Dear Tim,

Thank you for your quick review of the map, and your reply. Actually, I always check every water molecule manually one by one before finalizing my structures because I know that automated algorithms often get them wrong, and also often place waters in ligand density. I'm away from home and away from a graphics computer, and it will be some hours before I can log in remotely to check this again, but judging from the residues on the perimeter of the screen shot you sent, it looks as if the view you are showing is of the pore that is through the center of the homotrimer (the trimer is doughnut shaped). The trimer essentially has a three-fold non-crystallographic symmetry axis down the center of the pore, which is a very narrow pore, often filled with water. (The pore isn't the active site).

However, the final maps were calculated with CNS as I recall (it was almost 7 years ago when I was actually doing the refinement) in order to visualize both overlapping ligands. I mention that because I noticed a long time ago that when displaying CNS maps in Coot, the sigma level is represented by the e/A3 contour. I discovered this when seeing a contour difference between CNS maps displayed in O and Coot. Then when I displayed them in PyMol, they matched the O maps. However, when I displayed the CNS maps in Coot at 1 e/A3, then they looked like the 1 sigma CNS maps in O and PyMol. Many probably know about this already, but I just want to make sure that you are looking at the sigma level you think you have. That looks like a Coot screen shot you are showing, and this peculiar way Coot handles CNS contours may explain why you see that "extra ligand" in that screen shot, but not in your EDS server maps.

...and yes, I do want to get more experience using shelxl myself!

Gregg

On Fri, Nov 28, 2014 at 4:32 AM, Tim Gruene <[log in to unmask]> wrote:

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Dear Gregg,

your post raised my curiosity and I look at the map after a round of
refinement with shelxl.
I thought you might be interested in two observations:

1) shelxl refines the occupancy of the 1Q1 ligand to 60% rather than
78% (I don't think shelxl fills in missing reflections)

2) the second observation is the reason why I am posting this
publicly, because it illustrates the danger of automatic water
placement to hover out important information: there is strong
difference density for another ligand as you can see in the attached
screenshot (the Met on the left is residue A2 in the deposited PDB
file. The water network that was build instead has too high B-values
compared to the surround protein structures.

I assume you used an automated procedure to place these waters - the
map from the EDS sever looks very different on does not reveal an
extra ligand.

Once you model this extra ligand you should further decrease the noise
level and might get an even better indication of the two overlapping
ligands.

Did I mention I like shelxl ;-) (NB: these are 1.55A data!)

Best regards,
Tim

On 11/28/2014 06:22 AM, Gregg Crichlow wrote:
> My fault for not changing the thread subject! I'm sorry! Greetings
> to ccp4bb readers! This is a long-delayed reply to requests that I
> received after I posted an observation on the ccp4bb almost three
> years ago. (I think it was early 2012). The paper had not yet been
> published (pending finalization of experiments unrelated to
> structural biology) and so I was not free to distribute any of the
> structure factors to those who were interested. The paper now has
> been published, and the coordinates and structure factors have been
> released. The following is the peculiarity that made this of
> interest.
>
> I refined a structure with 1.55 A data of a homotrimeric protein
> bound to an inhibitor. Although the inhibitor added to the enzyme
> contained a five-membered ring, the inhibitor we observed in two of
> the three active sites had the ring opened. We therefore discovered
> that the ring-opened form is also an inhibitor. However, in the
> remaining active site, we discovered *both* the ring-opened and

> ring-closed forms of the inhibitor. (The active sites are
> identical, but crystal contacts create asymmetry among the three).
> The two forms of the inhibitor were positioned slightly differently
> in this active site, and so the electron density at this resolution
> clearly identified the presence of both forms, even though there
> was much overlap in their positions. I used the CNS occupancy
> refinement script, and then normalized the results so that the two
> occupancies added up to 1, and so I came up with occupancies of
> 0.88 (ring-opened) and 0.12 (ring-closed). Then having all
> inhibitor molecules in the model, I calculated maps with another
> program, which substituted Fcalc for missing reflections, and could
> no longer see the lower occupancy form in the electron density. I
> re-calculated maps in CNS, and again saw both forms in the electron
> density. So I calculated new maps in CNS the same way as before,
> but turned on the option to substitute missing reflections with
> Fcalc: and the lower occupancy form was missing from the density in
> the resulting maps. So, this was the difference - when I used zero
> for missing reflections, I was able to see both forms; but when I
> substituted Fcalc for missing reflections in maps, I could only see
> the predominant form. The data set was 99.6% complete overall,
> 99.2% in the highest resolution shell; with a multiplicity of 5.8
> (5.3 in the highest shell). So there didn't seem to be many missing
> reflections to be substituted. The test set contained roughly 2% of
> the total number of observed unique reflections.
>
> Some had replied to me, saying that they were interested in using
> the coordinates & structure factors for running tests; however, as
> I said, I was not free to distribute them at the time. Now they
> are publicly available: PDB ID 4K9G. I hope this helps all who are
> interested. Thank you.
>
> Gregg
>
> ***************************
>>

>> *Gregg Crichlow *Visiting Research Scientist Department of
>> Pharmacology Yale University School of Medicine
>>
>

- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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