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, PhDResearch AssociateMolecular and Cellular Biochemistry
Indiana University Bloomington
On Tuesday, July 3, 2018, 2:36:31 AM EDT, Eleanor Dodson
<[email protected]> 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 <[email protected]> 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 SinaiDepartment of Oncological Sciences1470
Madison AvenueNew York
On Mon, Jul 2, 2018 at 10:23 PM, Uma Gabale <00000ebb5dcf3eaa-dmarc-
[email protected]> 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, PhDResearch AssociateMolecular and Cellular Biochemistry
Indiana University Bloomington
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