Fo-Fc omit map for ligand in the protein-ligand structure

Wei Shi

13 Sep 2014 13 Sep '14

9:40 p.m.

Hi all, I am working with a protein dimer-ligand structure (resolution: 2.8Å) and am going to make a figure showing the bound ligand superimposed with the electron density (Fo-Fc) map obtained after omit refinement. What I did is to make the ligand-free structure model and then use Phenix.refine using the following parameters plus simulated annealing (torsion angle) or (Cartesian): Default: XYZ coordinates + Real-space+ Individual B factors + Occupancies. Also click: Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS. Contoured at 2σ, the Fo-Fc map generated show green density for most part of the ligand, but for several atoms in the middle of the ligand, the green density is missing... It's like this: some atoms have green density and the next few no green density and then a couple few has green density and then a couple more no green denisty.... The missing green density usually means that part is disordered in the structure, right? I am not sure why the density is missing for some atoms in the middle of the ligand. It would makes more sense if the missing green density is at the end of the ligand... Does anyone happen to have any clue about what it means for the green density missing in my case? Also, I am not sure whether I did the omit refinement right: (1). For simulated annealing, it has two options, one is Cartesian and the other is Torsion angle, and it's said that torsion angle is more suitable of low resolution data. Is there any difference about which one to choose in my case? (2). Is it Okay to run the omit refinement in the presence of the following parameters as I did: Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS. Thank you so much! Best, Wei

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Pavel Afonine

13 Sep 13 Sep

10:03 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Wei, I think I replied to a very similar question on this mailing list some time ago (in August). 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. At some point I've spent a day doing tests to exemplify all this. Here is how to do this right. You need to define a box around the ligand that covers the ligand itself and some volume around it, then omit all scattering from inside the box (ligand and bulk-solvent!), and then use this model with box omitted to calculate residual synthesis. I don't think we have a ready to use tool in Phenix to do this. It's possible to do this with some scripting using CCTBX. Pavel On 9/13/14 2:40 PM, Wei Shi wrote:

...

Hi all, I am working with a protein dimer-ligand structure (resolution: 2.8Å) and am going to make a figure showing the bound ligand superimposed with the electron density (Fo-Fc) map obtained after omit refinement. What I did is to make the ligand-free structure model and then use Phenix.refine using the following parameters plus simulated annealing (torsion angle) or (Cartesian): Default: XYZ coordinates + Real-space+ Individual B factors + Occupancies. Also click: Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS.

Contoured at 2σ, the Fo-Fc map generated show green density for most part of the ligand, but for several atoms in the middle of the ligand, the green density is missing... It's like this: some atoms have green density and the next few no green density and then a couple few has green density and then a couple more no green denisty.... The missing green density usually means that part is disordered in the structure, right? I am not sure why the density is missing for some atoms in the middle of the ligand. It would makes more sense if the missing green density is at the end of the ligand...

Does anyone happen to have any clue about what it means for the green density missing in my case?

Also, I am not sure whether I did the omit refinement right: (1). For simulated annealing, it has two options, one is Cartesian and the other is Torsion angle, and it's said that torsion angle is more suitable of low resolution data. Is there any difference about which one to choose in my case? (2). Is it Okay to run the omit refinement in the presence of the following parameters as I did: Optimize X-ray/stereochemistry weight + Optimize X-ray/ADP weight + Secondary structure restraints + NCS.

Thank you so much!

Best, Wei

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Nathaniel Echols

14 Sep 14 Sep

12:57 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Sat, Sep 13, 2014 at 3:03 PM, Pavel Afonine wrote:

...

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.

I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it. Wei: It's fine to use the options you mentioned for this - however, weight optimization is very slow and not really necessary since you are only running SA to remove phase bias, not to get the best possible model. I usually just use individual_sites+individual_adp plus whatever extra restraints are appropriate (NCS, secondary structure, etc.). I wouldn't worry if you end up with an overfit model with worse geometry - as long as it has been sufficiently shaken (but not too much!) and the ligand difference density is clear, you are fine. Just be careful that in the process of running SA, you haven't actually introduced new bias into the maps - but the mFo-DFc map is relatively safe (the 2mFo-DFc map much less so). Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect. Perhaps one day we will write up something more formal about map "validation", since there seem to be a lot of misconceptions about these methods and they are not always used appropriately. -Nat

Pavel Afonine

1:13 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On 9/13/14 5:57 PM, Nathaniel Echols wrote:

...

On Sat, Sep 13, 2014 at 3:03 PM, Pavel Afonine mailto:[email protected]> wrote:

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.

I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it.

It is bad both ways: 1) If you ignore zero occupancy atoms then you fill bulk-solvent into ligand region and therefore mask the ligand; 2) If you do not ignore zero occupancy atoms then what you compute is bulk-solvent-omit map in region around atoms with zero occupancy. This means that the "green" density you are going to see may be ligand or may be bulk-solvent or may be the mixture of the two. In fact, #1 above is better than #2 because if ligand density is strong enough it may not be masked by the bulk-solvent completely. Pavel

Wei Shi

1:36 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Thank you guys so much for your suggestions! Yes, I think it's true that "if ligand density is strong enough it may not be masked by the bulk-solvent completely". In my case, I have two different ligands in the protein-ligand structure, and using the method I described in the first email, one of the ligand has good, continuous green density but for the other ligand, as I described before, contoured at 2σ, the Fo-Fc map generated show green density for most part of the ligand, but for several atoms in the middle of the ligand, the green density is missing... Hi Nat, you mentioned that 1. "I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it." I am wondering how this could be done. 2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect." I am wondering how this could be done. Thank you guys so much! Best, Wei On Sat, Sep 13, 2014 at 9:13 PM, Pavel Afonine wrote:

...

On 9/13/14 5:57 PM, Nathaniel Echols wrote:

On Sat, Sep 13, 2014 at 3:03 PM, Pavel Afonine wrote:

...
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.

I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it.

It is bad both ways: 1) If you ignore zero occupancy atoms then you fill bulk-solvent into ligand region and therefore mask the ligand; 2) If you do not ignore zero occupancy atoms then what you compute is bulk-solvent-omit map in region around atoms with zero occupancy. This means that the "green" density you are going to see may be ligand or may be bulk-solvent or may be the mixture of the two.

In fact, #1 above is better than #2 because if ligand density is strong enough it may not be masked by the bulk-solvent completely.

Pavel

Pavel Afonine

3:21 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Wei,

...

Yes, I think it's true that "if ligand density is strong enough it may not be masked by the bulk-solvent completely".

indeed. You can verify it yourself by taking a few examples of protein-ligand complexes with ligand occupancy (or mobility or both) varying in some range.

...

Hi Nat, you mentioned that 1. "I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it."

Yes, results are expected to be very different for reasons I explained in previous email.

...

2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect."

I am wondering how this could be done.

I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it. Pavel

Tanner, John J.

3:05 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Might the feature enhanced map be an appropriate substitute for traditional simulated annealing omit maps? One could delete the ligand before calculating the FEM, although this might be redundant since the procedure systematically deletes sections of the map anyway. Jack On Sep 13, 2014, at 10:21 PM, Pavel Afonine wrote: Hi Wei, Yes, I think it's true that "if ligand density is strong enough it may not be masked by the bulk-solvent completely". indeed. You can verify it yourself by taking a few examples of protein-ligand complexes with ligand occupancy (or mobility or both) varying in some range. Hi Nat, you mentioned that 1. "I thought phenix.refine ignores zero-occupancy atoms when calculating the bulk solvent mask? In fact there is an option specifically to toggle this option, and I've noticed very different results with and without it." Yes, results are expected to be very different for reasons I explained in previous email. 2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect." I am wondering how this could be done. I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it. Pavel _______________________________________________ phenixbb mailing list [email protected]mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

15 Sep 15 Sep

6:38 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Jack,

...

Might the feature enhanced map be an appropriate substitute for traditional simulated annealing omit maps?

it might (by the way it is calculated), I think so. However, I have not done extensive tests to prove this specific point. Pavel

Nathaniel Echols

14 Sep 14 Sep

4:10 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Sat, Sep 13, 2014 at 8:21 PM, Pavel Afonine wrote:

...

2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues)

...
is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect."

I am wondering how this could be done.

I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it.

I think you're confusing what I wrote with the bulk solvent issue. It is also sometimes necessary to prevent actual atoms from being refined into the omit region - this is the purpose of adding harmonic restraints on the omitted atoms. (As far as I know this was first suggested in Hodel et al. 1992, although they don't go into a great deal of detail.) It will have no effect on the bulk solvent mask, which will still be extended over the zero-occupancy atoms. I'm sure there are plenty of cases where the restraints don't make a difference, but I just spent a great deal of time working with a structure where they were essential. Wei: I've attached a slide showing how to set up the restraints. But I agree with Pavel that changing the bulk solvent mask settings is dangerous. -Nat

Wei Shi

9:12 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Thank you so much! I guess I will do as Pavel first suggested to define a box around the ligand and omit all the scattering inside the box (ligand and bulk-solvent) using CCTBX. I now got CCTBX installed on my computer and are trying to figure out ... Let me know if any of you happen to know how to define a box and omit all the scattering using CCTBX. Thank you so much! Best, Wei On Sun, Sep 14, 2014 at 12:10 PM, Nathaniel Echols wrote:

...

On Sat, Sep 13, 2014 at 8:21 PM, Pavel Afonine wrote:

...
2. "Restraining the zero-occupancy ligand (and perhaps adjacent

...
residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect."

I am wondering how this could be done.

I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it.

I think you're confusing what I wrote with the bulk solvent issue. It is also sometimes necessary to prevent actual atoms from being refined into the omit region - this is the purpose of adding harmonic restraints on the omitted atoms. (As far as I know this was first suggested in Hodel et al. 1992, although they don't go into a great deal of detail.) It will have no effect on the bulk solvent mask, which will still be extended over the zero-occupancy atoms. I'm sure there are plenty of cases where the restraints don't make a difference, but I just spent a great deal of time working with a structure where they were essential.

Wei: I've attached a slide showing how to set up the restraints. But I agree with Pavel that changing the bulk solvent mask settings is dangerous.

-Nat

Nathaniel Echols

9:18 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Sun, Sep 14, 2014 at 2:12 PM, Wei Shi wrote:

...

Thank you so much! I guess I will do as Pavel first suggested to define a box around the ligand and omit all the scattering inside the box (ligand and bulk-solvent) using CCTBX. I now got CCTBX installed on my computer and are trying to figure out ... Let me know if any of you happen to know how to define a box and omit all the scattering using CCTBX. Thank you so much!

I'm skeptical that this is worth investing a lot of time in. Sophisticated map calculations can be very useful for interpretation and building an initial model, but you shouldn't need to resort to writing custom software just to demonstrate that something is bound. If I was a paper reviewer I would ask to see the standard omit map, without any tricks. (Actually, I'd ask for the model and experimental data so I could calculate the map myself, but I'm not sure how common this practice is.) -Nat

Wei Shi

9:31 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Nat, For standard omit map, do you mean using Phenix-> Maps -> Composite omit map. And omit map method: Anneal and map types: 2mFo-DFc. Thank you so much! Best, Wei On Sun, Sep 14, 2014 at 5:18 PM, Nathaniel Echols wrote:

...

On Sun, Sep 14, 2014 at 2:12 PM, Wei Shi wrote:

...
Thank you so much! I guess I will do as Pavel first suggested to define a box around the ligand and omit all the scattering inside the box (ligand and bulk-solvent) using CCTBX. I now got CCTBX installed on my computer and are trying to figure out ... Let me know if any of you happen to know how to define a box and omit all the scattering using CCTBX. Thank you so much!

I'm skeptical that this is worth investing a lot of time in. Sophisticated map calculations can be very useful for interpretation and building an initial model, but you shouldn't need to resort to writing custom software just to demonstrate that something is bound. If I was a paper reviewer I would ask to see the standard omit map, without any tricks. (Actually, I'd ask for the model and experimental data so I could calculate the map myself, but I'm not sure how common this practice is.)

-Nat

Nathaniel Echols

9:36 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Sun, Sep 14, 2014 at 2:31 PM, Wei Shi wrote:

...

For standard omit map, do you mean using Phenix-> Maps -> Composite omit map. And omit map method: Anneal and map types: 2mFo-DFc. Thank you so much!

All I meant by "standard" was "omit ligand, leave bulk solvent calculation alone". It doesn't need to be a composite omit map - what you were doing originally using phenix.refine was sufficient (if unnecessarily slow), and this is exactly what reviewers (or P.I.s) tend to ask for. -Nat

Pavel Afonine

15 Sep 15 Sep

7:23 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Wei, I'm glad this discussion gave you some food for thought! -:) If you have no programing experience with CCTBX then this may not be the best route to take. I mentioned CCTBX in my previous email just to identify this as a possible option. However the amount of scripting necessary to achieve this may be prohibitive for novices to CCTBX. At present we do not have tools in Phenix that compute local OMIT maps most optimally (and I'm not aware of such tools outside of Phenix). We do have great tools to calculate various kinds of OMIT maps but I believe they are suboptimal in contexts like yours for reasons I explained in one of my previous emails in this thread. In fact I'm preparing a manuscript that discusses this matter and hopefully makes my points clearer. Options you have are: 1) I'm going to add a tool that I plan to call phenix.difference_omit_map (or something similar) that will calculate OMIT map the way I described before. With some luck it may be available in nightly builds by end of this week. 2) If you can't wait you can just follow Nat's suggestions. If ligand density is sufficiently strong and you use tools correctly you may get what you want. However if ligand density is weak the OMIT map may not be useful as bulk-solvent may obscure the ligand density.. 3) You can send me files off list and I will calculate the OMIT map for you. All the best, Pavel On 9/14/14 2:12 PM, Wei Shi wrote:

...

Thank you so much! I guess I will do as Pavel first suggested to define a box around the ligand and omit all the scattering inside the box (ligand and bulk-solvent) using CCTBX. I now got CCTBX installed on my computer and are trying to figure out ... Let me know if any of you happen to know how to define a box and omit all the scattering using CCTBX. Thank you so much!

Best, Wei

On Sun, Sep 14, 2014 at 12:10 PM, Nathaniel Echols mailto:[email protected]> wrote:

On Sat, Sep 13, 2014 at 8:21 PM, Pavel Afonine mailto:[email protected]> wrote:

2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect."

I am wondering how this could be done.

I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it.

I think you're confusing what I wrote with the bulk solvent issue. It is also sometimes necessary to prevent actual atoms from being refined into the omit region - this is the purpose of adding harmonic restraints on the omitted atoms. (As far as I know this was first suggested in Hodel et al. 1992, although they don't go into a great deal of detail.) It will have no effect on the bulk solvent mask, which will still be extended over the zero-occupancy atoms. I'm sure there are plenty of cases where the restraints don't make a difference, but I just spent a great deal of time working with a structure where they were essential.

Wei: I've attached a slide showing how to set up the restraints. But I agree with Pavel that changing the bulk solvent mask settings is dangerous.

-Nat

Dale Tronrud

3:58 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 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. This map will have the bulk solvent issues discussed previously, but the ligand density must have been sufficiently strong and clear to overcome that obstacle in order for you to be convinced. Dale Tronrud On 9/15/2014 12:23 AM, Pavel Afonine wrote:

...

Hi Wei,

I'm glad this discussion gave you some food for thought! -:)

If you have no programing experience with CCTBX then this may not be the best route to take. I mentioned CCTBX in my previous email just to identify this as a possible option. However the amount of scripting necessary to achieve this may be prohibitive for novices to CCTBX.

At present we do not have tools in Phenix that compute local OMIT maps most optimally (and I'm not aware of such tools outside of Phenix). We do have great tools to calculate various kinds of OMIT maps but I believe they are suboptimal in contexts like yours for reasons I explained in one of my previous emails in this thread. In fact I'm preparing a manuscript that discusses this matter and hopefully makes my points clearer.

Options you have are:

1) I'm going to add a tool that I plan to call phenix.difference_omit_map (or something similar) that will calculate OMIT map the way I described before. With some luck it may be available in nightly builds by end of this week.

2) If you can't wait you can just follow Nat's suggestions. If ligand density is sufficiently strong and you use tools correctly you may get what you want. However if ligand density is weak the OMIT map may not be useful as bulk-solvent may obscure the ligand density..

3) You can send me files off list and I will calculate the OMIT map for you.

All the best, Pavel

On 9/14/14 2:12 PM, Wei Shi wrote:

...
Thank you so much! I guess I will do as Pavel first suggested to define a box around the ligand and omit all the scattering inside the box (ligand and bulk-solvent) using CCTBX. I now got CCTBX installed on my computer and are trying to figure out ... Let me know if any of you happen to know how to define a box and omit all the scattering using CCTBX. Thank you so much!

Best, Wei

On Sun, Sep 14, 2014 at 12:10 PM, Nathaniel Echols mailto:[email protected]> wrote:

On Sat, Sep 13, 2014 at 8:21 PM, Pavel Afonine mailto:[email protected]> wrote:

2. "Restraining the zero-occupancy ligand (and perhaps adjacent residues) is strongly recommended, especially if it contains heavier elements. (The newer versions of the phenix.refine GUI have a button in the Output tab to set this up.) Without restraints, you run the risk of refining the surrounding model into the ligand density. As always this is worse at lower resolution - at 2.8Å I'm not sure what to expect."

I am wondering how this could be done.

I am not sure why you may want to do this at all. If you keep the ligand in PDB file and "omit" it by setting occupancies to zero then the omit map you will get is useless if you ask the program to not fill bulk solvent into ligand region. If you let the program to fill bulk-solvent into ligand region (= ask mask calculation to ignore zero occupancy atoms) then why don't you just remove the ligand from PDB file (then you don't need to worry about restraining it etc.)? In fact you can even keep it in file as you don't care (for map calculation purpose) where restraints will move it.

I think you're confusing what I wrote with the bulk solvent issue. It is also sometimes necessary to prevent actual atoms from being refined into the omit region - this is the purpose of adding harmonic restraints on the omitted atoms. (As far as I know this was first suggested in Hodel et al. 1992, although they don't go into a great deal of detail.) It will have no effect on the bulk solvent mask, which will still be extended over the zero-occupancy atoms. I'm sure there are plenty of cases where the restraints don't make a difference, but I just spent a great deal of time working with a structure where they were essential.

Wei: I've attached a slide showing how to set up the restraints. But I agree with Pavel that changing the bulk solvent mask settings is dangerous.

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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Nathaniel Echols

6:41 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Mon, Sep 15, 2014 at 8:58 AM, Dale Tronrud wrote:

...

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.

Agreed - this is much easier than placing the ligand and then having to run SA. I suspect this is a case where "oral tradition" has been misinterpreted, since everyone is told that the SA omit map is the "standard" for showing unbiased difference density, and they don't realize that it's better to simply avoid the bias to begin with. -Nat

Pavel Afonine

16 Sep 16 Sep

4:22 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hi Dale, your points are all very good! Let's have a look at this at a different angle though. Bulk-solvent is part of the model: it is the model for the other "half" of the unit cell volume that is not interpreted in terms of individual atoms. If ligand is not built yet this means the program will use an incorrect model (bulk-solvent) to describe ligand's region. Isn't it introducing a bias?! I would think so. Pavel On 9/15/14 11:41 AM, Nathaniel Echols wrote:

...

On Mon, Sep 15, 2014 at 8:58 AM, Dale Tronrud mailto:[email protected]> wrote:

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.

Agreed - this is much easier than placing the ligand and then having to run SA. I suspect this is a case where "oral tradition" has been misinterpreted, since everyone is told that the SA omit map is the "standard" for showing unbiased difference density, and they don't realize that it's better to simply avoid the bias to begin with.

-Nat

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Dale Tronrud

6:36 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Absolutely! But the bias is against the hypothesis that you so desperately want to be true. If that map still showed density that convinced you that something is binding, that is strong evidence. Remember, this is the discovery map so it must have been convincing despite its flaws. If you could only "discover" the ligand by assuming its presence and then seeing its density you have a problem. If you built a ligand there must have been a map that convinced you. It may have been the original MR map where that ATP was so clear that you built it from the start. It may have been a map calculated from a nearly complete refinement of everything else whose precision allowed the visualization of some weak binding ligand. Whatever it was, there had to be such a map in the history of the project. If that map convinced you it should be convincing to the reader (and reviewer) otherwise you are not being skeptical enough. It is hard to calculate phases from a map without making an affirmative statement about the density at every point. The FFT doesn't have an input value for "I don't know". You could calculate a bunch of FFT's with different densities in this region and build a PDF in reciprocal space. Of course the PDF's for each reflection will be tightly coupled to all the others and no one really knows how to represent that. But it isn't the case that we know nothing about the density. We know it is not a uranium cluster and we know the density is not negative. In fact we have pretty specific ideas about what it there, bulk solvent, vacuum (yes, some cavities have nothing in them), or a ligand. Now we are into hypothesis testing - which model works best. If you assume vacuum (as a prior) and density comes back or you assume bulk solvent and weaker density comes back one would doubt the prior in each case. If vacuum is eliminated then you have to have a test between bulk solvent and a ligand (and we don't know which ligand yet). The difference is that the density for a ligand will have high resolution components (say >4 A) where bulk solvent will not. Now we want to say, what is the smoothest map we can construct that fits the data? This sounds like Bricogne's maximum entropy maps. If the data does not demand high resolution features you should not presume they exist and should not build a ligand. Dale Tronrud On 9/16/2014 9:22 AM, Pavel Afonine wrote:

...

Hi Dale,

your points are all very good!

Let's have a look at this at a different angle though. Bulk-solvent is part of the model: it is the model for the other "half" of the unit cell volume that is not interpreted in terms of individual atoms.

If ligand is not built yet this means the program will use an incorrect model (bulk-solvent) to describe ligand's region. Isn't it introducing a bias?! I would think so.

Pavel

On 9/15/14 11:41 AM, Nathaniel Echols wrote:

...
On Mon, Sep 15, 2014 at 8:58 AM, Dale Tronrud mailto:[email protected]> wrote:

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.

Agreed - this is much easier than placing the ligand and then having to run SA. I suspect this is a case where "oral tradition" has been misinterpreted, since everyone is told that the SA omit map is the "standard" for showing unbiased difference density, and they don't realize that it's better to simply avoid the bias to begin with.

-Nat

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Nathaniel Echols

17 Sep 17 Sep

3:52 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud wrote:

...

This sounds like Bricogne's maximum entropy maps.

Maybe I'm misunderstanding this reference, but I don't think Bricogne was the first person to come up with the idea: https://www.researchgate.net/publication/232761305_Electron_density_images_f... which I think is just an extension of the application to X-ray astronomy described by Gull & Daniel (1978). -Nat

Dale Tronrud

6:39 a.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

I don't believe I said he was the first. He did develop an implementation which has proven useful to a large number of people. Dale On 9/16/2014 8:52 PM, Nathaniel Echols wrote:

...

On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud mailto:[email protected]> wrote:

This sounds like Bricogne's maximum entropy maps.

Maybe I'm misunderstanding this reference, but I don't think Bricogne was the first person to come up with the idea:

https://www.researchgate.net/publication/232761305_Electron_density_images_f...

which I think is just an extension of the application to X-ray astronomy described by Gull & Daniel (1978).

-Nat

Wei Shi

20 Sep 20 Sep

3:39 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Thank you so much! Is 'Create maximum entropy map' in the latest nightly build that could create the omit map you guys suggested? I was trying to run it but got the error message 'No column labels specified'. In the columns labels it shows '---' and don't give me any option to change... I just loaded my mtz file and the ligand-free pdb file and left all other settings as default. Thank you so much! Best, Wei On Wed, Sep 17, 2014 at 2:39 AM, Dale Tronrud wrote:

...

I don't believe I said he was the first. He did develop an implementation which has proven useful to a large number of people.

Dale

On 9/16/2014 8:52 PM, Nathaniel Echols wrote:

...
On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud mailto:[email protected]> wrote:

This sounds like Bricogne's maximum entropy maps.

Maybe I'm misunderstanding this reference, but I don't think Bricogne was the first person to come up with the idea:

https://www.researchgate.net/publication/232761305_Electron_density_images_f...

...
which I think is just an extension of the application to X-ray astronomy described by Gull & Daniel (1978).

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

4:39 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Most likely this is because you loaded MTZ with with no Fourier map coefficients. Pavel On 9/20/14 8:39 AM, Wei Shi wrote:

...

Thank you so much! Is 'Create maximum entropy map' in the latest nightly build that could create the omit map you guys suggested? I was trying to run it but got the error message 'No column labels specified'. In the columns labels it shows '---' and don't give me any option to change... I just loaded my mtz file and the ligand-free pdb file and left all other settings as default. Thank you so much!

Best, Wei

On Wed, Sep 17, 2014 at 2:39 AM, Dale Tronrud mailto:[email protected]> wrote:

I don't believe I said he was the first. He did develop an implementation which has proven useful to a large number of people.

Dale

On 9/16/2014 8:52 PM, Nathaniel Echols wrote: > On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud mailto:[email protected] > mailto:[email protected]>> wrote: > > This sounds like Bricogne's maximum entropy maps. > > > Maybe I'm misunderstanding this reference, but I don't think Bricogne > was the first person to come up with the idea: > > https://www.researchgate.net/publication/232761305_Electron_density_images_f... > > which I think is just an extension of the application to X-ray astronomy > described by Gull & Daniel (1978). > > -Nat _______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Wei Shi

5:41 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

The mtz file I loaded is from Phenix -> Reflection file editor with default settings using the cns.hkl file from xds. I am not sure how to get the MTZ with Fourier map coefficients. Thank you! Best, Wei On Sat, Sep 20, 2014 at 12:39 PM, Pavel Afonine wrote:

...

Most likely this is because you loaded MTZ with with no Fourier map coefficients. Pavel

On 9/20/14 8:39 AM, Wei Shi wrote:

Thank you so much! Is 'Create maximum entropy map' in the latest nightly build that could create the omit map you guys suggested? I was trying to run it but got the error message 'No column labels specified'. In the columns labels it shows '---' and don't give me any option to change... I just loaded my mtz file and the ligand-free pdb file and left all other settings as default. Thank you so much!

Best, Wei

On Wed, Sep 17, 2014 at 2:39 AM, Dale Tronrud wrote:

...
I don't believe I said he was the first. He did develop an implementation which has proven useful to a large number of people.

Dale

On 9/16/2014 8:52 PM, Nathaniel Echols wrote:

...
On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud mailto:[email protected]> wrote:

This sounds like Bricogne's maximum entropy maps.

Maybe I'm misunderstanding this reference, but I don't think Bricogne was the first person to come up with the idea:

https://www.researchgate.net/publication/232761305_Electron_density_images_f...

...
which I think is just an extension of the application to X-ray astronomy described by Gull & Daniel (1978).

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Pavel Afonine

5:57 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

Hello Wei, Maps you see in Coot are three-dimensional functions calculated on the grid. These functions are the finite resolution and finite quality images of electron (or nuclear, depending experiment) density in the crystal. They are calculated as a Fourier transform of Fourier map coefficients. In most cases these Fourier map coefficients are {2mFobs-DFmodel, PhiFmodel} and {mFobs-DFmodel, PhiFmodel}, that are complex numbers. Refinement programs like Refmac or phenix.refine output these map coefficients in MTZ formatted files, or you can calculated them given Fobs and atomic model using standalone programs, such as phenix.maps for instance. Back to you original question: 'Create maximum entropy map' utility expected from you one of these MTZ files. Hope this helps, Pavel On 9/20/14 10:41 AM, Wei Shi wrote:

...

The mtz file I loaded is from Phenix -> Reflection file editor with default settings using the cns.hkl file from xds. I am not sure how to get the MTZ with Fourier map coefficients. Thank you!

Best, Wei

On Sat, Sep 20, 2014 at 12:39 PM, Pavel Afonine mailto:[email protected]> wrote:

Most likely this is because you loaded MTZ with with no Fourier map coefficients. Pavel

On 9/20/14 8:39 AM, Wei Shi wrote:

...
Thank you so much! Is 'Create maximum entropy map' in the latest nightly build that could create the omit map you guys suggested? I was trying to run it but got the error message 'No column labels specified'. In the columns labels it shows '---' and don't give me any option to change... I just loaded my mtz file and the ligand-free pdb file and left all other settings as default. Thank you so much!

Best, Wei

On Wed, Sep 17, 2014 at 2:39 AM, Dale Tronrud mailto:[email protected]> wrote:

I don't believe I said he was the first. He did develop an implementation which has proven useful to a large number of people.

Dale

On 9/16/2014 8:52 PM, Nathaniel Echols wrote: > On Tue, Sep 16, 2014 at 11:36 AM, Dale Tronrud mailto:[email protected] > mailto:[email protected]>> wrote: > > This sounds like Bricogne's maximum entropy maps. > > > Maybe I'm misunderstanding this reference, but I don't think Bricogne > was the first person to come up with the idea: > > https://www.researchgate.net/publication/232761305_Electron_density_images_f... > > which I think is just an extension of the application to X-ray astronomy > described by Gull & Daniel (1978). > > -Nat _______________________________________________ phenixbb mailing list [email protected] mailto:[email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Dale Tronrud

16 Sep 16 Sep

6:42 p.m.

New subject: Fo-Fc omit map for ligand in the protein-ligand structure

...

Hi Dale,

your points are all very good!

Let's have a look at this at a different angle though. Bulk-solvent is part of the model: it is the model for the other "half" of the unit cell volume that is not interpreted in terms of individual atoms.

If ligand is not built yet this means the program will use an incorrect model (bulk-solvent) to describe ligand's region. Isn't it introducing a bias?! I would think so.

Pavel

On 9/15/14 11:41 AM, Nathaniel Echols wrote:

...
On Mon, Sep 15, 2014 at 8:58 AM, Dale Tronrud mailto:[email protected]> wrote:

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.

Agreed - this is much easier than placing the ligand and then having to run SA. I suspect this is a case where "oral tradition" has been misinterpreted, since everyone is told that the SA omit map is the "standard" for showing unbiased difference density, and they don't realize that it's better to simply avoid the bias to begin with.

-Nat

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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Dale Tronrud
Nathaniel Echols
Pavel Afonine
Tanner, John J.
Wei Shi