[Nescafe]

Hello,

I am wondering why is it hard to get a crystal structure for the following protein.

Protein A has an inhibitor with 150nM potency. There is a crystal structure with the inhibitor bound for protein A.

Protein B is a close cousin of protein A with 77% identity similarity and a known inhibitor with a binding potency of 50nM. Noone has been able to get a crystal structure of it.


Is the main reason just inability to obtain a crystal due to what exactly? The 23% difference causes a drastic change in the physical parameters which make it impossible to be crystallized?

Cheers,

Nescafe.

[Arkcon]

I don't know the answer myself, but they seem to be giving you another hint -- the relative potencies of their inhibitors  Is that something you've just started learning in class?  Can you tell us something about that?

[Nescafe]

Hi Arkcon,

I should have mentioned this is not a question I got from class, I graduated a while ago. I am just curious to know. I work in a lab and I heard a colleague saying that the protein she is working does not have a crystal structure whereas the protein I am working on which is 77% identical to the one she is working on has a crystal structure. So we were discussing the reasons as to why it is so.

If I have any idea what I am talking about, an inhibitor that binds tightly to the protein helps the crystallization process and also is a good lee way to figuring out where the binding pocket is. But at the end of the day if you cannot get a good crystal for reasons x, y and z you wont get a X-ray structure. I am just curious to find out what those x, y and z might be for a protein that is 77% identical to another protein for which there is a crystal structure.

Thanks,

Nescafe.

[Babcock_Hall]

I don't think that there is a completely general answer to this question.  However, the N- and C-terminii of proteins are sometimes mobile (floppy), and truncating the protein in this region sometimes makes it easier to crystallize. So, one protein might have a floppier C-terminus than the other, for example.  In a protein crystal some (but not all) regions of protein molecule 1 come into contact with protein molecule 2.  If those interactions were more favorable for one protein than another similar protein, then the first protein would be easier to crystallize.  The two proteins may have different pI values, but of course the crystallization conditions can take that into account. 

[Yggdrasil]

Maybe no one has really tried to get the structure of this protein, especially if structures of close homologs exist.  It seems like you would be able to obtain a decent homology model of the unknowns structure using the known structure that is 77% identical.

[fledarmus]

Protein B is a close cousin of protein A with 77% identity similarity and a known inhibitor with a binding potency of 50nM.

Yggdrasil certainly has one possible answer - maybe nobody has tried. However, in crystallographic terms, 77% is not very similar. In biochemical terms, the binding sites may be nearly identical leading to similar substrates and similar SARs among known inhibitors, but the binding site is a very small portion of the protein. The change from a few very polar groups like aspartic acid or lysine on the protein surface to non polar groups like phenylalanine or valine on the protein surface can radically change the intermolecular forces that would hold the proteins together in a crystal structure. So could a change from very small groups like glycine and alanine to much larger groups like tyrosine and tryptophan.

Crystallizing proteins is a real art, and many proteins, especially those typically present in large amounts, tend to have features that make it difficult for them to crystallize. After all, how useful would the proteins be if they formed crystals in your body? Often, the secret to forming good protein crystals is to figure out how many of those features can be removed or other features can be added to promote crystallization, without affecting the activity or three-dimensional structure of the protein.

[Faris Salama]

Hello,

The 23% difference dose not make it impossible to crystallize but, it is enough to make crystallize (maybe) in a totally different conditions. During my master degree, I tried to find a relation between a protein structures ( using PDB structures), sequence, functions and crystallization conditions. after a very hard work I realize that there no relation at all yet ( maybe in the future and after we solve more protein structures we could find such a relation).  The 23% difference may cause a different arrangement of the protein in the solution ( dimer or oligomer instead of monomer).

the good news is: if she success to crystallize it and get a good diffraction data from the crystal, it is going to be very easy to solve it crystal structure using your protein structure and molecular replacement.

Faris Salama