Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
An Fo-Fc map is actually the real-space representation of the Fo-vs-Fc R factor (Rcryst), so the sigma of this map will continue to drop relative to the true electron scale as your model improves and the difference between Fo and Fc diminishes. The 2Fo-Fc map, however, is a best guess of the total electron density, and that is always on about the same scale, so the sigma of this map is pretty constant throughout refinement. Toward the end of refinement, when your R/Rfee are around 20%, the sigma of the Fo-Fc map will be about 1/5 of the 2Fo-Fc sigma level. So, eventually you will see things at the 3 sigma level in the Fo-Fc map that are not visible at the 1 sigma contour level of a 2Fo-Fc map. This does not mean they are noise. I'm not sure where that rule came from. The noise level is actually another 1/5th below the Fo-Fc sigma level, or 25-fold smaller than the 1 sigma contour of the 2Fo-Fc map. This is because the noise is not really related to R/Rfree but rather the actual error in the data. This is roughly half the value of redundancy-corrected Rmerge-like values such as Rpim. The factor of two is because these latter R statistics are for intensities and the maps are calculated from amplitudes. The situation where the sigma of the Fo-Fc map is the noise level really only arises when the difference between Fo and Fc (aka Rcryst) is comparable to Rpim, and that pretty much only happens for small molecules where all the atoms in the unit cell can be named and accounted for. Macromolecular models generally don't achieve that. Not yet anyway. So, I encourage you to build into Fo-Fc density if you can. Remember, there is always something there, the question is what that something is. And also what else could it be? The bunch of waters model is always an alternative hypothesis. It is fairly easy to show that one model fits better than another, but to show that the difference is significant requires error bars, and that's why we developed the RAPID procedure: http://dx.doi.org/10.1073/pnas.1302823110 -James Holton MAD Scientist On 3/10/2014 1:41 AM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amlan Roychowdhury *Gesendet:* Montag, 10. März 2014 09:09 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
Dear All, Thank you very much for your reply. I have an another doubt and I want to discuss with you. Sometimes for some structure during refinement and model building we have found a green blob (Fo-Fc and 5 sigma). If I scroll down the blue 2Fo-Fc map to a very low sigma level nearly at 0.5 a really non convincing density will appear. And from the structural point of view, it will be very difficult to put anything within it due to its position within the structure. as an example once I have found an elongated green density without any trace of blue in between a helix and a beta sheet. Is it due to noise? The structure was well refined. What should we do in such cases? Regards amlan. On Mon, Mar 10, 2014 at 4:41 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amlan Roychowdhury *Gesendet:* Montag, 10. März 2014 09:09 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
You dont say what resolution you are working at, or what the current R factor is. or how complete the model is. There are assumptions made in the refinement scaling algorithms and in their treatment of supposedly poorly ordered solvent which can generate false density (both positive and negative) on the boundaries of the model. And as well as Herman says the Sigma level is set globally whereas the actual density locally is affected by the B values. One of the strengths of COOT is that it is easy to adjust the sigma level at local regions. Eleanor On 11 March 2014 07:42, Amlan Roychowdhury amlan.iit...@gmail.com wrote: Dear All, Thank you very much for your reply. I have an another doubt and I want to discuss with you. Sometimes for some structure during refinement and model building we have found a green blob (Fo-Fc and 5 sigma). If I scroll down the blue 2Fo-Fc map to a very low sigma level nearly at 0.5 a really non convincing density will appear. And from the structural point of view, it will be very difficult to put anything within it due to its position within the structure. as an example once I have found an elongated green density without any trace of blue in between a helix and a beta sheet. Is it due to noise? The structure was well refined. What should we do in such cases? Regards amlan. On Mon, Mar 10, 2014 at 4:41 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amlan Roychowdhury *Gesendet:* Montag, 10. März 2014 09:09 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
Dear Sir May i know how to refine the group occupancy for a ligand??? Thanx in advance On Mon, Mar 10, 2014 at 2:11 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Amlan Roychowdhury Gesendet: Montag, 10. März 2014 09:09 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
Re: [ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
Dear Monica, for refmac5 this is nicely explained at http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#Occupancy Best, Tim On 03/11/2014 12:14 PM, Monica Mittal wrote: Dear Sir May i know how to refine the group occupancy for a ligand??? Thanx in advance On Mon, Mar 10, 2014 at 2:11 PM, herman.schreu...@sanofi.com wrote: Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Amlan Roychowdhury Gesendet: Montag, 10. März 2014 09:09 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] AW: [ccp4bb] regarding Fo-Fc map in coot
Dear Amlan, The sigma of an Fo-Fc map map depends on the residual noise in your map. In a well-refined structure, the sigma will be low, so at 3 sigma it will show very weak features. My guess is that your ligand is present in partial occupancy and that you will find it in your 2Fo-Fc map when you scroll down your contour level. If you see convincing Fo-Fc density without a ligand being fitted, the presence of the ligand must be real and you can fit it. However, I would refine a group occupancy for your ligand. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Amlan Roychowdhury Gesendet: Montag, 10. März 2014 09:09 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] regarding Fo-Fc map in coot Dear All, Some times during model building in coot we have found that at the position of ligand molecules and water, there is a good Fo-Fc map (above 3 sigma), devoid of any 2Fo-Fc map. 1.What does it physically mean and why the 2Fo-Fc map was not generated properly? 2. Can we fit ligand molecule there? Thanks in advance. Best Wishes Amlan. -- Amlan Roychowdhury. Senior Research Fellow. Protein Crystallography Lab. Dept. of Biotechnology, IIT Kharagpur. Kharagpur 721302 West Bengal. India.
