Hi Florian, I'm glad it works now. Regarding difference between Wilson B and mean refined B: well, it's hard to quantify what's "too low" or "too different". Average refined B may deviate from Wilson B and the difference in your case does not seem to be terribly large. Did the refinement converge? What if you do say 10 or 20 macro-cycles instead of default 3? Will B-factors further change between macro-cycles? Pavel On 3/19/13 7:37 AM, Florian Brückner wrote:

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita mailto:[email protected]>:

...

Dear Florian,

Maybe you are using phenix-dev-1311 or earlier? I am afraid that you have experienced the bug that overall B-factor is not applied to individual atomic B-factors in certain cases, which I have also experienced recently. That bug was fixed in dev-1312 if I recall correctly.

Hope it helps, Keitaro

2013/3/19 Florian Brückner mailto:[email protected]>:

...

Dear all

I am refining a structure at 3.2 A with phenix.refine using automatic optimization of target weights, solved by molecular replacement with a 2.1A structure of the same crystal form. Rwork=26%, Rfree=28%. I use individual atomic B-factor refinement. The obtained distribution of B-factors after refinement looks reasonable and quite similar to the distribution in the higher resolution model, which I suppose would be expected, since it is the same crystal form. However, the average B-factor does not change substantially, i.e. if I start with the B-factors from the high resolution model (average B-factor around 32 A**2) after 6 macrocycles the average B-factor is still around 32 A**2. If I set all B-factors to the Wilson B-factor determined by phenix.refine (66 A**2), after 6 macrocycles the distribution of B-factors looks again very similar to the high resolution MR model, but the average B-factor is still around 65 A**2. What should I make of this? Is this an expected behavior at this resolution, i.e. the fact that the absolute values of B-factors cannot be determined during refinement, although a reasonable distribution can be obtained as judged by plotting average B-factors vs. residue#? Since the average B-factor does not change much during refinement, which average B-factor should I start refinement with to end up with reasonable absolute values? I have manually determined the Wilson B-factor from the linear part of the Wilson plot (4.5-3.2 A) to be 97 A**2. This seems high to me. Would that be expected for the given resolution, given that the 2.1A structure of the same crystal form has 32 A**2? I should add, that there is not a substantial effect of the average refined B-factor on Rfree.

Thanks for any comments or suggestions.

Best regards

Florian

Hi Florian, I think it is pretty convincing that refinement went all right and the mean B you get is what it actually is. Also I just looked at the files that you sent me the other day and I did not notice anything suspicious. Pavel P.S.: Sorry if you received multiple copies of this email: I was trying to attach a tiny image with some PDB statistics but our server did not let me do it. On 3/21/13 1:00 AM, Florian Brückner wrote:

Dear Pavel

yes, the refinement did converge. I did 6 macrocycles and in separate runs started from different average atomic B-factors of 32, 66 and 97 keeping the distribution as in the original high resolution molecular replacement model from the same crystal form (i.e. adding 0, 34 and 65 to all atomic B-factors in the starting pdb). In all cases after 6 macrocycles I get an almost identical distribution of B-factors and also average B-factor of about 52. Also setting all B-factors to the same value of 66 gave a similar distribution after refinement with an average of 52. However, in the latter case the R-free was about 2% higher compared to starting from the original distribution, but it dropped by about 1% during refinement.

Best regards

Florian

Am 21.03.2013 um 06:40 schrieb Pavel Afonine mailto:[email protected]>:

...

Hi Florian,

I'm glad it works now. Regarding difference between Wilson B and mean refined B: well, it's hard to quantify what's "too low" or "too different". Average refined B may deviate from Wilson B and the difference in your case does not seem to be terribly large. Did the refinement converge? What if you do say 10 or 20 macro-cycles instead of default 3? Will B-factors further change between macro-cycles?

Pavel

On 3/19/13 7:37 AM, Florian Brückner wrote:

...

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita mailto:[email protected]>:

...

Dear Florian,

Maybe you are using phenix-dev-1311 or earlier? I am afraid that you have experienced the bug that overall B-factor is not applied to individual atomic B-factors in certain cases, which I have also experienced recently. That bug was fixed in dev-1312 if I recall correctly.

Hope it helps, Keitaro

2013/3/19 Florian Brückner mailto:[email protected]>:

...

Dear all

I am refining a structure at 3.2 A with phenix.refine using automatic optimization of target weights, solved by molecular replacement with a 2.1A structure of the same crystal form. Rwork=26%, Rfree=28%. I use individual atomic B-factor refinement. The obtained distribution of B-factors after refinement looks reasonable and quite similar to the distribution in the higher resolution model, which I suppose would be expected, since it is the same crystal form. However, the average B-factor does not change substantially, i.e. if I start with the B-factors from the high resolution model (average B-factor around 32 A**2) after 6 macrocycles the average B-factor is still around 32 A**2. If I set all B-factors to the Wilson B-factor determined by phenix.refine (66 A**2), after 6 macrocycles the distribution of B-factors looks again very similar to the high resolution MR model, but the average B-factor is still around 65 A**2. What should I make of this? Is this an expected behavior at this resolution, i.e. the fact that the absolute values of B-factors cannot be determined during refinement, although a reasonable distribution can be obtained as judged by plotting average B-factors vs. residue#? Since the average B-factor does not change much during refinement, which average B-factor should I start refinement with to end up with reasonable absolute values? I have manually determined the Wilson B-factor from the linear part of the Wilson plot (4.5-3.2 A) to be 97 A**2. This seems high to me. Would that be expected for the given resolution, given that the 2.1A structure of the same crystal form has 32 A**2? I should add, that there is not a substantial effect of the average refined B-factor on Rfree.

Thanks for any comments or suggestions.

Best regards

Florian

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

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

Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.:+41-(0)56-310-2332 Email:[email protected] mailto:[email protected]

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

Thanks, Pavel. So you don't think that the difference between Wilson B (66.45 according to phenix) and average B (52 after refinement) is still too large? Where did you take the statistics from? Is that published anywhere? Best regards Florian Am 22.03.2013 um 07:09 schrieb Pavel Afonine :

Hi Florian,

I think it is pretty convincing that refinement went all right and the mean B you get is what it actually is. Also I just looked at the files that you sent me the other day and I did not notice anything suspicious.

Pavel

P.S.: Sorry if you received multiple copies of this email: I was trying to attach a tiny image with some PDB statistics but our server did not let me do it.

On 3/21/13 1:00 AM, Florian Brückner wrote:

...

Dear Pavel

yes, the refinement did converge. I did 6 macrocycles and in separate runs started from different average atomic B-factors of 32, 66 and 97 keeping the distribution as in the original high resolution molecular replacement model from the same crystal form (i.e. adding 0, 34 and 65 to all atomic B-factors in the starting pdb). In all cases after 6 macrocycles I get an almost identical distribution of B-factors and also average B-factor of about 52. Also setting all B-factors to the same value of 66 gave a similar distribution after refinement with an average of 52. However, in the latter case the R-free was about 2% higher compared to starting from the original distribution, but it dropped by about 1% during refinement.

Best regards

Florian

Am 21.03.2013 um 06:40 schrieb Pavel Afonine :

...

Hi Florian,

I'm glad it works now. Regarding difference between Wilson B and mean refined B: well, it's hard to quantify what's "too low" or "too different". Average refined B may deviate from Wilson B and the difference in your case does not seem to be terribly large. Did the refinement converge? What if you do say 10 or 20 macro-cycles instead of default 3? Will B-factors further change between macro-cycles?

Pavel

On 3/19/13 7:37 AM, Florian Brückner wrote:

...

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita :

...

Dear Florian,

Maybe you are using phenix-dev-1311 or earlier? I am afraid that you have experienced the bug that overall B-factor is not applied to individual atomic B-factors in certain cases, which I have also experienced recently. That bug was fixed in dev-1312 if I recall correctly.

Hope it helps, Keitaro

2013/3/19 Florian Brückner :

...

Dear all

I am refining a structure at 3.2 A with phenix.refine using automatic optimization of target weights, solved by molecular replacement with a 2.1A structure of the same crystal form. Rwork=26%, Rfree=28%. I use individual atomic B-factor refinement. The obtained distribution of B-factors after refinement looks reasonable and quite similar to the distribution in the higher resolution model, which I suppose would be expected, since it is the same crystal form. However, the average B-factor does not change substantially, i.e. if I start with the B-factors from the high resolution model (average B-factor around 32 A**2) after 6 macrocycles the average B-factor is still around 32 A**2. If I set all B-factors to the Wilson B-factor determined by phenix.refine (66 A**2), after 6 macrocycles the distribution of B-factors looks again very similar to the high resolution MR model, but the average B-factor is still around 65 A**2. What should I make of this? Is this an expected behavior at this resolution, i.e. the fact that the absolute values of B-factors cannot be determined during refinement, although a reasonable distribution can be obtained as judged by plotting average B-factors vs. residue#? Since the average B-factor does not change much during refinement, which average B-factor should I start refinement with to end up with reasonable absolute values? I have manually determined the Wilson B-factor from the linear part of the Wilson plot (4.5-3.2 A) to be 97 A**2. This seems high to me. Would that be expected for the given resolution, given that the 2.1A structure of the same crystal form has 32 A**2? I should add, that there is not a substantial effect of the average refined B-factor on Rfree.

Thanks for any comments or suggestions.

Best regards

Florian

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

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

Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.: +41-(0)56-310-2332 Email: [email protected]

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

------------------------------------------------------------- Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland Tel.: +41-(0)56-310-2332 Email: [email protected]

Hi Florian, Wilson B is just an estimate, and your refined B reflect your data.. Assumptions made to derive Wilson B include uniformly distributed atoms in the unit cell, same atom type and B-factor (what else?). This is not quite true for protein crystals, which typically consist of atoms of different kind, have different degree of disorder (B-factors), and they kind of form some structure (definitely not randomly distributed in the unit cell). So to me it's a miracle that oftentimes Wilson B closely matches refined average B. We should acknowledge that when it doesn't that's a valid possibility. Regarding papers.. Exactly that plot I sent you before was not published (I guess it did not go to the phenixbb for the reason Nat explained yesterday), but the tools and methods are described here: Afonine, P.V., Grosse-Kunstleve, R.W., Chen, V.B.,Headd, J.J., Moriarty, N.W., Richardson, J.S., Richardson, D.C.,Urzhumtsev, A.,Zwart, P.H. & Adams, P.D. (2010).*phenix.model**_vs_data**: a high-level tool for the calculation of crystallographic model and data statistics*. J. Appl.Crystallogr. 43, 669-676. Urzhumtsev, A., Afonine, P.V. & Adams P.D. (2009).*On the use of logarithmic scales for analysis of diffraction data*.ActaCryst. D65, 1283-1291. Urzhumtseva, L., Afonine, P.V., Adams, P.D. &Urzhumtsev, A. (2009).*Crystallographic model quality at a glance*.ActaCryst. D65, 297-300. and the source of information used for all the analysis described in above papers is PDB. Also, I guess, you can obtain that plot from PHENIX GUI. Pavel On 3/23/13 6:53 AM, Florian Brückner wrote:

Thanks, Pavel.

So you don't think that the difference between Wilson B (66.45 according to phenix) and average B (52 after refinement) is still too large? Where did you take the statistics from? Is that published anywhere?

Best regards

Florian

Am 22.03.2013 um 07:09 schrieb Pavel Afonine mailto:[email protected]>:

...

Hi Florian,

I think it is pretty convincing that refinement went all right and the mean B you get is what it actually is. Also I just looked at the files that you sent me the other day and I did not notice anything suspicious.

Pavel

P.S.: Sorry if you received multiple copies of this email: I was trying to attach a tiny image with some PDB statistics but our server did not let me do it.

On 3/21/13 1:00 AM, Florian Brückner wrote:

...

Dear Pavel

yes, the refinement did converge. I did 6 macrocycles and in separate runs started from different average atomic B-factors of 32, 66 and 97 keeping the distribution as in the original high resolution molecular replacement model from the same crystal form (i.e. adding 0, 34 and 65 to all atomic B-factors in the starting pdb). In all cases after 6 macrocycles I get an almost identical distribution of B-factors and also average B-factor of about 52. Also setting all B-factors to the same value of 66 gave a similar distribution after refinement with an average of 52. However, in the latter case the R-free was about 2% higher compared to starting from the original distribution, but it dropped by about 1% during refinement.

Best regards

Florian

Am 21.03.2013 um 06:40 schrieb Pavel Afonine mailto:[email protected]>:

...

Hi Florian,

I'm glad it works now. Regarding difference between Wilson B and mean refined B: well, it's hard to quantify what's "too low" or "too different". Average refined B may deviate from Wilson B and the difference in your case does not seem to be terribly large. Did the refinement converge? What if you do say 10 or 20 macro-cycles instead of default 3? Will B-factors further change between macro-cycles?

Pavel

On 3/19/13 7:37 AM, Florian Brückner wrote:

...

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita mailto:[email protected]>:

...

Dear Florian,

Maybe you are using phenix-dev-1311 or earlier? I am afraid that you have experienced the bug that overall B-factor is not applied to individual atomic B-factors in certain cases, which I have also experienced recently. That bug was fixed in dev-1312 if I recall correctly.

Hope it helps, Keitaro

2013/3/19 Florian Brückner mailto:[email protected]>: > Dear all > > I am refining a structure at 3.2 A with phenix.refine using > automatic > optimization of target weights, solved by molecular replacement > with a 2.1A > structure of the same crystal form. Rwork=26%, Rfree=28%. I use > individual > atomic B-factor refinement. The obtained distribution of > B-factors after > refinement looks reasonable and quite similar to the > distribution in the > higher resolution model, which I suppose would be expected, > since it is the > same crystal form. However, the average B-factor does not change > substantially, i.e. if I start with the B-factors from the high > resolution > model (average B-factor around 32 A**2) after 6 macrocycles the > average > B-factor is still around 32 A**2. If I set all B-factors to the > Wilson > B-factor determined by phenix.refine (66 A**2), after 6 > macrocycles the > distribution of B-factors looks again very similar to the high > resolution MR > model, but the average B-factor is still around 65 A**2. What > should I make > of this? Is this an expected behavior at this resolution, i.e. > the fact that > the absolute values of B-factors cannot be determined during > refinement, > although a reasonable distribution can be obtained as judged by > plotting > average B-factors vs. residue#? Since the average B-factor does > not change > much during refinement, which average B-factor should I start > refinement > with to end up with reasonable absolute values? I have manually > determined > the Wilson B-factor from the linear part of the Wilson plot > (4.5-3.2 A) to > be 97 A**2. This seems high to me. Would that be expected for > the given > resolution, given that the 2.1A structure of the same crystal > form has 32 > A**2? I should add, that there is not a substantial effect of > the average > refined B-factor on Rfree. > > Thanks for any comments or suggestions. > > Best regards > > Florian

It is even more "mathematical", only linear part of Wilson statistics contribute to determination of Wilson B factor. Linear part usually start around 3.2 Angstrom, it is more or less what Mark sa, but from a slightly different perspective. I am sure also a scattering/diffraction physics is involved…. :-\ FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: [email protected] Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Mar 23, 2013, at 20:10 , "VAN RAAIJ , MARK JOHAN" wrote:

At 3.2 A resolution I don't think Phenix (or any other program, or anyone) can reliably determine a Wilson B beyond giving a rough idea of the range, because you have too few data points and the data points you have are "contaminated" by influences of the peaks and troughs due to regular repeats in secondary structure elements (and you don't know the amount of these secondary structure elements...), so I wouldn't worry about it. Mark PS Perhaps Phenix should not quote it with apparent four-digit precision, i.e. 66.45, at least at this resolution, in case people take this number too seriously?

Quoting Florian Brückner :

...

Thanks, Pavel.

So you don't think that the difference between Wilson B (66.45 according to phenix) and average B (52 after refinement) is still too large? Where did you take the statistics from? Is that published anywhere?

Best regards

Florian

Am 22.03.2013 um 07:09 schrieb Pavel Afonine :

...

Hi Florian,

I think it is pretty convincing that refinement went all right and the mean B you get is what it actually is. Also I just looked at the files that you sent me the other day and I did not notice anything suspicious.

Pavel

P.S.: Sorry if you received multiple copies of this email: I was trying to attach a tiny image with some PDB statistics but our server did not let me do it.

On 3/21/13 1:00 AM, Florian Brückner wrote:

...

Dear Pavel

yes, the refinement did converge. I did 6 macrocycles and in separate runs started from different average atomic B-factors of 32, 66 and 97 keeping the distribution as in the original high resolution molecular replacement model from the same crystal form (i.e. adding 0, 34 and 65 to all atomic B-factors in the starting pdb). In all cases after 6 macrocycles I get an almost identical distribution of B-factors and also average B-factor of about 52. Also setting all B-factors to the same value of 66 gave a similar distribution after refinement with an average of 52. However, in the latter case the R-free was about 2% higher compared to starting from the original distribution, but it dropped by about 1% during refinement.

Best regards

Florian

Am 21.03.2013 um 06:40 schrieb Pavel Afonine :

...

Hi Florian,

I'm glad it works now. Regarding difference between Wilson B and mean refined B: well, it's hard to quantify what's "too low" or "too different". Average refined B may deviate from Wilson B and the difference in your case does not seem to be terribly large. Did the refinement converge? What if you do say 10 or 20 macro-cycles instead of default 3? Will B-factors further change between macro-cycles?

Pavel

On 3/19/13 7:37 AM, Florian Brückner wrote:

...

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita :

> Dear Florian, > > Maybe you are using phenix-dev-1311 or earlier? > I am afraid that you have experienced the bug that overall B-factor is > not applied to individual atomic B-factors in certain cases, which I > have also experienced recently. That bug was fixed in dev-1312 if I > recall correctly. > > Hope it helps, > Keitaro > > 2013/3/19 Florian Brückner : >> Dear all >> >> I am refining a structure at 3.2 A with phenix.refine using automatic >> optimization of target weights, solved by molecular replacement with a 2.1A >> structure of the same crystal form. Rwork=26%, Rfree=28%. I use individual >> atomic B-factor refinement. The obtained distribution of B-factors after >> refinement looks reasonable and quite similar to the distribution in the >> higher resolution model, which I suppose would be expected, since it is the >> same crystal form. However, the average B-factor does not change >> substantially, i.e. if I start with the B-factors from the high resolution >> model (average B-factor around 32 A**2) after 6 macrocycles the average >> B-factor is still around 32 A**2. If I set all B-factors to the Wilson >> B-factor determined by phenix.refine (66 A**2), after 6 macrocycles the >> distribution of B-factors looks again very similar to the high resolution MR >> model, but the average B-factor is still around 65 A**2. What should I make >> of this? Is this an expected behavior at this resolution, i.e. the fact that >> the absolute values of B-factors cannot be determined during refinement, >> although a reasonable distribution can be obtained as judged by plotting >> average B-factors vs. residue#? Since the average B-factor does not change >> much during refinement, which average B-factor should I start refinement >> with to end up with reasonable absolute values? I have manually determined >> the Wilson B-factor from the linear part of the Wilson plot (4.5-3.2 A) to >> be 97 A**2. This seems high to me. Would that be expected for the given >> resolution, given that the 2.1A structure of the same crystal form has 32 >> A**2? I should add, that there is not a substantial effect of the average >> refined B-factor on Rfree. >> >> Thanks for any comments or suggestions. >> >> Best regards >> >> Florian

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

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

Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.: +41-(0)56-310-2332 Email: [email protected]

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

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

Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.: +41-(0)56-310-2332 Email: [email protected]

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

B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

I think the Wilson B calculated from the linear part (say beyond 3A) should be _lower_ than the average atomic B, because well-ordered parts of the structure contribute disproportionately to the high-resolution scattering and the disordered bits contribute nothing in that range, while the average atomic B weights each atoms contribution the same. Sort of like log(average) is not the same as average(log). But then if Wilson B is 97 I wouldn't expect diffraction to extend much beyond 3A. OK, this is 3.2A data. In that case I think there is no linear part- from the ideal wilson plot that arp-warp shows us, in the region 4-3A the value is coming down off a peak due to secondary structure, and is considerably steeper than in 3-2 A range. So the value of 97 may be an overestimate. Would the following sanity check with another suite make sense? Calculate Fc,Phic from the refined model with sfall, and scale the raw data against that and Fmask,PhiFmask from phenix.refine with ccp4 icoefl program. If the overall B applied to the raw data is far from zero, it suggests something is fishy about the atomic B's? Florian Brückner wrote:

Dear Keitaro and Pavel,

thank you for your suggestions. I have now used the latest nightly build (1326). With this version, the average B-factor converges at about 52 A**2 and also the distribution is very similar, regardless with what B-factors I start the refinement (I tried average B-factors between 32 and 97). So clearly the behavior of this newest version is different from the version I have been using before (1.8.1 stable release 1168) and also from the latest stable release (1.8.2 stable release 1309), which behaves the same as 1168. To me the B-factors still look a bit too low, since the Wilson B-factor calculated by phenix is 66.45, the Wilson B-factor I determined manually from the linear part of the Wilson plot is 97.

Best regards

Florian

Am 19.03.2013 um 00:06 schrieb Keitaro Yamashita mailto:[email protected]>:

...

Dear Florian,

Maybe you are using phenix-dev-1311 or earlier? I am afraid that you have experienced the bug that overall B-factor is not applied to individual atomic B-factors in certain cases, which I have also experienced recently. That bug was fixed in dev-1312 if I recall correctly.

Hope it helps, Keitaro

2013/3/19 Florian Brückner mailto:[email protected]>:

...

Dear all

I am refining a structure at 3.2 A with phenix.refine using automatic optimization of target weights, solved by molecular replacement with a 2.1A structure of the same crystal form. Rwork=26%, Rfree=28%. I use individual atomic B-factor refinement. The obtained distribution of B-factors after refinement looks reasonable and quite similar to the distribution in the higher resolution model, which I suppose would be expected, since it is the same crystal form. However, the average B-factor does not change substantially, i.e. if I start with the B-factors from the high resolution model (average B-factor around 32 A**2) after 6 macrocycles the average B-factor is still around 32 A**2. If I set all B-factors to the Wilson B-factor determined by phenix.refine (66 A**2), after 6 macrocycles the distribution of B-factors looks again very similar to the high resolution MR model, but the average B-factor is still around 65 A**2. What should I make of this? Is this an expected behavior at this resolution, i.e. the fact that the absolute values of B-factors cannot be determined during refinement, although a reasonable distribution can be obtained as judged by plotting average B-factors vs. residue#? Since the average B-factor does not change much during refinement, which average B-factor should I start refinement with to end up with reasonable absolute values? I have manually determined the Wilson B-factor from the linear part of the Wilson plot (4.5-3.2 A) to be 97 A**2. This seems high to me. Would that be expected for the given resolution, given that the 2.1A structure of the same crystal form has 32 A**2? I should add, that there is not a substantial effect of the average refined B-factor on Rfree.

Thanks for any comments or suggestions.

Best regards

Florian

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Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.: +41-(0)56-310-2332 Email: [email protected] mailto:[email protected]

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

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

Dr. Florian Brückner Laboratory of Biomolecular Research (LBR) OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland

Tel.:+41-(0)56-310-2332 Email:[email protected] mailto:[email protected]

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