[ccp4bb] Is it possible to install stereo view in labtop or desktop?
Dear Everyone, I have to work at home because of the budget cut. However, I still want to do some research at this difficult time. I have been used to viewing the crystal structures with stereo view with program O. I want to find out is it possible to install the stereo option in my labtop. If possible, how to install and what facilities I have to buy. Must I have installed the Linux operation system in my labtop first? If not possible for labtop, how about a desktop? Is the program O better than COOT? Thank you very much for your suggestions. Best wishes, Sun
Re: [ccp4bb] Why NCS doesn't help?
Dear Eleanor, Thank you very much for your reply. I think that the NCS operator is very close to the crystallographic two fold symmetry because when I searched for the solution with PHASER with one molecule as the model I could not find the solution so I have to use two molecules as the search model but I did not expect that will affect the NCS refinement. How to deal with this problem? Thank you very much for your great help! I also greatly appreciated all the replies to my question which help me clarify a lot. Thank you All! Best wishes, Sun From: Eleanor Dodson c...@ysbl.york.ac.uk To: Sun Tang suntang2...@yahoo.com Cc: CCP4BB@jiscmail.ac.uk Sent: Wednesday, July 8, 2009 10:06:41 AM Subject: Re: [ccp4bb] Why NCS doesn't help? There are many reasons why this could have ocurred. One is that your NCS operator is very close to being a crystallographic one. But you need to give more details of the problem before I can comment sensibly Eleanor Sun Tang wrote: Dear All, In refining my structure (two molecules in au) that was solved with molecular replacement, refinement starting with restrained lowered the R-free to about 0.35 while with NCS increased the R-free to 0.55. Does it imply that two molecules are quite different or something wrong with the refinement? Thank you very much for your suggestions? Best wishes, Sun Tang
[ccp4bb] Why NCS doesn't help?
Dear All, In refining my structure (two molecules in au) that was solved with molecular replacement, refinement starting with restrained lowered the R-free to about 0.35 while with NCS increased the R-free to 0.55. Does it imply that two molecules are quite different or something wrong with the refinement? Thank you very much for your suggestions? Best wishes, Sun Tang
Re: [ccp4bb] How to refine a solution obtained by molecular replacement
Dear All, Thank you very much for all your suggestions. They are very helpful in my further refinements. I am adding some more information about the problem: 1) The Z-score is 11.2 and LLG is 125. 2) The model has 390 aa while my structure has about 440 aa. Please let me know of any further suggestions. I will try all your suggestions and let you know the updated information about the refinement. Best regards, Sun From: Ho-Leung Ng hole...@berkeley.edu To: CCP4 bulletin board CCP4BB@jiscmail.ac.uk; suntang2...@yahoo.com Sent: Wednesday, March 18, 2009 12:10:29 AM Subject: Re: How to refine a solution obtained by molecular replacement I've found CNS's simulated annealing composite omit maps to be very useful in situations like this to avoid phase bias. RESOLVE's prime and switch offers similar functionality, but I've had less experience with it. ho UC Berkeley
Re: [ccp4bb] question about getting rid of model bias in refinement
Hello Eleanor, Thank you very much for your reply. I don't know whether or not the conformation is model biased or not. I just want to make sure the conformation is model free of model bias because these residues are important. The refinement moved the residue back may indicate the previous conformation is correct. I want to know whether there are any other methods to cross-verify the result. Thank you very much for your opinions. Best, Sun --- On Thu, 7/31/08, Eleanor Dodson [EMAIL PROTECTED] wrote: From: Eleanor Dodson [EMAIL PROTECTED] Subject: Re: [ccp4bb] question about getting rid of model bias in refinement To: [EMAIL PROTECTED] Date: Thursday, July 31, 2008, 11:41 AM Sun Tang wrote: Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun This seems rather strange! The bias would usually disappear especially if you have a) set occs to 0.0 for selected residues, b) done several cycles of refinement. Could there be 2 conformations for these residues? One where it was originally and one where you have built them? Eleanor
Re: [ccp4bb] question about getting rid of model bias in refinement
Hello Charlie, Thank you very much for your comments. I mostly agree with you. However, as far as I know most of the complexes structures are solved with MR with the their apo-enzyme as search model and refined the structures with CCP4 or CNS. I tried the simulated annealing omitting the residue and 4 neighboring residues on each side and I found the conformation are essentially the same. I also tried to use composite omit-map calculation in CSN but I gave it because it took several days of computer time but only finished only 1/4 of the calculation. I understand the starting from the beginning is one choice. I wonder whether there are other easier ways in CCP4 to deal with this situation because this problem is quite common in refinement. I appreciate all the replies to my questions and I say Thank you very much here. Best, Sun --- On Sat, 7/26/08, Charles W. Carter Jr. [EMAIL PROTECTED] wrote: From: Charles W. Carter Jr. [EMAIL PROTECTED] Subject: Re: [ccp4bb] question about getting rid of model bias in refinement To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, July 26, 2008, 3:15 PM Sun, I'm most of the way to one side of this debate: I believe that it is not possible to emerge fully from model bias without avoiding it in the first place with experimental phases. I may be overly pessimistic, but have considerable experience supporting at least skepticism. My interpretation of the experimental result you describe is that the covariances among the parts of the structure you left in place and those side chains you omitted is so strong and extensive that you'll never see the correct density coming back upon refinement, because other parts of the structure are ever so slightly off their true mean positions to compensate for the (evidently false) positions of the residues you omitted. Bill's suggestion that you actually refine the structure using simulated annealing without the omitted residues is an improvement over what you did, but it will require many cycles to get a much better approximation, and there is really no way to be sure when you can be confident. Starting the entire refinement over is a more aggressive strategy. If you decide to try this, you should examine the projection of the residue by residue real-space correlation coefficients across the entire sequence to ensure that you have only one population of values and delete all residues that comprise any population that has a distinctly different real-space correlation coefficient, building them back into the structure as it refines. That is, you should ensure that you don't begin refining any residues at the very beginning for which there is evidence that they might be different from their positions in your molecular replacement model. Charlie On Jul 26, 2008, at 2:12 PM, William G. Scott wrote: Hi Sun: It might be worth doing a simulated annealing omit refinement in phenix or CNS, with the residues in question omitted. CNS also allows you to make a composite-omit map. I haven't seen that in phenix yet but presumably it is doable. Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 25, 2008, at 10:53 PM, Sun Tang wrote: Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun **UNCrystallographers NOTE new website url** [EMAIL PROTECTED] http://xtal.med.unc.edu/CARTER/Welcome.html Department of Biochemistry and Biophysics CB 7260 UNC Chapel Hill, Chapel Hill, NC 27599-7260 Tel: 919 966-3263 FAX 919 966-2852
[ccp4bb] question about getting rid of model bias in refinement
Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun
[ccp4bb] How to generate two molecules of proteins and two chains of RNA in CNS 1.2
Dear All, When I used CNS 1.2 to generate the pdb file for two molecules of proteins and two chains of RNA (with different chain ids in format of CCP4), only one RNA chain is generated. Can anyone tell me how to correctly generate the files used for refinement with CNS 1.2? I downloaded the input files from the CNS website and I tried true and false for the renaming segid but all did not work. Thank you very much! Best, Sun Tang - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
[ccp4bb] anomalous signal of Mn and Ca ions
Dear All, In my structures, I want to assign Mn or Ca ions for some densities. But when I did not have anomalous density in CCP4i. I am not sure whether I was correct. The following was what I did: I processed the data with HKL2000 and select anomalous signal in scaling. In CCP4i, I selected Run FFT-Creat Map in the Map Mask Utilities. I select O format to cover asymmetric unit and Plot section on Z axis from 0 to 1 in steps on 10. All others were by default values. I display in ono10. I collected the data at the wavelength of 1 A. Do I need to adjust the wavelength to maximize the anomalous signal from Mn or Ca? Any ideas and suggestions are greatly appreciated! Sun Tang - Never miss a thing. Make Yahoo your homepage.
[ccp4bb] Check the conformation of one important amino acid
Dear All experts, I want to check the conformation of one important amino acid in the structure by looking the difference density map. Should I just omit that amino acid in the refinement or should I also omit its flanking amino acids? Which one is better? Thank you very much for your suggestions! Best wishes, Sun Tang - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
Re: [ccp4bb] an over refined structure
Hi Tim, Thank you for your and information and suggestions. There are two indepdent molecules in the asymmetric unit and one molecule does not have very good density, especially in the N-terminus. Do you think that I should remove the region in the refinement? Best, Sun Tim Gruene [EMAIL PROTECTED] wrote: I would agree that the difference is suspiciously high. I. Tickle and others have published analytical expressions for how to estimate the ratio between R and Rfree, just google for tickle rfree to find the references. You easily achieve a large difference by adding too many waters which just model noise. There may be other reasons for which more knowledge about the structure is required. Do you have large unmodelled regions, like loops that do not show in the density map? Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 4 Feb 2008, Sun Tang wrote: Hello All, I refined a structure with Refmac in CCP4i and the R/Rfree is 0.215/0.277. The difference between R and Rfree is too much even though I used 0.01 for weighting term in the refinement (the default value is 0.3). The RMSD for bond length and bond angle is 0.016 A and 1.7 degree. What may be wrong with the over-refined structure? What is the reason for leading to an over-refined structure? How to avoid it? Best wishes, Sun Tang - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
[ccp4bb] an over refined structure
Hello All, I refined a structure with Refmac in CCP4i and the R/Rfree is 0.215/0.277. The difference between R and Rfree is too much even though I used 0.01 for weighting term in the refinement (the default value is 0.3). The RMSD for bond length and bond angle is 0.016 A and 1.7 degree. What may be wrong with the over-refined structure? What is the reason for leading to an over-refined structure? How to avoid it? Best wishes, Sun Tang - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
Re: [ccp4bb] an over refined structure
Hi Ian, Thank you very much for your detailed information. I checked the effect of weighter term (wa) in CCP4i for the R/Rfree. When I used wa=0.01 , the value is 0.225/0.277 FOM =0.799. The values changed to 0.204/0.269 (FOM=0.806) for wa= 0.05, 0.195/0.268 (FOM=0.807) for wa=0.1 and 0.186/0.267 (FOM=0.807) for wa=0.2, respectively. It seemed that increase in wa decreases both R and Rfree with R more than Rfree. Which wa value is the best one in this case? Thank you very much for your valuable help. Best, Sun Ian Tickle [EMAIL PROTECTED] wrote: Hi Sun Tang Unfortunately there's no such thing as a fixed value for the maximum acceptable Rfree-Rwork difference that applies in all circumstances, because the 'normal' difference depends on a number of factors, mainly the observation/parameter ratio, which depends in turn on the resolution and the solvent content (a greater solvent content means a bigger cell volume which means more reflections for a given number of ordered atoms in the a.u. and hence a bigger obs/param ratio). The Rfree-Rwork difference also depends on Rwork itself (i.e. you tend to get higher values of Rfree-Rwork for higher values of Rwork), so it's better to think in terms of the Rfree/Rwork ratio (which is independent of Rwork). So for example at very high resolution a 'normal' value for Rfree-Rwork might be only 0.02 (so 0.05 which is what many people consider acceptable would actually be unacceptably high), whereas at low resolution it might be 0.1 (so 0.05 would be unacceptably low). Also you need to bear in mind that Rfree tends to have a quite high uncertainty, particularly at low resolution (because it's usually based on a relatively small number of observations), so the deviation has to be quite big (e.g. 3 SU) before it can be considered to be statistically significant. So Rfree needs to be compared not with Rwork at all but with the value of the optimal Rfree/Rwork expected on the basis that the model parameterisation and weighting of X-ray terms and restraints are optimal and the errors in the model have the same effect as the random experimental errors in the data (i.e. a statistical 'null hypothesis'). As Tim just pointed out we tried to do this in our Acta D (1998) papers: there you can compare your observed Rfree/Rwork ratio either with the theoretical value or with the value found for 'typical' structures in the PDB at the same resolution. An abnormal Rfree/Rwork ratio could arise from a number of causes, not just over-fitting (I assume that's what you mean by 'over-refinement' - it's not clear to me how a structure can be 'over-refined' since a fundamental requirement of the maximum likelihood method is that the structure is always refined to convergence, and refining beyond that will by definition produce no further statistically significant changes in the parameters). For example the number of parameters being refined may be either too low, or too high (over-fitting), or the values of the weighting parameters may not be appropriate, or there may be something badly wrong with the atomic model (e.g. mistraced chain). Given the values you are reporting I think the latter is very unlikely, possibly you just need to tweak the X-ray and/or restraint weights. HTH Cheers -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Sun Tang Sent: 04 February 2008 16:56 To: Boaz Shaanan Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] an over refined structure Hi Boaz, Thank you for your opinions. The resolution is 2.8A and I remembered some people may think the structure is over-refined when the difference between Rfree/Rwork is greater than 6. What do you think the greatest acceptable difference between the two? Best, Sun Boaz Shaanan wrote: Hi, Why do you think this structure is over-refined ? The Rfree/Rwork difference of 6.2% seems fine, although you didn't mention resolution. If anything, an over-refined structure would show a smaller difference, as far as I know. If all the other criteria (Ramachandran outliers, etc., map) are OK you should just be happy with your structure. Cheers, Boaz - Original Message - From: Sun Tang Date: Monday, February 4, 2008 18:41 Subject: [ccp4bb] an over refined structure To: CCP4BB@JISCMAIL.AC.UK Hello All, I refined a structure with Refmac in CCP4i and the R/Rfree is 0.215/0.277. The difference between R and Rfree is too much even though I used 0.01 for weighting term in the refinement (the default value is 0.3). The RMSD for bond length and bond angle is 0.016 A and 1.7 degree. What may be wrong with the over-refined structure? What is the reason for leading to an over-refined structure? How to avoid it? Best wishes, Sun Tang
Re: [ccp4bb] an over refined structure
Hi Anastassis, Thank you very much for your suggestions. I answered the questions as follows. I used NCS before rigid body refinement. After that I did not put NCS restraints in the restrained refinement and TLS+restrained refinement because it raised the R/Rfree quite a lot. The resolution is 2.8 A. I did not check twinning. I will do that soon. I used PHASER to solve the structure and the density of the N-domain (~ 50 a.a) in one molecule is not good, with a lot of broken density for the backbone. I used the TLS in the refinement. I usually used the initial TLS parameters (with only residues in group, no coordinates for the center) for all the TLS refinement. When I used the refined TLS parameters, the refinement would go divergence. I only added about 120 water molecules for the whole structures. I will update the information after I try further refinement. Best wishes, Sun Anastassis Perrakis [EMAIL PROTECTED] wrote: Hi - I don't think there is something necessarily wrong with the values you report. A few questions to see *if* something is wrong are: - as you wrote to Tim you have NCS: do you use NCS restraints ? - what is the resolution / B factor of the data ? - have the data been checked for twining ? (phenix.xtriage) - is the N-term domain of one copy really invisible (then indeed do remove ...!) - has TLS been used ? - did you add waters ? (too many?) I guess then we can make better suggestions if something is wrong and if so how its best to fix. A. I refined a structure with Refmac in CCP4i and the R/Rfree is 0.215/0.277. The difference between R and Rfree is too much even though I used 0.01 for weighting term in the refinement (the default value is 0.3). The RMSD for bond length and bond angle is 0.016 A and 1.7 degree. - Looking for last minute shopping deals? Find them fast with Yahoo! Search.
Re: [ccp4bb] Why there are difference density when occupancy is 1.00
Hi James, I did check teh B-factors and they are similar to the flanking regions (about 40). The difference density appeared at the later stage of refinement (TLS and restrained in CCP4i). What do you think and how to do it? Best, Sun Tang James Irving [EMAIL PROTECTED] wrote: Hi Sun, I suggest checking that the b-factors for those residues aren't unexpectedly high compared to those in the flankinh regions. Cheers, James On 1/23/08, Sun Tang wrote: Hello Everyone, When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? Thank you very much for your opinions and suggestions! Best wishes, Sun Tang - Never miss a thing. Make Yahoo your homepage. -- Sent from Gmail for mobile | mobile.google.com Dr. James Irving NHMRC C.J. Martin Fellow School of Biomedical Sciences Building 13D Monash University Wellington Road Melbourne 3800 Australia - Never miss a thing. Make Yahoo your homepage.
[ccp4bb] Why there are difference density when occupancy is 1.00
Hello Everyone, When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? Thank you very much for your opinions and suggestions! Best wishes, Sun Tang - Never miss a thing. Make Yahoo your homepage.
[ccp4bb] How to refine a structure with ATP and AMP and pyrophosphate sharing the same density in CCP4i?
Hello everyone, I have a structure of intermediate state in which about half amount of ATP decomposed to AMP and pyrophosphate. The ATP and AMP + pyrophosphate have little difference in conformation, sharing the same electron density. I just gave them different residue ID and did the TLS and restrained refinement in CCP4i. It is hard to tell from the R-factor because they are only a very small part of the whole structure. Can anyone tell whether it is the correct way to do? Any suggestions are greatly appreciated. Thank you very much! Sincerely, Sun Tang - Never miss a thing. Make Yahoo your homepage.
[ccp4bb] How to improve the density of another molecule in Asymmetric unit
Hello Everyone, I have a question about how to improve the electron density. I have two indepedent molecules in the asymmetric unit (1.9 A resolution). I solved the structure with PHASER and refined with CCP4i (Rfree = 0.29). For the first molecule, the density is very good, but for the second one, the density is much worse than the first one. Are there any ways to improve the density of the second molecule, such as some kinds of averaging? Why that happens? Thank you very much for your help! Sincerely, Sun Tang [EMAIL PROTECTED] - Never miss a thing. Make Yahoo your homepage.
