Re: [ccp4bb] Picking water molecules at 4A structure.
Dear Bert That is a limitation, I agree. Suffice to say the clarity of details seen, or not seen, will not get better in the 'real' situation. The resolution 'limit' based on CC 1/2 also now needs to be considered (in addition to I/sigI criterion). John On 17 Apr 2015, at 17:59, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: John, the lower-resolution datasets in your paper were generated by truncating a high-res dataset, i.e. the lo-res datasets are of great quality. Would the conclusions still be valid if the data are true low-res? (i.e. I/sigI 1.5-2 in last shell)? Tx Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of John R Helliwell [jrhelliw...@gmail.com] Sent: Friday, April 17, 2015 5:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Picking water molecules at 4A structure. Hi, This paper:- doi:10.1107/S0907444903004219 I think will be of interest. Whilst 4 Angstrom resolution is not covered the article will indicate the tests you could make to evaluate your 'possible water like densities'. Best wishes, John On Mon, Apr 13, 2015 at 7:14 PM, Sudipta Bhattacharyya sudiptabhattacharyya.iit...@gmail.com wrote: Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta. -- Professor John R Helliwell DSc
Re: [ccp4bb] Picking water molecules at 4A structure.
Good morning Pavel, That's interesting. In our study 'ghosts' of waters in our truncated maps did not occur. Waters and hydrogens behave differently as ghost objects presumably? Greetings, John On 17 Apr 2015, at 20:10, Pavel Afonine pafon...@gmail.com wrote: Hello, John, the lower-resolution datasets in your paper were generated by truncating a high-res dataset, i.e. the lo-res datasets are of great quality. Would the conclusions still be valid if the data are true low-res? (i.e. I/sigI 1.5-2 in last shell)? genuinely low-res data set is clearly not the same as one obtained by truncation of high-res reflections. Some time ago I did a test where I truncated an ultra-high resolution data set (0.6A resolution) at 2A, and I could still see H atoms in 2A resolution map! Pavel
Re: [ccp4bb] Picking water molecules at 4A structure.
If you are seeing waters at 4 A you are probably looking at ions. Those are OK to model, but definitely try occupancy refinement from a variety of starting points to make sure you are not fooling yourself. Occupancy refinement might also help you assign which ion it is. That, and some prior knowledge about what ions are in your buffers. As for random noise, if you re-collect the data and re-refine your model, and find that the feature is still there, then it is NOT random noise. Random noise (by definition) changes every time you measure something. As for Fourier ripples, there is an easy check for this: 1) calculate structure factors from your refined model to 1.0 A or so (even if you have 4A data). 2) flag all your observed hkls, set the FC of those hkls to zero (use SFTOOLS for this). 3) use the remaining FC values to calculate an electron density map. This map will be the difference between a perfect map (essentially no missing reflections), and one that has the same missing Fourier terms as your observed map. This is the best estimation you can make of where you expect to see Fourier ripples, given your particular model and resolution cutoff. You will find that the ripples are generally very very small. Unless your observations cut out way too much good data. If you look at a calculated map at 4A with waters in it, such as found in my movie here: http://bl831.als.lbl.gov/~jamesh/movies/ You will find then you will find that the waters disappear below 1 sigma at around 2.8A resolution. It doesn't mean they don't exist, they just drop below the 1 sigma contour level. If you lower the contour you will see them again. At what point does lowering the contour level get you to noise peaks? Well, that's around -1.0 sigma. Yes, negative sigmas. Everything above that is real (Lang et al. 2014 http://dx.doi.org/10.1073/pnas.1302823110). Just difficult to build into. -James Holton MAD Scientist On 4/13/2015 12:12 PM, Phoebe A. Rice wrote: At 4A, I wouldn't unless I had an exceptionally good reason to. There will always be some blobs, due to random noise and fourier ripples as well as due to an imperfect model. Unless a blob makes nice H-bonds to something else that is nicely ordered, I wouldn't model at water into it. If you can't see nice density for side chains then you probably aren't really seeing density for waters either. ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago pr...@uchicago.edu mailto:pr...@uchicago.edu *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sudipta Bhattacharyya [sudiptabhattacharyya.iit...@gmail.com] *Sent:* Monday, April 13, 2015 1:14 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Picking water molecules at 4A structure. Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta.
Re: [ccp4bb] Picking water molecules at 4A structure.
John, the lower-resolution datasets in your paper were generated by truncating a high-res dataset, i.e. the lo-res datasets are of great quality. Would the conclusions still be valid if the data are true low-res? (i.e. I/sigI 1.5-2 in last shell)? Tx Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of John R Helliwell [jrhelliw...@gmail.com] Sent: Friday, April 17, 2015 5:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Picking water molecules at 4A structure. Hi, This paper:- doi:10.1107/S0907444903004219http://dx.doi.org/10.1107/S0907444903004219 I think will be of interest. Whilst 4 Angstrom resolution is not covered the article will indicate the tests you could make to evaluate your 'possible water like densities'. Best wishes, John On Mon, Apr 13, 2015 at 7:14 PM, Sudipta Bhattacharyya sudiptabhattacharyya.iit...@gmail.commailto:sudiptabhattacharyya.iit...@gmail.com wrote: Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta. -- Professor John R Helliwell DSc
Re: [ccp4bb] Picking water molecules at 4A structure.
Hi, This paper:- doi:10.1107/S0907444903004219 http://dx.doi.org/10.1107/S0907444903004219 I think will be of interest. Whilst 4 Angstrom resolution is not covered the article will indicate the tests you could make to evaluate your 'possible water like densities'. Best wishes, John On Mon, Apr 13, 2015 at 7:14 PM, Sudipta Bhattacharyya sudiptabhattacharyya.iit...@gmail.com wrote: Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta. -- Professor John R Helliwell DSc
Re: [ccp4bb] Picking water molecules at 4A structure.
Hello, John, the lower-resolution datasets in your paper were generated by truncating a high-res dataset, i.e. the lo-res datasets are of great quality. Would the conclusions still be valid if the data are true low-res? (i.e. I/sigI 1.5-2 in last shell)? genuinely low-res data set is clearly not the same as one obtained by truncation of high-res reflections. Some time ago I did a test where I truncated an ultra-high resolution data set (0.6A resolution) at 2A, and I could still see H atoms in 2A resolution map! Pavel
Re: [ccp4bb] Picking water molecules at 4A structure.
Dear all, I thank you all for your kind suggestions and remarks. So the bottom line appeared to me is - one should not pick water molecules at low resolution (grater than 3.0/3.5A) data (not a truncated data I guess) unless there is sufficient reasons/evidences (like presence of water molecules attached to a electron dense molecule such as metal) supporting its presence. Please correct me, if I have wrongly concluded. Regards, Sudipta. On Fri, Apr 17, 2015 at 1:10 PM, Pavel Afonine pafon...@gmail.com wrote: Hello, John, the lower-resolution datasets in your paper were generated by truncating a high-res dataset, i.e. the lo-res datasets are of great quality. Would the conclusions still be valid if the data are true low-res? (i.e. I/sigI 1.5-2 in last shell)? genuinely low-res data set is clearly not the same as one obtained by truncation of high-res reflections. Some time ago I did a test where I truncated an ultra-high resolution data set (0.6A resolution) at 2A, and I could still see H atoms in 2A resolution map! Pavel
[ccp4bb] Picking water molecules at 4A structure.
Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta.
Re: [ccp4bb] Picking water molecules at 4A structure.
At 4A, I wouldn't unless I had an exceptionally good reason to. There will always be some blobs, due to random noise and fourier ripples as well as due to an imperfect model. Unless a blob makes nice H-bonds to something else that is nicely ordered, I wouldn't model at water into it. If you can't see nice density for side chains then you probably aren't really seeing density for waters either. ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago pr...@uchicago.edumailto:pr...@uchicago.edu From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sudipta Bhattacharyya [sudiptabhattacharyya.iit...@gmail.com] Sent: Monday, April 13, 2015 1:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Picking water molecules at 4A structure. Dear community, Recently we have been able to solve a crystal structure of a DNA/protein complex at 4A resolution. After almost the final cycles of model building and refinement (with R/Rfree of ~ 22/27) we could see some small water like densities...all throughout the complex. Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Many thanks in advance...!!! My best regards, Sudipta.
Re: [ccp4bb] Picking water molecules at 4A structure.
Now my query is, whether one should pick water molecules at this low resolutions or it is totally unscientific to do so? Your question is justified in intent, but ill phrased. The question you are faced with is “How plausible would the assignment of a given electron density reconstruction feature as a water molecule be?” The answer depends on observational evidence and chemical plausibility. You have the most knowledge about your protein complex and should have some knowledge about chemical plausibility of your proposal. (a)A few questions to consider re. evidence: What is the noise level in your map? How do normal 2Fo-Fc densities compare to difference densities? Density shape? Other isolated mystery density of same levels somewhere? If your maps are excellent and low noise it is not impossible to see a very well bound water molecule at 4A. (b) Plausibility based on prior expectations: Was Mg in the cocktail? Being isoelectronic with HOH (and a favorite companion of DNA in crystallization), it might be a plausible candidate. Anything else heavier, perhaps? SO4, PO4? Perhaps any clues from anomalous data/ano diff maps? Fragments of PEGs? What does the refinement tell you? How did you refine? Does your protocol match the low resolution of the data? Even at the low resolution, do bond length and coordination support a discrete moiety? Distances, geometry, B-factors? If everything points in your favor, you can justify the proposition of a discrete moiety. Your scientific credibility depends on how well your proposition is supported by reasoning from (a) and (b) – probably with heavy emphasis on (b) as you are poorly determined – and not whether you are ultimately right or not. Although I doubt that a water molecule without biological relevance assigned to it has any effect on global refinement stats nor on your career, you can always invoke the rule of parsimony for your model – no explanation is better than an unsupported one. “I don’t know” is a perfectly scientific answer. LGBR
