Hi All, the fixed version will be available in the next installer made after version 1.4-51: http://www.phenix-online.org/download/nightly_builds.cgi Pavel. On 4/30/09 1:01 PM, Tom Terwilliger wrote:

Hi Pavel, I'm having trouble using your tool...Can you unpack

~terwill/fofomap.tgz

and try:

ls coords.pdb native.mtz native.sca peak.mtz peak.sca [ttmbpro:transfer/test/testit] terwill% phenix.fobs_minus_fobs_map peak.mtz native.mtz phase_source=coords.pdb

========================== phenix.fobs_minus_fobs_map =========================

Command line arguments: ['peak.mtz', 'native.mtz', 'phase_source=coords.pdb']

Sorry: No PDB file found.

Also:

phenix.fobs_minus_fobs_map peak.sca native.sca coords.pdb

========================== phenix.fobs_minus_fobs_map =========================

Command line arguments: ['peak.sca', 'native.sca', 'coords.pdb']

*** Parameters: f_obs_1_file_name = None f_obs_1_label = None f_obs_2_file_name = None f_obs_2_label = None high_resolution = None low_resolution = None sigma_cutoff = None phase_source = None scattering_table = *xray neutron

Sorry: No reflection data file found.

Thanks! -Tom T

Thomas C. Terwilliger Mail Stop M888 Los Alamos National Laboratory Los Alamos, NM 87545

Tel: 505-667-0072 email: [email protected] mailto:[email protected] Fax: 505-665-3024 SOLVE web site: http://solve.lanl.gov PHENIX web site: http:www.phenix-online.org http://www.phenix-online.org ISFI Integrated Center for Structure and Function Innovation web site: http://techcenter.mbi.ucla.edu TB Structural Genomics Consortium web site: http://www.doe-mbi.ucla.edu/TB CBSS Center for Bio-Security Science web site: http://www.lanl.gov/cbss

On Apr 30, 2009, at 11:39 AM, Pavel Afonine wrote:

...

fobs_minus_fobs_map

------------------------------------------------------------------------

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Hi Maia, - I'm wondering why this puzzles you? - The gap Rfree-Rwork seems suspiciously small, although I can't tell without knowing the resolution. - Overall, better R-factors (Rwork, Rfree and Rfree-Rwork) mean that among many possible refinement strategies you have chosen the one that is better than the others. What is the resolution? What are the R-factor before using NCS? What will be the R-factors after you turn NCS back off? Pavel. On 5/4/09 12:27 PM, Maia Cherney wrote:

Hi everybody, I am wondering why my final structure refined with NCS=True has lower R factors (19.2% and 21.2%) than without NCS by approximately 1.5%.

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

I see. I suggest to run two refinements: - with NCS and "optimize_wxc=true optimize_wxu=true" options, and - without NCS and with "optimize_wxc=true optimize_wxu=true". This will make it weight-choice independent and so easier to understand. Otherwise, since NCS term is included in weights calculation, using or not using NCS may change the X-ray/Restrains weight which may have larger effects than using/not using NCS itself. Pavel. On 5/4/09 6:48 PM, Maia Cherney wrote:

Hi Pavel, The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints. There are five restraint groups in the asym. unit.

Maia

Pavel Afonine wrote:

...

Hi Maia,

- I'm wondering why this puzzles you?

- The gap Rfree-Rwork seems suspiciously small, although I can't tell without knowing the resolution.

- Overall, better R-factors (Rwork, Rfree and Rfree-Rwork) mean that among many possible refinement strategies you have chosen the one that is better than the others.

What is the resolution? What are the R-factor before using NCS? What will be the R-factors after you turn NCS back off?

Pavel.

On 5/4/09 12:27 PM, Maia Cherney wrote:

...

Hi everybody, I am wondering why my final structure refined with NCS=True has lower R factors (19.2% and 21.2%) than without NCS by approximately 1.5%.

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

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

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Hi Boaz, The two options are equivalent and no twin law needs to be specified in either case. SA should be possible with twin refinement. Cheers Peter 2009/5/6 Boaz Shaanan :

Hi,

If I understand correctly, the Phenix manual suggests two options for selecting the R-free set for twin refinement. One is to do this using the phenix.reflection_file_converter with the parameter

--use-lattice-symmetry-in-r-free-flag-generation

which I suppose does not require input of the twin law. The second is in the actual refinement using the parameter

xray_data.r_free_flags.generate=True

in which case the twin law is part of the input. My first question is whether the two options are truly equivalent. The second question is whether one can go into either of them with an mtz file in which R-free set reflections have already been selected (e.g. in scala or XDS) without any twinning assumption, or should one use a file without any R-free set altogether and let the phenix script choose it from scratch, under the twinning assumption.

Another (related) question is whether simulated annealing is possible in conjunction with twin refinement.

Thanks,

Boaz

----- Original Message ----- From: Pavel Afonine Date: Tuesday, May 5, 2009 4:53 Subject: Re: [phenixbb] ncs To: PHENIX user mailing list

...

I see. I suggest to run two refinements:

- with NCS and "optimize_wxc=true optimize_wxu=true" options, and

- without NCS and with "optimize_wxc=true optimize_wxu=true".

This will make it weight-choice independent and so easier to understand. Otherwise, since NCS term is included in weights calculation, using or not using NCS may change the X-ray/Restrains weight which may have larger effects than using/not using NCS itself.

Pavel.

On 5/4/09 6:48 PM, Maia Cherney wrote:

...

Hi Pavel, The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints. There are five restraint groups in the asym. unit.

Maia

Pavel Afonine wrote:

...

Hi Maia,

- I'm wondering why this puzzles you?

- The gap Rfree-Rwork seems suspiciously small, although I can't tell without knowing the resolution.

- Overall, better R-factors (Rwork, Rfree and Rfree-Rwork) mean that among many possible refinement strategies you have chosen the one that is better than the others.

What is the resolution? What are the R-factor  before using NCS? What will be the R-factors after you turn NCS back off?

Pavel.

On 5/4/09 12:27 PM, Maia Cherney wrote:

...

Hi everybody, I am wondering why my final structure refined with NCS=True has lower  R factors (19.2% and 21.2%) than without NCS by approximately 1.5%.

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

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

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

Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan‎ _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------

Okai, when the twin law is not specified, it will not do a twin based refinement, but does take all possible twin laws into considerations when making an free set. when specifying the twin law, a twin based refinement is carried out. I am not sure about making a new test set when you already have one. Pavel, Ralf, any clue? P 2009/5/6 Boaz Shaanan :

Hi Peter,

I went ahead and tried your suggestion in phenix.refine. With this parameter in the command line

xray_data.r_free_flags.generate=True

 but without twin_law="whatever" parameter, the program does not have any clue about twinning and the refinement indeed ignores it, at least by the look of the progress (I didn't let it go to the end and then check the resulting mtz file). On the other hand, with twin_law="whatever" all is OK. In fact I also got an answer to my second question in the previous message, i.e. that a new R-free set is selected even thought the incoming mtz file had a previously selected R-free set (by scala). I guess I should use the new mtz file from now on and not use xray_data.r_free_flags.generate=True anymore, is this correct ?

 Cheers,

             Boaz ----- Original Message ----- From: Peter Zwart Date: Wednesday, May 6, 2009 17:52 Subject: Re: [phenixbb] Selection of R-free set for twin refinement To: PHENIX user mailing list

...

Hi Boaz,

The two options are equivalent and no twin law needs to be specified in either case.

SA should be possible with twin refinement.

Cheers

Peter

...

Hi,

If I understand correctly, the Phenix manual suggests two

...

selecting the R-free set for twin refinement. One is to do

...

phenix.reflection_file_converter with the parameter

--use-lattice-symmetry-in-r-free-flag-generation

which I suppose does not require input of the twin law. The second is in the actual refinement using the parameter

xray_data.r_free_flags.generate=True

in which case the twin law is part of the input. My first question is whether the two options are truly equivalent. The second question is whether one can go into either of them with an mtz file in which R-free set reflections have already been selected (e.g. in scala or XDS) without any twinning assumption, or should one use a file without any R-free set altogether and let the phenix script choose it from scratch, under the twinning assumption.

Another (related) question is whether simulated annealing is

2009/5/6 Boaz Shaanan : options for this using the possible in

...

conjunction with twin refinement.

     Thanks,

                Boaz

----- Original Message ----- From: Pavel Afonine Date: Tuesday, May 5, 2009 4:53 Subject: Re: [phenixbb] ncs To: PHENIX user mailing list

...

I see. I suggest to run two refinements:

- with NCS and "optimize_wxc=true optimize_wxu=true" options, and

- without NCS and with "optimize_wxc=true optimize_wxu=true".

This will make it weight-choice independent and so easier to understand. Otherwise, since NCS term is included in weights calculation, using or not using NCS may change the X-ray/Restrains weight which may have larger effects than using/not using NCS itself.

Pavel.

On 5/4/09 6:48 PM, Maia Cherney wrote:

...

Hi Pavel, The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints. There are five restraint groups in the asym. unit.

Maia

Pavel Afonine wrote:

...

Hi Maia,

- I'm wondering why this puzzles you?

- The gap Rfree-Rwork seems suspiciously small, although I can't tell without knowing the resolution.

- Overall, better R-factors (Rwork, Rfree and Rfree-Rwork) mean that among many possible refinement strategies you have chosen the one that is better than the others.

What is the resolution? What are the R-factor  before using NCS? What will be the R-factors after you turn NCS back off?

Pavel.

On 5/4/09 12:27 PM, Maia Cherney wrote:

> Hi everybody, > I am wondering why my final structure refined with NCS=True has lower  R > factors (19.2% and 21.2%) than without NCS by approximately 1.5%. > > Leo > _______________________________________________ > phenixbb mailing list > [email protected] > http://www.phenix-online.org/mailman/listinfo/phenixbb > > > _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

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

Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan‎ _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB:     http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX:  http://cctbx.sf.net -----------------------------------------------------------------

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

Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan‎ _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------

No twin law is needed for rfree flags: it uses the lattice symmetry from the unit cell for this purpose (which includes the twin law you gave) Adding the twin law only activates the use of the twin target. P 2009/5/7 Boaz Shaanan :

Hi Peter,

As far as I can see (by comparing the input & output mtz files) this command:

phenix.refine H1pk.mtz H1pk.pdb xray_data.r_free_flags.generate=True twin_law="-k,-h,-l"

does generate a new set of R-free reflections in the newly generated mtz file, hopefully taking into account the twin law.

Cheers,

Boaz ----

----- Original Message ----- From: Peter Zwart Date: Thursday, May 7, 2009 0:08 Subject: Re: [phenixbb] Selection of R-free set for twin refinement To: PHENIX user mailing list

...

Okai,

when the twin law is not specified, it will not do a twin based refinement, but does take all possible twin laws into considerations when making an free set.

when specifying the twin law, a twin based refinement is carried out.

I am not sure about making a new test set when you already have one. Pavel, Ralf, any clue?

P

...

Hi Peter,

I went ahead and tried your suggestion in phenix.refine. With

...

in the command line

xray_data.r_free_flags.generate=True

but without twin_law="whatever" parameter, the program does not have any clue about twinning and the refinement indeed ignores it, at least by the look of the progress (I didn't let it go to the end and then check the resulting mtz file). On the other hand, with twin_law="whatever" all is OK. In fact I also got an answer to my second question in the

...

i.e. that a new R-free set is selected even thought the incoming mtz file had a previously selected R-free set (by scala). I guess I should use the new mtz file from now on and not use xray_data.r_free_flags.generate=True> anymore, is this correct ?

Cheers,

Boaz ----- Original Message ----- From: Peter Zwart Date: Wednesday, May 6, 2009 17:52 Subject: Re: [phenixbb] Selection of R-free set for twin refinement To: PHENIX user mailing list

...

Hi Boaz,

The two options are equivalent and no twin law needs to be specified>> in either case.

SA should be possible with twin refinement.

Cheers

Peter

...

Hi,

If I understand correctly, the Phenix manual suggests two

...

selecting the R-free set for twin refinement. One is to do

...

phenix.reflection_file_converter with the parameter

--use-lattice-symmetry-in-r-free-flag-generation

which I suppose does not require input of the twin law. The second is in the actual refinement using the parameter

xray_data.r_free_flags.generate=True

in which case the twin law is part of the input. My first question is whether the two options are truly equivalent. The second question is whether one can go into either of them with an mtz file in which R-free set reflections have already been selected (e.g. in scala or XDS) without any twinning assumption, or should one use a file without any R-free set altogether and let the phenix script choose it from scratch, under the twinning assumption.>> > Another (related) question is whether simulated annealing is

2009/5/6 Boaz Shaanan : options for this using the possible in

...

conjunction with twin refinement.

Thanks,

Boaz

----- Original Message ----- From: Pavel Afonine Date: Tuesday, May 5, 2009 4:53 Subject: Re: [phenixbb] ncs To: PHENIX user mailing list

...

I see. I suggest to run two refinements:

- with NCS and "optimize_wxc=true optimize_wxu=true"

2009/5/6 Boaz Shaanan : this parameter previous message, options, and

...

...

...

...

- without NCS and with "optimize_wxc=true optimize_wxu=true".

This will make it weight-choice independent and so easier to understand. Otherwise, since NCS term is included in weights calculation, using or not using NCS may change the X-ray/Restrains weight which may have larger effects than using/not using NCS itself.

Pavel.

On 5/4/09 6:48 PM, Maia Cherney wrote: > Hi Pavel, > The resolution is 2.15 A. The NCS was always on during the refinement > until we got low R factors (19.2% and 21.2%). Then the

NCS was

...

turned > off for the final refinement and the R factors increased, which is > strange as they should be going down when you apply less restraints. > There are five restraint groups in the asym. unit. > > Maia > > > > Pavel Afonine wrote: > >> Hi Maia, >> >> - I'm wondering why this puzzles you? >> >> - The gap Rfree-Rwork seems suspiciously small, although I can't tell >> without knowing the resolution. >> >> - Overall, better R-factors (Rwork, Rfree and Rfree-Rwork) mean that >> among many possible refinement strategies you have chosen the one that >> is better than the others. >> >> What is the resolution? >> What are the R-factor before using NCS? >> What will be the R-factors after you turn NCS back off? >> >> Pavel. >> >> >> On 5/4/09 12:27 PM, Maia Cherney wrote: >> >> >>> Hi everybody, >>> I am wondering why my final structure refined with NCS=True has lower R >>> factors (19.2% and 21.2%) than without NCS by approximately 1.5%. >>> >>> Leo >>> _______________________________________________ >>> phenixbb mailing list >>> [email protected] >>> http://www.phenix-online.org/mailman/listinfo/phenixbb >>> >>> >>> >> _______________________________________________ >> phenixbb mailing list >> [email protected] >> http://www.phenix-online.org/mailman/listinfo/phenixbb >> >> >> > > _______________________________________________ > phenixbb mailing list > [email protected] > http://www.phenix-online.org/mailman/listinfo/phenixbb >

Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- --------------------------------------------------------------

---

...

...

P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net --------------------------------------------------------------

---

...

...

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Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------

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

Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan‎ _______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb

-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------

Thank you Nat, Pavel, Actually, the NCS approaches a 3-fold crystallographic symmetry axis with 3 types of protein molecules plus 2 restraint groups for DNA molecules. The result is the same with "optimize_wxc=true optimize_wxu=true" options, Rfree is going up without NCS by ~1.5%. Rwork is practically the same. Should I increase the test set (now it's 5%)? or should I leave NCS=true for the final pdb? Maia Nathaniel Echols wrote:

On May 4, 2009, at 6:48 PM, Maia Cherney wrote:

...

The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints.

This isn't necessarily true - regardless of which program you use to refine, R-work isn't actually the refinement target, and minimization algorithms aren't foolproof either. When you remove the restraints you are effectively decreasing the observation:parameter ratio, which increases the risk of overfitting. If nothing else I would expect the gap between Rwork and Rfree to increase, but I've also seen both increase when NCS restraints were removed.

It may be possible that the NCS restraints are biasing your test set, leading to the suspiciously low Rfree-Rwork gap, but I thought this only applied when the NCS approached crystallographic symmetry, and there's no such thing as fivefold crystallographic symmetry. Hopefully someone else on the list can clarify this.

-Nat

------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

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This might be good to mention:

Remove all waters, reset all B-factors to average one, shake coordinates to achieve rmsd~1A and then run 10-20 macro-cycles of refinement in phenix.refine (or until the R-factors stop changing),

you can perform all these manipulations using phenix.pdbtools. Example: phenix.pdbtools model.pdb remove="water" or equivalently: phenix.pdbtools model.pdb keep="not water" phenix.pdbtools model.pdb set_b_iso=25 phenix.pdbtools model.pdb sites.shake=0.75 For more examples and general information see phenix.pdbtools manual: http://phenix-online.org/documentation/pdbtools.htm Pavel.

Hi Maia, you can easily do it by editing your parameter file: - change NCS group selections the way you want; - make sure that "main.ncs = True" and "ncs.find_automatically = False" If you have any problem with this: send me the parameters file and tell which residues you want to exclude and I can edit it, so you have an example for future reference. Pavel. On 6/27/09 9:12 AM, Maia Cherney wrote:

Hi Pavel, could you please tell me how to exclude several residues from ncs refinement. Should I make several restraint_groups or I can just give what to exclude?

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

Hi Maia,

I made all restraint_groups in my parameter file. It's just too many of them. I thought that may be there is an option to say exclude residues 125, 248 etc.

No - there is no such command: you need to edit selections manually.

By the way, I have excessive_distance_limit = None. If I change it to some number, how does it work? the violating residues will be excluded or the whole molecule will?

I'm not sure, Ralf knows this better. Ralf, could you comment on this please? My guess, that all this parameter does is it checks the distances between atoms in NCS copies and if there is a distance larger than excessive_distance_limit, then phenix.refine stops and tells you this. Pavel.

Hi Engin, thank you for your reply. That is exactly what I wanted. I just forgot the correct syntax. Actually, I could not find these words in the documentaion. I guess, they are very well hidden. Maia Engin Ozkan wrote:

Am I missing something? How about this

refinement.ncs.restraint_group { reference = chain A and not (resid 125 or resid 248 or ...) selection = chain B and not (resid 125 or resid 248 or ...) }

as it is in http://www.phenix-online.org/documentation/ I don't think this is harder than excluding residues with an "option".

Engin

On 6/27/09 10:28 AM, Pavel Afonine wrote:

...

Hi Maia,

...

I made all restraint_groups in my parameter file. It's just too many of them. I thought that may be there is an option to say exclude residues 125, 248 etc.

No - there is no such command: you need to edit selections manually.

...

By the way, I have excessive_distance_limit = None. If I change it to some number, how does it work? the violating residues will be excluded or the whole molecule will?

I'm not sure, Ralf knows this better. Ralf, could you comment on this please?

My guess, that all this parameter does is it checks the distances between atoms in NCS copies and if there is a distance larger than excessive_distance_limit, then phenix.refine stops and tells you this.

Pavel.

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

...

By the way, I have excessive_distance_limit = None. If I change it to some number, how does it work? the violating residues will be excluded or the whole molecule will?

I'm not sure, Ralf knows this better. Ralf, could you comment on this please?

My guess, that all this parameter does is it checks the distances between atoms in NCS copies and if there is a distance larger than excessive_distance_limit, then phenix.refine stops and tells you this.

Yes, that's correct. The distance check is just to alert you to the problem; it is not trying to be smart about fixing it. You have to do it yourself with the atom selection syntax ("and", "or", "not"). Ralf

Thank you, Dale. To clarify the 2 restraint groups for DNA, I meant two types of DNA sequences as the DNAs are double stranded; due to 3-fold there are totally 6 strands. Maia Dale Tronrud wrote:

If you have nearly C3 ncs symmetry parallel to the crystallographic 3-fold, you very likely also have some form of pseudo-translational symmetry. If present this will lead to quite a few of your intensities being very small, and poorly measured. This situation can require you to continue restraining ncs even at 2.1A resolution, particularly if many of your weak reflections are "unobserved".

It is quite possible that the free R is telling you exactly what you need to know, that your structure factors alone are insufficient to define your model.

Dr. Echols' comment:

...

...

It may be possible that the NCS restraints are biasing your test set, leading to the suspiciously low Rfree-Rwork gap, but I thought this only applied when the NCS approached crystallographic symmetry, and

is the reverse of my understanding. The cross-talk between the working set and test set is minimal when the ncs approaches crystallographic symmetry, and at its worst when the two are unrelated. When the ncs and "cs" are similar the ncs images of a test set reflections falls very near its proper symmetry images, which are also in the test set by definition. When the two types of symmetry are unrelated, the ncs image of a test set reflection will fall near a reflection which only has a 5% chance of being in the test set. Now you have a direct relationship between a reflection in the test set and one in the working set.

With care, a test set can be selected that minimizes this crosstalk. Since you have ncs symmetry that is masked by cs symmetry this is not much of a problem for you. (I don't quite understand how the 2 copy ncs of the DNA relates to the crystallographic symmetry, so it might be a problem.)

Dale Tronrud

Maia Cherney wrote:

...

Thank you Nat, Pavel,

Actually, the NCS approaches a 3-fold crystallographic symmetry axis with 3 types of protein molecules plus 2 restraint groups for DNA molecules.

The result is the same with "optimize_wxc=true optimize_wxu=true" options, Rfree is going up without NCS by ~1.5%. Rwork is practically the same.

Should I increase the test set (now it's 5%)? or should I leave NCS=true for the final pdb?

Maia

Nathaniel Echols wrote:

...

On May 4, 2009, at 6:48 PM, Maia Cherney wrote:

...

The resolution is 2.15 A. The NCS was always on during the refinement until we got low R factors (19.2% and 21.2%). Then the NCS was turned off for the final refinement and the R factors increased, which is strange as they should be going down when you apply less restraints.

This isn't necessarily true - regardless of which program you use to refine, R-work isn't actually the refinement target, and minimization algorithms aren't foolproof either. When you remove the restraints you are effectively decreasing the observation:parameter ratio, which increases the risk of overfitting. If nothing else I would expect the gap between Rwork and Rfree to increase, but I've also seen both increase when NCS restraints were removed.

It may be possible that the NCS restraints are biasing your test set, leading to the suspiciously low Rfree-Rwork gap, but I thought this only applied when the NCS approached crystallographic symmetry, and there's no such thing as fivefold crystallographic symmetry. Hopefully someone else on the list can clarify this.

-Nat

------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

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No problems, I read emails with any subject -:) I just sent an attempt to answer your question, although your question is too abstract so the reply... Pavel. On 5/4/09 6:21 PM, Maia Cherney wrote:

Hi everybody, I am sorry to send this message under wrong subject, but now I have changed the subject according to the question.

I am wondering why our final structure refined with NCS=True has lower R factors (19.2% and 21.2%) than without NCS by approximately 1.5%.

Maia

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