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Hi Tanya,<br>
<br>
thanks for sending the data and model files. Here are some results.<br>
<br>
1) Starting values corresponding to the model you sent me:<br>
<br>
r_work = 0.2891 r_free = 0.3267<br>
MOLPROBITY STATISTICS.<br>
�ALL-ATOM CLASHSCORE : 67.04 <br>
�RAMACHANDRAN PLOT:<br>
�� OUTLIERS : 6.04� %<br>
�� ALLOWED� : 16.58 %<br>
�� FAVORED� : 77.38 %<br>
�ROTAMER OUTLIERS : 14.80 %<br>
�CBETA DEVIATIONS : 13<br>
<br>
2) I ran six refinement jobs using input model with and without H
atoms, and for each model I tried 3 ways of using NCS in refinement:
the new option defining NCS in torsion angle space, let
phenix.refine define NCS automatically and apply it in Cartesian
space, and finally I used the NCS selections that you sent me. So 2
(model with and w/o H) * 3 (NCS options) = 6 refinements in total.<br>
<br>
Here are the results:<br>
<br>
MODEL with H:<br>
<br>
- torsion NCS:<br>
<br>
r_work = 0.2951 r_free = 0.3319<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 19.91 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 1.99� %<br>
REMARK�� 3���� ALLOWED� : 6.99� %<br>
REMARK�� 3���� FAVORED� : 91.02 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 0.45 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 0<br>
<br>
- Cartesian NCS defined automatically:<br>
<br>
r_work = 0.3260 r_free = 0.3427<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 12.79 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 1.02� %<br>
REMARK�� 3���� ALLOWED� : 5.97� %<br>
REMARK�� 3���� FAVORED� : 93.01 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 14.07 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 45<br>
<br>
- Cartesian NCS using your selections for NCS groups:<br>
<br>
r_work = 0.2792 r_free = 0.3245<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 15.29 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 0.90� %<br>
REMARK�� 3���� ALLOWED� : 7.77� %<br>
REMARK�� 3���� FAVORED� : 91.33 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 12.87 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 36<br>
<br>
<br>
MODEL without H:<br>
<br>
- torsion NCS:<br>
<br>
r_work = 0.2938 r_free = 0.3301<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 30.12 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 1.89� %<br>
REMARK�� 3���� ALLOWED� : 7.01� %<br>
REMARK�� 3���� FAVORED� : 91.11 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 0.22 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 2<br>
<br>
- Cartesian NCS defined automatically:<br>
<br>
r_work = 0.3174 r_free = 0.3354<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 33.76 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 0.63� %<br>
REMARK�� 3���� ALLOWED� : 6.31� %<br>
REMARK�� 3���� FAVORED� : 93.06 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 12.60 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 142<br>
<br>
- Cartesian NCS using your selections for NCS groups:<br>
<br>
r_work = 0.2762 r_free = 0.3233<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 41.83 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 0.73� %<br>
REMARK�� 3���� ALLOWED� : 8.32� %<br>
REMARK�� 3���� FAVORED� : 90.95 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 18.36 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 118<br>
<br>
3) Looking at the results above, I would say we have two overall
equally good results (in my interpretation):<br>
<br>
MODEL with H (torsion NCS):<br>
<br>
r_work = 0.2951 r_free = 0.3319<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 19.91 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 1.99� %<br>
REMARK�� 3���� ALLOWED� : 6.99� %<br>
REMARK�� 3���� FAVORED� : 91.02 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 0.45 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 0<br>
<br>
and<br>
<br>
MODEL without H (torsion NCS):<br>
<br>
r_work = 0.2938 r_free = 0.3301<br>
REMARK�� 3� MOLPROBITY STATISTICS.<br>
REMARK�� 3�� ALL-ATOM CLASHSCORE : 30.12 <br>
REMARK�� 3�� RAMACHANDRAN PLOT:<br>
REMARK�� 3���� OUTLIERS : 1.89� %<br>
REMARK�� 3���� ALLOWED� : 7.01� %<br>
REMARK�� 3���� FAVORED� : 91.11 %<br>
REMARK�� 3�� ROTAMER OUTLIERS : 0.22 %<br>
REMARK�� 3�� CBETA DEVIATIONS : 2<br>
<br>
compare it to your original structure:<br>
<br>
r_work = 0.2891 r_free = 0.3267<br>
MOLPROBITY STATISTICS.<br>
�ALL-ATOM CLASHSCORE : 67.04 <br>
�RAMACHANDRAN PLOT:<br>
�� OUTLIERS : 6.04� %<br>
�� ALLOWED� : 16.58 %<br>
�� FAVORED� : 77.38 %<br>
�ROTAMER OUTLIERS : 14.80 %<br>
�CBETA DEVIATIONS : 13<br>
<br>
4) To be able to reproduce these numbers you need to use the most
recent PHENIX version from the nightly builds:<br>
<a class="moz-txt-link-freetext" href="http://www.phenix-online.org/download/nightly_builds.cgi">http://www.phenix-online.org/download/nightly_builds.cgi</a><br>
use dev-838 and up.<br>
<br>
5) I'm sending the relevant files off-list.<br>
<br>
6) The six commands I used are:<br>
<br>
phenix.refine data.mtz model_H.pdb ramachandran_restraints=true
main.number_of_mac=5 optimize_xyz_weight=true
optimize_adp_weight=true --overwrite main.ncs=true ncs.type=torsion
output.prefix=H secondary_structure_restraints=true *cif
xray_data.high_res=3.6 > & zlogH &<br>
<br>
phenix.refine data.mtz model.pdb ramachandran_restraints=true
main.number_of_mac=5 optimize_xyz_weight=true
optimize_adp_weight=true --overwrite main.ncs=true ncs.type=torsion
output.prefix=noH secondary_structure_restraints=true *cif
xray_data.high_res=3.6 > & zlognoH &<br>
<br>
<br>
phenix.refine data.mtz model_H.pdb excessive_distance_limit=None
ramachandran_restraints=true main.number_of_mac=5
optimize_xyz_weight=true optimize_adp_weight=true --overwrite
main.ncs=true output.prefix=H_ncsC
secondary_structure_restraints=true *cif xray_data.high_res=3.6 >
& zlogH_ncsC &<br>
<br>
phenix.refine data.mtz model.pdb�� excessive_distance_limit=None
ramachandran_restraints=true� main.number_of_mac=5
optimize_xyz_weight=true optimize_adp_weight=true --overwrite
main.ncs=true output.prefix=noH_ncsC
secondary_structure_restraints=true *cif xray_data.high_res=3.6 >
& zlognoH_ncsC &<br>
<br>
<br>
phenix.refine data.mtz model_H.pdb excessive_distance_limit=None
ncs_groups_H.params ramachandran_restraints=true
main.number_of_mac=5 optimize_xyz_weight=true
optimize_adp_weight=true --overwrite main.ncs=true
output.prefix=H_ncsC_cust secondary_structure_restraints=true *cif
xray_data.high_res=3.6 > & zlogH_ncsC_cust &<br>
<br>
phenix.refine data.mtz model.pdb�� excessive_distance_limit=None
ncs_groups_H.params ramachandran_restraints=true�
main.number_of_mac=5 optimize_xyz_weight=true
optimize_adp_weight=true --overwrite main.ncs=true
output.prefix=noH_ncsC_cust secondary_structure_restraints=true *cif
xray_data.high_res=3.6 > & zlognoH_ncsC_cust &<br>
<br>
It may not be necessary to use weights optimization, although I did
not try without it. I recommend you try it: if it turns out to be
not necessary than it may save you many hours of time.<br>
<br>
Also, the amount of CB-outliers and Ramachandran outliers in your
original model probably indicates that it is far from final and
requires some careful analysis manually.<br>
<br>
As Nat mentioned before, fit_rotamers will not work at resolutions
lower than 2.8-3.0A, as it relies on "reasonably good" density. I
plan to extend it to lower resolutions, but it will take some time.<br>
<br>
You don't need to run tools like phenix.ramalyze, etc separately
after refinement, since most of Molprobity statistics is reported in
REMARK 3 records by phenix.refine as in examples above.<br>
<br>
Finally, you don't really have to type all these long commands.
Using parameter files is easy, and can reduce the amount of typing
to something like:<br>
<br>
phenix.refine params.eff<br>
<br>
See
<a class="moz-txt-link-freetext" href="https://www.phenix-online.org/presentations/latest/pavel_phenix_refine.pdf">https://www.phenix-online.org/presentations/latest/pavel_phenix_refine.pdf</a><br>
for details and examples.<br>
<br>
7) Finally finally, at 3.6A resolution or so, the R-factors withing
~2% can be considered the same. For example, I would not say that
r_free = 0.3245 is better than 0.3319.<br>
For discussion see<br>
<a class="moz-txt-link-freetext" href="http://phenix-online.org/newsletter/CCN_2011_07.pdf">http://phenix-online.org/newsletter/CCN_2011_07.pdf</a><br>
article "<span class="Apple-style-span" style="border-collapse:
separate; color: rgb(0, 0, 0); font-family: Times; font-style:
normal; font-variant: normal; font-weight: normal; letter-spacing:
normal; line-height: normal; orphans: 2; text-indent: 0px;
text-transform: none; white-space: normal; widows: 2;
word-spacing: 0px; font-size: medium;"><span
class="Apple-style-span" style="font-family:
verdana,helvetica,arial,sans-serif; font-size: 14px;">Improved
target weight optimization in phenix.refine</span></span>". <br>
<br>
Please let me know if you have any questions or need any help with
this.<br>
<br>
Pavel.<br>
<br>
<br>
On 7/30/11 11:35 AM, Tatyana Sysoeva wrote:
<blockquote
cite="mid:CAGikDO7iCFgb=fP=LyM1mGQzZ1++HY=FVoJT5SfAbE-166SbYg@mail.gmail.com"
type="cite">I ran the same commands with the build dev-833.<br>
Below are the results. <br>
These parameters are obtained by running phenix.ramalyze, cbetdev,
and clashscore.<br>
I am almost done repeating fix-rotamers run. First time it gave a
way worse validation results then before running phenix without
fix_rotamers=true. <br>
I don't understand what I am doing wrong in the runs and would
appreciate your help!<br>
<br>
Thanks,<br>
Tanya<br>
<br>
<br>
<p class="MsoNormalCxSpFirst" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">I used
phenix-dev-833 to test the
riding hydrogens refinement at 3.6A.</span></p>
<p class="MsoNormalCxSpLast" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">I ran two
indentical commands:</span></p>
<p class="MsoPlainText" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">Command line
arguments: "model.pdb"
"data.mtz" "main.ncs=true"
"ncs.find_automatically=false" "ncs_groups.params"
"refinement.input.xray_data.high_resolution=3.6"
"mgadpbef.cif"
"refinement.ncs.excessive_distance_limit=None"
"strategy=individual_sites+individual_sites_real_space+group_adp+occupancies"</span></p>
<p class="MsoNormalCxSpFirst" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";"><span style="">�</span>with
the only difference in the input files �
ncs.params and model.pdb</span></p>
<p class="MsoNormalCxSpMiddle" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">Model pdb contained
the model
identical to the control run but with added H atoms. I have
not add H to the
ADP molecules since I did not know how to write a correct CIF
file for it. NCS
definitions were changed by addition of �and not (element H)�
to each line. It
was done to exclude H from the NCS groups.</span></p>
<table class="MsoTableGrid" style="border-collapse: collapse;
border: medium none;" border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr style="">
<td style="width: 239.4pt; border: 1pt solid windowtext;
padding: 0in 5.4pt;" valign="top" width="319">
<p class="MsoNormalCxSpMiddle" style="margin-bottom:
0.0001pt; text-align: justify;"><u><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">no
hydrogens</span></u></p>
</td>
<td style="width: 239.4pt; border-width: 1pt 1pt 1pt medium;
border-style: solid solid solid none; border-color:
windowtext windowtext windowtext -moz-use-text-color;
padding: 0in 5.4pt;" valign="top" width="319">
<p class="MsoNormalCxSpMiddle" style="margin-bottom:
0.0001pt; text-align: justify;"><u><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">with
hydrogens</span></u><span style="font-size: 12pt;
font-family:
"Arial","sans-serif";"></span></p>
</td>
</tr>
<tr style="">
<td style="width: 239.4pt; border-width: medium 1pt 1pt;
border-style: none solid solid; border-color:
-moz-use-text-color windowtext windowtext; padding: 0in
5.4pt;" valign="top" width="319">
<p class="MsoPlainTextCxSpFirst" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";"># Date
2011-07-29 Time 19:09:58 EDT -0400 (1311980998.46 s)</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">wall clock
time: 2646.75 s</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">�</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">Start
R-work = 0.2891, R-free = 0.3267</span></p>
<p class="MsoPlainTextCxSpLast" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">Final
R-work = 0.2868, R-free = <span style="background:
none repeat scroll 0% 0% yellow;">0.3304</span></span></p>
<p class="MsoNormal" style="margin-bottom: 0.0001pt;
text-align: justify;"><span style="font-size: 12pt;
font-family:
"Arial","sans-serif";">�</span></p>
</td>
<td style="width: 239.4pt; border-width: medium 1pt 1pt
medium; border-style: none solid solid none; border-color:
-moz-use-text-color windowtext windowtext
-moz-use-text-color; padding: 0in 5.4pt;" valign="top"
width="319">
<p class="MsoPlainTextCxSpFirst" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";"># Date
2011-07-29 Time 20:14:20 EDT -0400 (1311984860.16 s)</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">wall clock
time: 6517.60 s</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">�</span></p>
<p class="MsoPlainTextCxSpMiddle" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">Start
R-work = 0.2910, R-free = 0.3273</span></p>
<p class="MsoPlainTextCxSpLast" style="text-align:
justify;"><span style="font-size: 12pt; font-family:
"Arial","sans-serif";">Final
R-work = 0.2720, R-free = <span style="background:
none repeat scroll 0% 0% yellow;">0.3353</span></span></p>
<p class="MsoNormalCxSpFirst" style="margin-bottom:
0.0001pt; text-align: justify;"><span style="font-size:
12pt; font-family:
"Arial","sans-serif";">�</span></p>
</td>
</tr>
<tr style="">
<td style="width: 239.4pt; border-width: medium 1pt 1pt;
border-style: none solid solid; border-color:
-moz-use-text-color windowtext windowtext; padding: 0in
5.4pt;" valign="top" width="319">
<p class="MsoPlainText" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">clashscore
<span style="background: none repeat scroll 0% 0%
yellow;">31.77</span></span></p>
</td>
<td style="width: 239.4pt; border-width: medium 1pt 1pt
medium; border-style: none solid solid none; border-color:
-moz-use-text-color windowtext windowtext
-moz-use-text-color; padding: 0in 5.4pt;" valign="top"
width="319">
<p class="MsoNormalCxSpFirst" style="margin-bottom:
0.0001pt; text-align: justify;"><span style="font-size:
12pt; font-family:
"Arial","sans-serif";">clashscore
<span style="background: none repeat scroll 0% 0%
yellow;">96.12</span></span></p>
</td>
</tr>
<tr style="">
<td style="width: 239.4pt; border-width: medium 1pt 1pt;
border-style: none solid solid; border-color:
-moz-use-text-color windowtext windowtext; padding: 0in
5.4pt;" valign="top" width="319">
<p class="MsoPlainText" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">cbeta <span
style="background: none repeat scroll 0% 0% yellow;">0</span></span></p>
</td>
<td style="width: 239.4pt; border-width: medium 1pt 1pt
medium; border-style: none solid solid none; border-color:
-moz-use-text-color windowtext windowtext
-moz-use-text-color; padding: 0in 5.4pt;" valign="top"
width="319">
<p class="MsoNormalCxSpFirst" style="margin-bottom:
0.0001pt; text-align: justify;"><span style="font-size:
12pt; font-family:
"Arial","sans-serif";">cbeta <span
style="background: none repeat scroll 0% 0% yellow;">151</span></span></p>
</td>
</tr>
<tr style="">
<td style="width: 239.4pt; border-width: medium 1pt 1pt;
border-style: none solid solid; border-color:
-moz-use-text-color windowtext windowtext; padding: 0in
5.4pt; background-color: rgb(255, 255, 255);" valign="top"
width="319">
<p class="MsoPlainText" style="text-align: justify;"><span
style="font-family: "Courier New";
background-image: none; background-repeat: repeat;
background-attachment: scroll; background-position: 0%
0%; -moz-background-clip: border;
-moz-background-origin: padding; -moz-background-size:
auto auto;">rama 12.62</span><span style="font-family:
"Courier New";">% outliers</span><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";"></span></p>
</td>
<td style="width: 239.4pt; border-width: medium 1pt 1pt
medium; border-style: none solid solid none; border-color:
-moz-use-text-color windowtext windowtext
-moz-use-text-color; padding: 0in 5.4pt;" valign="top"
width="319">
<p class="MsoNormalCxSpFirst" style="margin-bottom:
0.0001pt; text-align: justify;"><span style="font-size:
10.5pt; font-family: "Courier New";
background: none repeat scroll 0% 0% rgb(255, 255,
255);">rama 19.67</span><span style="font-size:
10.5pt; font-family: "Courier New";
background-color: rgb(255, 255, 255);">% outliers</span><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";"></span></p>
</td>
</tr>
</tbody>
</table>
<p class="MsoNormalCxSpMiddle" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">�</span></p>
<p class="MsoNormalCxSpMiddle" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">Interestingly in
previous release
version the same refinement runs produced:</span></p>
<p class="MsoNormalCxSpMiddle" style="text-align: justify;"><span
style="font-size: 12pt; font-family:
"Arial","sans-serif";">without H
clashscore = 77.580195/cbeta
=8</span></p>
<span style="font-size: 12pt; font-family:
"Arial","sans-serif";">with H clashscore =
119.729960/cbeta=330</span><br>
</blockquote>
<br>
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