Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Dear Dale, For me, a high quality map is a map which clearly shows where the model is correct and where adjustments are needed, e.g. in places where the search model is different from the structure to be solved and in places where the model is missing. This is not the same as a difference in contour level. The most impressive example I have seen was with proTAFI. Here we had 3 molecules in the asymmetric unit. 2 molecules were well-defined, one was somewhat disordered. The prodomain had 91 residues, the catalytic domain 309. The resolution was 2.5Å (optimistic estimate) and the solvent content was 72%. The structure was solved with phaser using a model of the catalytic domain only (~3/4 of the total). The resulting maps clearly showed positive difference density for the complete 2 well-ordered prodomains (92 residues), which could be fitted in one go. I have never seen something like that with crystals with a lower solvent content (say 50% or less). In these cases we would need several cycles of model building and refinement before the complete missing parts could be fitted. Best regards, Herman -Original Message- From: Dale Tronrud [mailto:det...@uoxray.uoregon.edu] Sent: Tuesday, May 24, 2011 6:33 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio On 5/24/2011 2:35 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off If you choose your contour level based on the map rms (often inappropriately called sigma) the 2Fo-Fc density of a high-solvent-content map will appear stronger even when the absolute quality is the same. All that flat space will cause the overall rms to be low even if the rms calculated over the protein is the same. Dale Tronrud course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Hi Seema, Small addition to the already abundant suggestions, if you have high solvent content or significant portion of non-observable density, you normally get higher R-free. Clement Clement Angkawidjaja, PhD. G30 Assistant Professor --- Chemistry-Biology Combined Major Program International College, Osaka University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax +81-6-6850-5961 --- Laboratory of Molecular Biotechnology Graduate School of Engineering Osaka University 2-1 Yamadaoka U1E-804 Suita, Osaka 565-0871, japan http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html Tel/Fax +81-6-6879-4157
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 10:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi Seema, Small addition to the already abundant suggestions, if you have high solvent content or significant portion of non-observable density, you normally get higher R-free. Clement Clement Angkawidjaja, PhD. G30 Assistant Professor --- Chemistry-Biology Combined Major Program International College, Osaka University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax +81-6-6850-5961 --- Laboratory of Molecular Biotechnology Graduate School of Engineering Osaka University 2-1 Yamadaoka U1E-804 Suita, Osaka 565-0871, japan http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html Tel/Fax +81-6-6879-4157
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
I think you are just confused. The solvent flattening is just a step to make your map clearer. You do not carry the modified phases from solvent flattening to refinement (and I sincerely hope you don't refine against the solvent flattened amplitudes but against the original data!) Herman's observation is right, i think. One of the reasons that high solvent content will give better Rfree is also that this wY you have more reflections than usual per atom: considering two asymmetric units with the same volume, they have the same number of reflections at a given resolution, but if one has higher solvent it has less atoms so you refine better. A. Sent from my iPhone On 24 May 2011, at 11:18, Clement Angkawidjaja clem...@bio.mls.eng.osaka-u.ac.jp wrote: But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
It's also possible that a lower Rfree is the result of reduced overfitting, because increased solvent content pushes up the obs/param ratio, i.e. the unit-cell volume is greater so you get more reflections for a given resolution cutoff, while the no of parameters stays about the same (i.e. there's a greater volume of solvent but you use the same no of parameters to describe it). Of course this assumes you get data to the same resolution with the increased solvent content, which may not be the case if there's increased thermal motion/disorder. Cheers -- Ian On Tue, May 24, 2011 at 10:01 AM, herman.schreu...@sanofi-aventis.comwrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 10:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi Seema, Small addition to the already abundant suggestions, if you have high solvent content or significant portion of non-observable density, you normally get higher R-free. Clement Clement Angkawidjaja, PhD. G30 Assistant Professor --- Chemistry-Biology Combined Major Program International College, Osaka University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax +81-6-6850-5961 --- Laboratory of Molecular Biotechnology Graduate School of Engineering Osaka University 2-1 Yamadaoka U1E-804 Suita, Osaka 565-0871, japan http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html Tel/Fax +81-6-6879-4157
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Dear Herman, You are right. Thank you for the explanation. Clement Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
On 5/24/2011 2:35 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off If you choose your contour level based on the map rms (often inappropriately called sigma) the 2Fo-Fc density of a high-solvent-content map will appear stronger even when the absolute quality is the same. All that flat space will cause the overall rms to be low even if the rms calculated over the protein is the same. Dale Tronrud course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Seema, I agree completely with Eleanor. You need to take a step back
here. When you say that 'Rfree got stuck at 29-30' what makes you so
sure that this isn't the correct Rfree? Who told you that there's a
problem to be solved in the first place?
If you look at our paper (Acta Cryst., 1998. D54, 547-557) you'll see
how to estimate the expected Rfree; then you can compare that with the
Rfree you actually obtained and use that objective information to
decide if any remedial action is necessary. If we take your values,
say Rwork = 0.205, Rfree = 0.295 we can compute the ratio ('y' in the
paper) Rfree/Rwork = 1.44 (note that it's the ratio that matters NOT
the difference!). From this we compute 'z' = (y^2-1)/(y^2+1) = 0.35
(see Fig 2). Now look at the constellation of points (purple squares)
representing structures in the PDB in the resolution range 3.0-2.5
Ang. You''ll see that your z value 0.35 falls pretty well in the
middle of those. To pinpoint the exact value of z (hence y, and hence
Rfree) that you would expect to get, you would need make use of the
ratio no of atoms/no of reflections.
You can also see that, depending on the resolution and the obs/param
ratio, the z value could go as low as 0.1 or indeed as high as 0.5.
The latter corresponds to y = 1.73, which for Rwork = 0.20 corresponds
to expected Rfree = 0.35 (in other words an Rfree much less than this
would lead one to conclude there's something wrong); it all depends on
the resolution and the observation/parameter ratio.
So I really don't think you have anything to worry about (except maybe
referees who don't understand the statistics of cross-validation!).
As others have pointed out, you are likely to do more damage to your
structure by cutting out perfectly good data in a vain attempt to
solve a non-existent problem!
Cheers
-- Ian
On Mon, May 23, 2011 at 9:54 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote:
I dont think there is an Rfree problem..
At 2.7A you expect quite a big difference between R and Rfree
Reducing the resolution will a) probably makethe Rfree/R difference greater,
and b) degrade the quality of your maps and model.
Eleanor
On 05/21/2011 02:28 AM, Seema Mittal wrote:
Hi Ethan,
You are absolutely right. As a matter of fact, I had initially processed
the data to 2.7A and it looked pretty decent with R symm less than 10%.
The maps looked good too.
The problem arose during second round of refinement. The Rfree got stuck
at around 29-30 while the Rfactor kept decreasing to about 20-21. The
bond length and angle values are fine too.
I cut down the resolution to 3A hoping to improve the data quality by
removing some noise. But, it did not work. i also tried to put restrains
on the backbone B factors with limited success.
Any thoughts on how i can resolve this Rfree issue?
Thanks much,
Seema
On May 20, 2011, at 5:38 PM, Ethan Merritt wrote:
On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote:
Hi All,
I am currently working on a 3A resolution dataset. The scaled file
shows the following statistics (scroll down to the end of this
email). It is P212121 space group with R merge of 8.8%.
Your data statistics look fine. In fact, it looks to me that your
crystal is
probably yielding good data to considerably better resolution than 3A.
Why did you choose to cut it there?
My question is : Is there a way to selectively use only the data with
I/Sigma value of 2 and more for refinement?
That is a bad idea. By removing data you are throwing away information.
Noisy data is still better than no data.
good luck with your [probably better than 3A] structure,
Ethan
And how do i achieve this using refmac? I am aware that this would
come at the cost of compromising data completeness. Any
suggestions/help would be greatly appreciated.
Thanks much,
Seema Mittal
Department of Biochemistry Molecular Pharmacology
970L Lazare Research Building
University of Massachusetts Medical School
364 Plantation Street
Worcester, MA 01605
Shell I/Sigma in resolution shells:
Lower Upper % of of reflections with I / Sigma less than
limit limit 0 1 2 3 5 10 20 20 total
50.00 6.46 2.0 3.8 5.3 6.2 7.6 12.5 34.3 65.0 99.3
6.46 5.13 0.7 2.2 3.9 5.3 8.2 15.7 36.6 63.4 100.0
5.13 4.48 1.3 2.8 4.0 5.8 9.3 13.8 27.3 72.7 100.0
4.48 4.07 0.7 1.7 4.0 5.4 7.9 13.9 35.4 64.1 99.5
4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9
3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2
3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6
3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5
3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9
3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4 4.5 96.9
All hkl 1.7 4.3 7.1 9.8 15.3 28.0 58.1 39.9 98.0
Shell Lower Upper Average Average Norm. Linear Square
limit Angstrom I error stat. Chi**2 R-fac R-fac
50.00 6.46 511.7 20.0 8.8 1.098 0.065 0.073
6.46 5.13 284.6 10.1 6.3 1.047 0.062 0.064
5.13 4.48 500.9 17.0 8.8 1.007 0.062 0.069
4.48 4.07
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Thank you all for your thoughts and suggestions in response to my query. The rotamers, peptide omega angles, mol-probity data all are perfectly fine. There are 3 outliers in Ramachandran plot ( may be the ones causing the problem). The protein has two engineered cysteines involved in disulfide bond formation which restrict the conformation around the disulfide bond. One of these cys residues is followed by pro which seems to be constrained a bit. Nevertheless, the overall geometry is good. There certainly are a couple of big positive density peaks which i haven't been able to account for yet. The usual suspects from my crystallization conditions do not seem to fit in those blobs. I shall go back to 2.7A resolution and continue to try to resolve these positive peaks. Thanks for your suggestions. Any other thoughts on the matter would be greatly appreciated. Thanks much, Best, Seema On May 20, 2011, at 10:41 PM, Ethan Merritt wrote: On Friday, 20 May 2011, you wrote: Hi Ethan, You are absolutely right. As a matter of fact, I had initially processed the data to 2.7A and it looked pretty decent with R symm less than 10%. The maps looked good too. The problem arose during second round of refinement. The Rfree got stuck at around 29-30 while the Rfactor kept decreasing to about 20-21. That can simply mean there are many little things wrong with the structure - rotamers, phi/psi, perhaps even a register shift. I suggest that you use the 2.7A data, so that there is more local information to drive the refinement, and take seriously any hints that Molprobity provides about bad local geometry. Another thought - are there any large positive peaks in the difference density? Perhaps you are missing a solvent ion or something of that sort. Even one missing sulfate could make a noticeable difference in Rfree. regards, Ethan The bond length and angle values are fine too. I cut down the resolution to 3A hoping to improve the data quality by removing some noise. But, it did not work. i also tried to put restrains on the backbone B factors with limited success. Any thoughts on how i can resolve this Rfree issue? Thanks much, Seema On May 20, 2011, at 5:38 PM, Ethan Merritt wrote: On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4 4.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
I would also make sure that the spacegroup is correct. If you have instead P222, P2212, etc, you might find the solution at low resolution but the problem would become evident during advanced refinement steps, such as a stuck high Rfree or a noisy difference map. Good luck, Javier. On Sat, May 21, 2011 at 4:44 PM, Seema Mittal seema.mit...@umassmed.eduwrote: Thank you all for your thoughts and suggestions in response to my query. The rotamers, peptide omega angles, mol-probity data all are perfectly fine. There are 3 outliers in Ramachandran plot ( may be the ones causing the problem). The protein has two engineered cysteines involved in disulfide bond formation which restrict the conformation around the disulfide bond. One of these cys residues is followed by pro which seems to be constrained a bit. Nevertheless, the overall geometry is good. There certainly are a couple of big positive density peaks which i haven't been able to account for yet. The usual suspects from my crystallization conditions do not seem to fit in those blobs. I shall go back to 2.7A resolution and continue to try to resolve these positive peaks. Thanks for your suggestions. Any other thoughts on the matter would be greatly appreciated. Thanks much, Best, Seema On May 20, 2011, at 10:41 PM, Ethan Merritt wrote: On Friday, 20 May 2011, you wrote: Hi Ethan, You are absolutely right. As a matter of fact, I had initially processed the data to 2.7A and it looked pretty decent with R symm less than 10%. The maps looked good too. The problem arose during second round of refinement. The Rfree got stuck at around 29-30 while the Rfactor kept decreasing to about 20-21. That can simply mean there are many little things wrong with the structure - rotamers, phi/psi, perhaps even a register shift. I suggest that you use the 2.7A data, so that there is more local information to drive the refinement, and take seriously any hints that Molprobity provides about bad local geometry. Another thought - are there any large positive peaks in the difference density? Perhaps you are missing a solvent ion or something of that sort. Even one missing sulfate could make a noticeable difference in Rfree. regards, Ethan The bond length and angle values are fine too. I cut down the resolution to 3A hoping to improve the data quality by removing some noise. But, it did not work. i also tried to put restrains on the backbone B factors with limited success. Any thoughts on how i can resolve this Rfree issue? Thanks much, Seema On May 20, 2011, at 5:38 PM, Ethan Merritt wrote: On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4 4.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2
[ccp4bb] how to remove part of data with bad signal to noise ratio
Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 20 20 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Hi Ethan, You are absolutely right. As a matter of fact, I had initially processed the data to 2.7A and it looked pretty decent with R symm less than 10%. The maps looked good too. The problem arose during second round of refinement. The Rfree got stuck at around 29-30 while the Rfactor kept decreasing to about 20-21. The bond length and angle values are fine too. I cut down the resolution to 3A hoping to improve the data quality by removing some noise. But, it did not work. i also tried to put restrains on the backbone B factors with limited success. Any thoughts on how i can resolve this Rfree issue? Thanks much, Seema On May 20, 2011, at 5:38 PM, Ethan Merritt wrote: On Friday, May 20, 2011 02:28:26 pm Mittal, Seema wrote: Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. Your data statistics look fine. In fact, it looks to me that your crystal is probably yielding good data to considerably better resolution than 3A. Why did you choose to cut it there? My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? That is a bad idea. By removing data you are throwing away information. Noisy data is still better than no data. good luck with your [probably better than 3A] structure, Ethan And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 2020 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.4 4.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
The only thing you can do is to move the detector in and collect higher resolution. Then you can determine what the I/sigI ratio is for each of your resolution shells and manually set the resolution limit once you run HKL2000. As far as the R/Rfree values go, you will need to manually set the weighting factor if you are using refmac. I have found that during the initial rigid body refinement and the first few rounds of restrained refinement, it works well if you keep the weighting factor low (0.01-0.9), then as you go through a few rounds, gradually increase the weighting factor. But keep in mind that as you increase the weighting factor, the difference between R/Rfree will also increase. -- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 -- -- From: Mittal, Seema seema.mit...@umassmed.edu Sent: Friday, May 20, 2011 5:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi All, I am currently working on a 3A resolution dataset. The scaled file shows the following statistics (scroll down to the end of this email). It is P212121 space group with R merge of 8.8%. My question is : Is there a way to selectively use only the data with I/Sigma value of 2 and more for refinement? And how do i achieve this using refmac? I am aware that this would come at the cost of compromising data completeness. Any suggestions/help would be greatly appreciated. Thanks much, Seema Mittal Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 Shell I/Sigma in resolution shells: LowerUpper % of of reflections with I / Sigma less than limit limit 0 1 2 3 5 10 20 20 total 50.00 6.46 2.0 3.8 5.3 6.27.6 12.5 34.3 65.0 99.3 6.46 5.13 0.7 2.2 3.9 5.38.2 15.7 36.6 63.4 100.0 5.13 4.48 1.3 2.8 4.0 5.89.3 13.8 27.3 72.7 100.0 4.48 4.07 0.7 1.7 4.0 5.47.9 13.9 35.4 64.1 99.5 4.07 3.78 1.8 3.6 5.1 6.9 11.8 20.8 49.6 47.3 96.9 3.78 3.56 1.5 3.8 6.7 8.7 13.3 26.7 65.4 30.8 96.2 3.56 3.38 0.8 3.2 7.1 8.9 12.9 31.1 76.6 20.0 96.6 3.38 3.23 2.0 4.8 8.1 14.8 23.4 44.8 84.7 12.7 97.5 3.23 3.11 4.1 9.2 13.8 18.4 29.6 51.0 86.0 11.0 96.9 3.11 3.00 2.4 8.6 13.9 18.8 30.6 53.9 92.44.596.9 All hkl1.7 4.3 7.19.8 15.3 28.0 58.1 39.9 98.0 Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 6.46 511.720.0 8.8 1.098 0.065 0.073 6.46 5.13 284.610.1 6.3 1.047 0.062 0.064 5.13 4.48 500.917.0 8.8 1.007 0.062 0.069 4.48 4.07 446.117.4 9.2 1.032 0.069 0.070 4.07 3.78 307.114.5 8.4 1.065 0.089 0.092 3.78 3.56 243.413.8 7.9 1.033 0.108 0.112 3.56 3.38 182.312.0 8.3 1.083 0.132 0.134 3.38 3.23 136.510.4 7.7 1.048 0.155 0.151 3.23 3.11 107.4 9.2 7.3 1.096 0.184 0.163 3.11 3.0091.0 8.7 7.3 1.0440.215 0.201 All reflections287.713.5 8.0 1.055 0.088 0.082
