-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Pavel has already responded to most of these points but I would like to add a few notes. The fact that after you build a ligand you see that the density greatly "improves" is quite scary to me. This sounds more like the map is echoing back your model and not confirming it. I would delay building the ligand until everything else in the model has been corrected to get the most reliable and unbiased map possible and then try to figure out what is there. There is no reason to rush the interpretation if there is more work to do on the back side of the protein. The mistake you appear to be making is to ask the question "Does my ligand fit the map?" Even if something has bound, you don't know for sure that it is your ligand. The fact that you built your ligand into the pocket and your stats improved does not mean that building something else wouldn't have improved them more. You can't simply see density and assume it is density for your ligand. The fact that you have to contour at lower than usual levels indicates that whatever is there is not at full occupancy. This makes identifying the true occupant of your site much more difficult. First you have to decide what possible things might be binding to your protein and identify the most visible characteristics of each. I'm used to working with chlorophyll so I'll use that as an example. A chlorophyll is a big and flat dinner plate. Unlike most other big conjugated molecules, a porphyrin usually has a ligand that bind right in the center. While heme is also a common porphyrin the metal at its center usually is much more electron rich, while the chlorophyll has a "flag" stiking up from one of its ringlets. These are the sorts of details you have to have in mind when looking at your density. Also remember that if whatever is in your binding pocket is there only 60% of the time, something else is probably there 40% of the time. Your density will include that too. It is very handy to have an isomorphous apo crystal form so you can see what that 40% looks like. If you have that apo data you can get a clearer view of the difference upon binding with an Fo-Fo map. Pavel has listed a number of types of maps to calculate that are all good possibility but each has its drawbacks (which he listed). I am a big advocate of the "discovery map" because if it doesn't show clearly that your ligand is the best interpretation than you shouldn't be writing the paper to begin with. Holding off on building the ligand as long as possible will give the best map possible and you the best shot at not making a mistake. On the other hand the discovery map is just a difference map and suffers the same limitation Pavel mentioned about the limitations of bulk solvent models. One solution is an idea advocated by Andy Karplus that he calls the "clean difference map". This will sound odd from coming from a person who has spent decades trying to convince people to refine their models against all their data, but just because you refine against all the data doesn't mean you have to include it all in your map calculation. A distinction between the bulk solvent model which may be inappropriately flooding into your pocket and the density of a true ligand is that the true ligand will have high resolution features. You can filter out the undesired bulk solvent contribution by leaving out the low resolution reflections when calculating your Fo-Fc map. This is what Andy calls a "clean difference map". The clean difference map will have artifacts due to the low resolution series termination ripples, but they will be different than the artifacts due to the inappropriate bulk solvent model in your pocket. The features that are common to the two maps should be unrelated to either set of artifacts and more likely due to the true contents of the pocket. Give it a shot, it can't hurt. We usually cut off at around 6A resolution because between 5 and 6 is when the bulk solvent scattering really starts to kick in. You also asked if burning your crystals could improve the map. If you have more crystals I would recommend soaking them in a higher concentration of ligand. No amount of fancy data collection is going to make the density of a low occupancy compound stronger. Getting the occupancy up will do you worlds of good. Dale Tronrud On 11/13/2014 10:40 PM, Ivan IVANOV wrote:

Dear All,

Would you mind if I disrupt your conversation about the omit map or composite omit map ?? I would like to share with you I'm in the same situation as Bishal. I want to see a ligand in the structure I have. I am using Phenix for refinement. When I do a molecular replacement with the model or part of the model, it looks like there is continues density of the ligand on 2Fo-Fc and Fo-Fc map at lower counter level.

1st QUESTION: Once we got the phases from the model which serves for MR, what is the counter level limit for a 2Fo-Fc and Fo-Fc maps we can look at ?? Long time ago I saw some papers in the literature telling about a sigma of 0.4-0.5 there is still useful information. (This first map after MR is not biased by ligand I want to see). Further going on the structure refinement, at some point I add the compound I poorly see and I do 3 different refinements !! One with compound occupancy 0.8, another one with 0.7 and another one with 0.6. Then the density for the compound after refinement appears. At that point I am on the second option in Pavel's suggestions below - I get probably a biased positive density in the map. BUT, I would like to notice that my Fwork and Ffree go significantly down. 2nd QUESTION: Does it mean the compound is there if statistics do down??

About first Pavel's suggestion below when I am doing refinement in absence of the ligand it is the same situation Fwork and Free go down and the ligand density it looks like to be still there and it is NOT completely masked by bulk solvent (but I have to go still to lower counter lever for the 2Fo-Fc map).

Another point I would like to arise. I've done a composite omit map with Phenix (because as I told you I like Phenix). One thing it is worth noticing when you are in a hurry or you want to see quick result because you are not patient enough, do not use Phenix for omit maps (it is slow :-) :-) :-) ). Except that I like very much Phenix. In comparison I did also the same maps with CCP4 and at the end it does not seem to change that much the result when you compare both omit maps coming from Phenix or CCP4.

About my case the omit map (no biased) gives the same quality of density for the compound as 2Fo-Fc map after MR. I have to go in a lower counter lever to see something.

3rd QUESTION: What is the limit for a composite omit map to look at ?? 0.5-0.6 sigma ??

4th QUESTION: Do you think I can improve the quality of the density if I burn my crystals (collect several multiplicity) ?? or probably murge different crystals data??

I like very much Dale's offer about the discovery map. I believe there is something there.

Sorry for the long mail,

Kind regards, Ivan

On Thu, 2014-11-13 at 08:38 -0800, Pavel Afonine wrote:

...

Hello Bishal,

as discussed before, computing ligand-omit map is tricky.

Quoting my previous email:

""" There are two commonly used options for omitting the ligand in order to demonstrate its presence/absence in Fo-Fc OMIT map:

1) Physically remove the ligand from PDB file. Then do some refinement and calculate Fo-Fc map.

2) Keep ligand in the file, set its occupancy to zero. Then, again, do some refinement and calculate Fo-Fc map. In this case you may want to ask refinement program to not move the ligand or move it only a little.

Now, here is why these two options are poor and will not give you what you want.

In the first case the bulk-solvent mask will be set in the ligand region and therefore it will mask ligand density (bulk-solvent will be filled into the ligand region). Depending on the strength of ligand density it may be masked completely or deteriorated.

If you follow the second option you will always get positive density in ligand area. This density may correspond to bulk-solvent, ligand or mixture of both. That is there will be no simple way to differentiate whether this density arises from the ligand or bulk-solvent. """

Dale Tronrud offered a great alternative option:

""" An alternative you might want to consider is what I call the "discovery map". At some point in the refinement process there was a map that convinced YOU that this ligand was present. You should be the hardest person to be convinced so that map will be both an omit map (because the model had been refined without the ligand prior to this) and clear enough to satisfy the reader. """

Now, recently I improved composite OMIT map calculation in

phenix.composite_omit_map

tool, see picture that illustrates how it works:

https://www.dropbox.com/s/dbp8e0348v5p78h/fig_6.png?dl=0

I think at present that's the best option to follow if for some reason you cannot follow Dale's suggestion.

Pavel

...

Hello everybody, I am generating figures showing the electron density map around the ligands. I deleted the ligands from final co-ordinate file and then performed refinement with identical parameters as before while keeping simulated annealing=true. I shall be thankful if someone could suggest me whether I consider mFo-DFc map or 2mFo-DFc map or both. Kindly also tell me the minimum acceptable contour level required for showing difference map. All structures are at 1.4 - 2.0 angstrom resolutions.

Regards, Bishal _______________________________________________ phenixbb mailing

On 11/13/14 6:58 AM, Singh, Bishal wrote: list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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Hi Nat, high-resolution data won't help you much and certainly much less than accurate low resolution reflections. "Burning the crystal" also contradicts "as accurately as possible", and what Dale suggests will help much more than high resolution reflections. High resolution reflections get lost first upon radiation damage and for partially occupied ligands, and accurate low resolution reflections are worth much more. Regards, Tim On 11/15/2014 12:28 AM, Nathaniel Echols wrote:

On Fri, Nov 14, 2014 at 2:06 PM, Dale Tronrud wrote:

...

You also asked if burning your crystals could improve the map. If you have more crystals I would recommend soaking them in a higher concentration of ligand. No amount of fancy data collection is going to make the density of a low occupancy compound stronger. Getting the occupancy up will do you worlds of good.

But won't measuring the weak high-resolution reflections as accurately as possible help resolve map details like partial-occupancy atoms? (Although I think it's better to do this by collecting more redundant data than increasing the exposure time.)

-Nat

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