Dear all,
I have got a protein crystals which diffracted very well (~0.9A) and during solving the structure I have noticed that around my protein in few places I have a electron density of the small molecule. I have checked all buffer compounds and potential other reagents that were used during purification but unfortunately I couldn't fit any compound. I used Roche protease inhibitors but none of popular protease inhibitors didn't fit. Electron density is present outside the protein. It seems that it has very big atom and one in the middle which connects all together. It seems like sp3 hybridisation (tetrahedral). I have ~5 places where I can find this electron density, so it doesn't seem to be an artifact. Please check the attached pictures with the density (1.8 I/sigma) from back/front/side and up.
Kind regards,
Dawid
it sounds interesting maybe protease inhibitors u fit are not the actual inhbitor?
The problem with Roche inhibitors is that you don't have any list of ingredients. I have compared inhibitors from Sigma Aldrich tablets but nothing fitted to the density (inhibitors are mainly peptides and aromatic compounds like PMSF).
Yes, 20% glycerol was a cryoprotectant. Do you think it might be glycerol or some compound which could be present in glycerol stock?
Hi,
Can you explain the main density with an Oxygen atom?
Have this atom any interaction with the protein?
The distances between the 3 density peaks are? (a picture could help)
All theses 5 places lock the same?
If O atom explain the main density, could be, ethylene glygol (EDO), glycerol (GOL) or PEG ???
Did you finish the protein refinement already? I will try to explain that at the end of.
Cheers
Hi,
The density is bigger than single oxygen atom, even two sub densities I think are bigger than oxygen.
I can find it in close proximity of ASN and ARG of my protein main chain. So in total I have only 2 densities like this - rest of them are related to the symmetry. Density which pictures I posted is in ~2A from the ASP so it is very likely that it forms hydrogen bonds.
I don't understand the question - if you mean the size of this density you have in every picture above a crosshair - (the ticks are at 1.54Å, 2.7Å and 3.8Å). The density has ~4.5A length.
Like before, 1 of them is near ASN, second near ARG.
As previous
Yes, protein refinement is ready.
Thank you,
Dawid
If you could send me the files (I'm Nicole Wheatley, from UCLA, I won't steal the data) I could give you my opinion. My main concern is that modeled density doesn't seem to be near the ligand. If you have a ligand, it will be supported by several close contacts with your protein. If this isn't the case, you might consider other possibilities, such as the density representing a a portion of a disordered region of your molecule that happens to be creating a crystal contact. These can be very difficult to model. Do the ligands lay between molecules in the crystal? What is the protein? To what is it homologous? That density looks like a very low resolution ADP/AMP molecule, but you say the data is high resolution. So, until I can dink around with it, I can't say too much.
Hi Dawid
From the pictures, it seems that you are in a center of symmetry?
If it it the case could be an artifact because of the crystal packing.
Which space group you defined? Did you tried a lower one?
Could you try to refine 3 O atoms inside and post here pictures again with distance between them and the resulting B factors?
Cheers
Carlos
Are all 5 areas similarly occupied (roughly same appearance at each I/sigma) and all within H bonding distance the protein? Looks similar to density I've modeled as acetate, but that was in my buffers
Thank for your answers.
Carlos - Space group is P2(1) and I am very sure that this is the space group. In the picture you can see 2 water molecules and one sodium - otherwise water molecules were to close to each other and were connecting in coot.
William - in total I can see 4 of them - every two are related in symmetry. I can see some differences in the densities between them but size and shape is exactly the same. The distance between density blob and protein chain is ~2.8/3A. Please check the picture with acetyl group inside the density (I used also acetate in my buffer) - still one density lacks an atom.
Hi Dawid
Ok, now for me it is more clear the sp3 that you mention.
Could you measure the distances and the angle between you O-Na-O, please...
Could be ...
OCl2 with two water molecules H-bonding (the two littlest densities)?
See : https://www.youtube.com/watch?v=QwWQX0CyO3Y
Cheers
Carlos
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