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Thanks Zaighum, Artem, and Eleanor, for your replies.

@Zaighum: I could not find a pdb file for PFPE- it is not listed in common ligand dictionaries of Refmac, Phenix or even HIC-Up server, or in general. It is fairly big molecule  to fit into this small cavity. Also I couldn't find any protein structures with PFPE in PDB database.
The green blobs do have blue density, it is just not visible at the current contour level. At slightly decreased contour level (1.81 vs 1.51 rmsd), it is perfectly visible. We have tried updating waters during refinement, even then, the green blobs would not completely go away. Yes I can add waters manually, and they might make sense, but we thought that the density might be something else we have missed to identify- so decided to ask here on BB.

@Artem and Eleanor: The structure is well refined (R and Rfree 19% and 23% respectively, at 1.9A) except for this density. As I mentioned above, decreasing the contour level of 2Fo-Fc map would show that the glutamate you suggested to remodel is sitting in the blue density (although slightly off). I have tried to move it in the green density, however, it does not stay there. Perhaps it has an alternate conformation, or lower occupancy. Even then that would satisfy a small part of the green density, not all. The remaining residues surrounding the density are well modeled.

Thanks again, and regards,
Uma.

--
Uma Gabale, PhD
Research Associate
Molecular and Cellular Biochemistry
Indiana University Bloomington


On Tuesday, July 3, 2018, 2:36:31 AM EDT, Eleanor Dodson <[log in to unmask]> wrote:


Hmm - is the refinement complete? Maybe the GLU on the right could be moved to use some of that greenness? 
I try to make all sensible corrections, then check blobs.. 
Eleanor

On 3 July 2018 at 04:22, zaigham mahmood khan <[log in to unmask]> wrote:
Uma, that is not something that we see regularly in the crystal structures. But i never used PFPE either. So, you may try PFPE. If this is not PFPE, then you may read the biology of the protein.

Also I can see green density, but these green blobs are devoid of blue density. So i will be cautious. May be just add water molecule in each blob, and re-run refinement. You will see a change in color of the blobs, that may indicate something.. If red blobs appear around the water molecules, i will consider it just a noise. Alternatively, you will see green density connecting the water molecules...





Best wishes

-Z


Zaigham Mahmood Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences

On Mon, Jul 2, 2018 at 10:23 PM, Uma Gabale <00000ebb5dcf3eaa-dmarc- [log in to unmask]> wrote:
Dear all,

We came across a blob of unidentified electron density in a shallow cavity of a bacterial protein structure (pictures attached). It is surrounded by residues Asp, Arg, Gln, His, G lu, Thr, and Trp.

The protein was expressed in E. coli BL21(DE3) and purified on Ni-NTA followed by gel filtration. The purification buffers included Tris, crystallization condition had HEPES and PEG3350; perfluoropolyether was used as a cryoprotectant.

We would appreciate any help in identifying it.​

Thanks and regards,

Uma.

--
Uma Gabale, PhD
Research Associate
Molecular and Cellular Biochemistry
Indiana University Bloomington


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