein and realised that some amino acids doesn't fit to the electron density in the electron density map. Is it possible to have different amino acid in the protein crystal structure that doesn't match with its real sequence?
if this is possible, can you explain how?
thanks,
Did you check the molecular mass of your protein by mass spectroscopy? The amino acid sequences of a protein does not suddenly change upon crystallization. A mismatch between the expected sequence and what you see in your structure may be due to a sequence error in the expression construct (if you are working with recombinant protein) or an unexpected postranslational modification (dependent on the expression host). Are you sure you can exclude a mis-interpretation of your density, e.g. a wrong alignment of your sequence to the specific segment of the density? How pure was your protein? Can you exclude the possibility that your crystals represent an impurity that crystallized out of your protein prep?
Shymaa Damfo
Annemarie pretty much covered everything relevant here, just let me add a few minor things.
If you have radiation damage the electron density on certain amino acid sidechains will not look like what you expect. Multiple alternate rotamer conformations in poorly contoured map regions can also be misleading.
Secondly, if you express proteins that are toxic for the host you can have natural selection in your expression system that accumulates less toxic mutants of your target protein. I once had a project where we had to sequence the vector in every batch of biomass before we would purify it.
And yes, always get a mass conformation!
Stefan
Annemarie Honegger Thank you for your reply. I confirmed the molecular size in the SDS gel page but did not check the protein molecular mass by mass spectroscopy. Do you recommend to do mass spectroscopy?
It's a recombinant protein, and E.coli was used as an expression host. The protein is pure (99%), so impurities are not expected. I did DNA sequencing so sequence error is excluded. I'm wondering now if it's an unexpected posttranslational modification or misinterpretation of the electron density map as the resolution is 2.9 Å.
Thank you for bringing all these to my attention.
Stefan Gajewski Thank you for answer. Is there any way to relate the affect of the radiation damage on any specific residue?
Than you for mentioning your experience here and It seems that mass confirmation is a must!
Mass spec of the full-length protein is a powerful tool to detect a variety of problems: from proteolytic loss of terminal fragments to sequencing errors to post-translational modification. It is therefore a powerful tool to confirm that the purifies protein is exactly what you expect it to be. If not, the mass difference narrows down the possible origins of the deviation.
Dear Shymaa,
Annemarie and Stefan should have started from a question: How did you solve your protein and at what stage of the refinement it is at the present time?
I only guess that you solved it by MR and that the stage is mid- to high 20%, maybe low 30%. Before the answer to this question is provided any speculations about anything else are highly speculative. In essence you have to have sufficient certainty in your electron density as you have in your sequence to claim what you claimed.
General caveats. There is a lot of mistracing errors in PDB. Every other protein I worked on in this resolution range had multiple shift errors. For the last two years I am trying to publish a paper with corrections to the structure with ~15% of residues mistraced, and had 3 rejections so far. Errors have a tendency to survive and multiply. People protect their mistakes sometimes vigorously.
Besides these few caveats, I completely agree with all and any suggestions made by other participants.
Bog
Proteins are made of matter and matter does not disappear easily. X-rays can alter the structure of residues but it is rare, and a lot depends on their exposure and mobility.
It's not clear to me what you see on X-rays, and how long is the sequence of your protein. Are amino acids missing from the sequence encoded in the DNA or the total number of residues is the same but the electron densities of one or more of them do not match? Either way, you need to be in control of the molecular mass. The starting point is the control of the molecular mass of the sequence got in e.coli regarding the molecular mass calculated from the sequence encoded in the DNA. Without a mass spectrometry check, you will never have a solid foundation to argue about.
I advise you to do a simple check of the net charge of your protein calculated at pH 7. If the calculated value is around zero ± 1, then I would assume that I am dealing with a rather compact protein, but if it is beyond these limits, I would evaluate the possibility that the protein may exhibit post-translational modifications. Nothing excludes that the metabolic system of e.coli may have modified the protein.
Post-translational modification (PTM) of proteins is central across all domains of life, but, despite years of study and a wealth of genomic and proteomic data, the Spatio-temporal function of many PTMs remains unknown. This is especially true for prokaryotic. A minimal change in the concentration of glucose or other nutrients in the culture medium is enough to have distinct PTM changes.
Greetings
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