Just a correction: "adp" is not anisotropic displacement parameters, but "Atomic Displacement Parameters". ADP is actually the proper name for B-factors. In phenix.refine, "adp" step can include isotropic ADP refinement, anisotropic ADP refinement, TLS refinement, or combinations of those. There is also "nhq" or something like that among the steps phenix.refine performs. It is for flipping sidechains (by 180 degrees) of residues that are hard to distinguish one way or the other based on clashes and hydrogen bonding. Obviously, it involves Asn, Gln and His. This step, while time consuming sometimes, is useful and should be performed, to prevent yourself from embarrassment by claiming hydrogen bonds with no donors and two acceptors, for example. Engin On 1/20/15 4:17 AM, Christian Roth wrote:

Hi,

bss: bulk solvent scaling rsrl: real space rotamer refinement? Did you select rotamer correction or realspace refinement in your strategy? adp: ainsotropic displacement parameters means simple B-Factor refinement

Cheers

Christian

Am 20.01.2015 um 01:49 schrieb Smith Lee:

...

Dear All.

In the Phenix refine "run status" window, with the progression of the refinement, it occurs 3' bss, 3' rsrl, 3' xyz, 3' adp (for 3rd round of refinement). I guess xyz means the strategy is for x, y, z coordinates? Then what do bss, rsrl and adp mean?

I am looking forward to getting your reply.

Smith

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Hi Georg, I would say probably neither a bug nor exactly "normal"... If everything were ideal, then an MR solution, even if very distant and contributing very little phase information, should improve the Phaser identification of the sub-structure. Therefore as you imagine, including it should have improved the situation. In real life there is a lot of noise in the system, so small changes can sometimes make the difference between finding sites and not. Also the default parameters for Phaser sub-structure identification are not exactly the same for MR-SAD and for extreme defaults in autosol. I am guessing that autosol, with extreme defaults, managed to find something that was partially correct, then the iteration of the substructure search (using density from the density-modified map and/or partial model that was built) resulted in additional sites, while MR-SAD did not happen to identify any convincing sites and autosol stopped. On your second question...the HL coefficients from MR-SAD are actually produced twice in the output MTZ file; once with information from the model (HLA HLB etc) and once without information from the model. (HLanomA HLanomB etc). I think in general your intuition is right and the best map will come from including model information. However if you would like to reduce model bias, you might want to exclude model information and use the HLanomA etc. The map coefficients (FWT PHWT) in the overall_best_denmod_map_coeffs.mtz from MRSAD will include or not include the model information based on how you set the parameter use_hl_anom_in_denmod (default is False, use HL coeffs and include model information). I'll pass on the other requests! All the best, Tom T ________________________________________ From: [email protected] [[email protected]] on behalf of Georg Mlynek [[email protected]] Sent: Sunday, January 25, 2015 4:54 AM To: [email protected] Subject: [phenixbb] MR-SAD, SAD Dear phenix-developers, I have collected a high redundancy dataset on our bruker home-source to get good anomalous data to confirm if a ligandbound near the active site is really the one I think. 1. If I do MR with phaser-MR a solution is found easiliy. If I then continue with MR-SAD the program does not give me a solution. No file named overall_best.pdb is present in the output directory. Warnings: FOM < 0.05 ... skipping solution 1 However Autosol with extreme density modification and Iterate substructure search is able to solve the structure. Is this a bug or normal. 2. A follow up on this: Will be the MTZ file (phases) produced from MR-SAD always better (compared to SAD or MR alone) because it will contain phases that combine the information from both the MR model and the SAD data. Or can there be cases where bad phases form either MR or SAD will mess up the other one. 3. There is a small bug in merging statistics. cc_ano is just writen out in the log output tab but not in the summary tab. 4. It would be nice, if xtriage could also print how many % of the reflections are Rfree flagged. Thanks in advance, best regards, Georg. _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

Dear Almudena, If AutoSol is using the Hendrickson-Lattman coefficients from the SAD phasing, then both the refinement and the map calculation should be using that phase information, so running MR-SAD shouldn’t necessarily improve things. However, what can happen is that, with the addition of a partial protein model, the MR-SAD procedure finds a more complete and more accurate substructure, which then can yield significantly better phases and maps. We have a nice example of this from a test case (in the appendix of this paper: http://journals.iucr.org/d/issues/2010/04/00/ba5142/index.html), where a substructure containing 57 atoms allowed a model of 70% of nitrate reductase to be built. MR-SAD with this model found a substructure containing 105 atoms, and the next step of model-building built a substantially more complete model. In this case, there are 21 Fe atoms (mostly in Fe-S clusters), one Mo atom, and over 120 S or P atoms, so there are a lot of minor sites to find. As Tom mentioned, Phaser produces two types of Hendrickson Lattman coefficients, the “HL” ones that contain all phase information (including that from the partial structure) and the “HLanom” ones that contain only the phase information from the anomalous scattering. The HL variant should be used when you need the complete phase information, such as for density modification. However, if you are refining a model that includes the atoms in the partial structure used for MR-SAD, you want to use the HLanom coefficients to avoid including the partial structure phase information twice. An average FOM of 0.49 is very good for SAD phasing, because the 2-fold phase ambiguity tends to limit phase accuracy. We’re looking into how to interpret the absolute LLG values, but at the moment I can’t tell you how good that number is (though it looks fairly high, which is probably good)! Best wishes, Randy Read ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 26 Jan 2015, at 08:40, Almudena Ponce Salvatierra wrote:

HI!

George gave me an idea actually... I did SAD phasing with Co sites and with AutoSol I got a partial model, that I then edited. However, since you were suggesting the phases should be better when you combine those from the model with the experimental ones I ran Phaser-EP in SAD-MR mode. I have provided the model I edited after AutoSol, my HA sites, and the data, and I got a better map than even just with AutoSol!!! I mean, when I submit it to Resolve, after phaser. :-)

Is it a good solution if the FOM is 0.49 and LLG is 36449.63 after Phaser?

Sooo thanks a lot for the inspiration! (it's not so easy on Monday mornings!!)

Best wishes,

Almudena.

2015-01-25 21:06 GMT+01:00 Terwilliger, Thomas Charles : Hi Georg,

I would say probably neither a bug nor exactly "normal"...

If everything were ideal, then an MR solution, even if very distant and contributing very little phase information, should improve the Phaser identification of the sub-structure. Therefore as you imagine, including it should have improved the situation. In real life there is a lot of noise in the system, so small changes can sometimes make the difference between finding sites and not. Also the default parameters for Phaser sub-structure identification are not exactly the same for MR-SAD and for extreme defaults in autosol.

I am guessing that autosol, with extreme defaults, managed to find something that was partially correct, then the iteration of the substructure search (using density from the density-modified map and/or partial model that was built) resulted in additional sites, while MR-SAD did not happen to identify any convincing sites and autosol stopped.

On your second question...the HL coefficients from MR-SAD are actually produced twice in the output MTZ file; once with information from the model (HLA HLB etc) and once without information from the model. (HLanomA HLanomB etc). I think in general your intuition is right and the best map will come from including model information. However if you would like to reduce model bias, you might want to exclude model information and use the HLanomA etc. The map coefficients (FWT PHWT) in the overall_best_denmod_map_coeffs.mtz from MRSAD will include or not include the model information based on how you set the parameter use_hl_anom_in_denmod (default is False, use HL coeffs and include model information).

I'll pass on the other requests!

All the best, Tom T

________________________________________ From: [email protected] [[email protected]] on behalf of Georg Mlynek [[email protected]] Sent: Sunday, January 25, 2015 4:54 AM To: [email protected] Subject: [phenixbb] MR-SAD, SAD

Dear phenix-developers,

I have collected a high redundancy dataset on our bruker home-source to get good anomalous data to confirm if a ligandbound near the active site is really the one I think.

1. If I do MR with phaser-MR a solution is found easiliy. If I then continue with MR-SAD the program does not give me a solution.

No file named overall_best.pdb is present in the output directory.

Warnings: FOM < 0.05 ... skipping solution 1

However Autosol with extreme density modification and Iterate substructure search is able to solve the structure.

Is this a bug or normal.

2. A follow up on this: Will be the MTZ file (phases) produced from MR-SAD always better (compared to SAD or MR alone) because it will contain phases that combine the information from both the MR model and the SAD data. Or can there be cases where bad phases form either MR or SAD will mess up the other one.

3. There is a small bug in merging statistics. cc_ano is just writen out in the log output tab but not in the summary tab.

4. It would be nice, if xtriage could also print how many % of the reflections are Rfree flagged.

Thanks in advance, best regards, Georg. _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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

-- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany

Dear Tom, 1. Your answer " I would say probably neither a bug nor exactly "normal"..." was completely right. I was putting 100 in the *Expected RMSD* field, because I am so used that phaser asks about *variance*. So it was a user mistake.(thanks Randy) 2. I was running MR-SAD again and it works perfect. However if I tick Autobuild model in the Autosol gui, the MR-model is rebuild and the oligomeric arrangement is changed. If I untick, no model pdb is written. Is there somewhere a parameter hidden which would do the same like in autobuild Rebuild in place =False? Thanks again, best regards, Georg. On 01/26/2015 10:44 AM, Randy Read wrote:

Dear Georg,

I agree with what Tom says in general about MR-SAD not necessarily always being better than ab initio substructure determination, particularly if the MR model is marginal in quality. In your case it depends on what you mean by saying that an MR solution was found easily. Usually when there’s a good signal in the MR search, the model is good enough to prime substructure determination with MR-SAD. You could send me the MR and MR-SAD log files off-line, and I could check whether there was any problem in the way that these were run.

Best wishes,

Randy Read

----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

On 25 Jan 2015, at 20:06, Terwilliger, Thomas Charles wrote:

...

Hi Georg,

I would say probably neither a bug nor exactly "normal"...

If everything were ideal, then an MR solution, even if very distant and contributing very little phase information, should improve the Phaser identification of the sub-structure. Therefore as you imagine, including it should have improved the situation. In real life there is a lot of noise in the system, so small changes can sometimes make the difference between finding sites and not. Also the default parameters for Phaser sub-structure identification are not exactly the same for MR-SAD and for extreme defaults in autosol.

I am guessing that autosol, with extreme defaults, managed to find something that was partially correct, then the iteration of the substructure search (using density from the density-modified map and/or partial model that was built) resulted in additional sites, while MR-SAD did not happen to identify any convincing sites and autosol stopped.

On your second question...the HL coefficients from MR-SAD are actually produced twice in the output MTZ file; once with information from the model (HLA HLB etc) and once without information from the model. (HLanomA HLanomB etc). I think in general your intuition is right and the best map will come from including model information. However if you would like to reduce model bias, you might want to exclude model information and use the HLanomA etc. The map coefficients (FWT PHWT) in the overall_best_denmod_map_coeffs.mtz from MRSAD will include or not include the model information based on how you set the parameter use_hl_anom_in_denmod (default is False, use HL coeffs and include model information).

I'll pass on the other requests!

All the best, Tom T

________________________________________ From: [email protected] [[email protected]] on behalf of Georg Mlynek [[email protected]] Sent: Sunday, January 25, 2015 4:54 AM To: [email protected] Subject: [phenixbb] MR-SAD, SAD

Dear phenix-developers,

I have collected a high redundancy dataset on our bruker home-source to get good anomalous data to confirm if a ligandbound near the active site is really the one I think.

1. If I do MR with phaser-MR a solution is found easiliy. If I then continue with MR-SAD the program does not give me a solution.

No file named overall_best.pdb is present in the output directory.

Warnings: FOM < 0.05 ... skipping solution 1

However Autosol with extreme density modification and Iterate substructure search is able to solve the structure.

Is this a bug or normal.

2. A follow up on this: Will be the MTZ file (phases) produced from MR-SAD always better (compared to SAD or MR alone) because it will contain phases that combine the information from both the MR model and the SAD data. Or can there be cases where bad phases form either MR or SAD will mess up the other one.

3. There is a small bug in merging statistics. cc_ano is just writen out in the log output tab but not in the summary tab.

4. It would be nice, if xtriage could also print how many % of the reflections are Rfree flagged.

Thanks in advance, best regards, Georg. _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

-- Mlynek Georg University of Vienna Department of Computational and Structural Biology Max F. Perutz Laboratories Campus Vienna Biocenter 5 level -2 1030 Vienna Austria e-mail: [email protected] mobil: +43 660 42 195 07 office: +43-1-4277-52263

Hi Tom, How is the input MR model treated when computing the HL's for the substructure search? That is to say, how are errors in the MR solution taken into account or is the input model treated as being correct? Errors being: positional errors, or model incompleteness errors? Thanks, F On Jan 25, 2015, at 3:06 PM, Terwilliger, Thomas Charles wrote:

If everything were ideal, then an MR solution, even if very distant and contributing very little phase information, should improve the Phaser identification of the sub-structure. Therefore as you imagine, including it should have improved the situation. In real life there is a lot of noise in the system, so small changes can sometimes make the difference between finding sites and not. Also the default parameters for Phaser sub-structure identification are not exactly the same for MR-SAD and for extreme defaults in autosol.

Hi, This rms value is used to calculate the initial values of some error estimates for structure factors from the partial model as a function of resolution, which are then refined. After those error estimates are refined, they will account for a combination of incompleteness and coordinate error. The default value of 1A is going to be fine most of the time, because the refinement will converge most of the time from the initial values computed from this. However, if you want to you can look at what the MR step in Phaser estimated as the RMS error of the model after the final refinement (the refined VRMS value at the bottom of the log file) and give that number instead. Best wishes, Randy On 26 Jan 2015, at 15:19, Terwilliger, Thomas Charles wrote:

Hi Francis,

An estimated RMSD is used in the calculation in Phaser. You can specify

partpdb_rms = 1

to define the estimated RMSD between model and true structure that is used when Phaser is called by autosol. The default is 1 A. I think there is no way at present to tell Phaser about incompleteness, at least autosol does not have a keyword for it (Randy may be able to comment on that...)

All the best, Tom T

________________________________________ From: Francis Reyes [[email protected]] on behalf of Francis Reyes [[email protected]] Sent: Monday, January 26, 2015 8:13 AM To: Terwilliger, Thomas Charles Cc: [email protected] Subject: Re: [phenixbb] MR-SAD, SAD

Hi Tom,

How is the input MR model treated when computing the HL's for the substructure search?

That is to say, how are errors in the MR solution taken into account or is the input model treated as being correct?

Errors being: positional errors, or model incompleteness errors?

Thanks,

F

On Jan 25, 2015, at 3:06 PM, Terwilliger, Thomas Charles wrote:

...

If everything were ideal, then an MR solution, even if very distant and contributing very little phase information, should improve the Phaser identification of the sub-structure. Therefore as you imagine, including it should have improved the situation. In real life there is a lot of noise in the system, so small changes can sometimes make the difference between finding sites and not. Also the default parameters for Phaser sub-structure identification are not exactly the same for MR-SAD and for extreme defaults in autosol.

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

------ Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

Dear All,

Then what is rsrg?

Smith

I'm puzzled too: what you mean by rsrg ? I can't remember introducing a shortcut resembling anything like this! Pavel