Dear Colin, Thank you for the email. To be honest, I don't know exactly which map they are. From my understanding (and according to http://www.ccp4.ac.uk/schools/India-2010/tutorials/refmac/Murshudov30th.pdf), Refmac5 outputs 'normal map' 2mF(obs)-DF(calc) and 'difference map' 2mF(obs)-DF(calc). I believe this is better than looking at the typical 2F(obs)-F(calc), although there still appears to be a fair amount of model bias.
Best, Peter Subject: RE: [ccp4bb] Help with model bias in merihedral twin Date: Wed, 27 Oct 2010 17:38:41 +0100 From: [email protected] To: [email protected] Peter Regarding the question "Although the electron density map looks good, I am not sure if I should have too much confidence in it because I was not able to obtain 'strong electron densities' from omitted sections of the model in a refinement. I don't know if this is an indicator for bias introduced somewhere. I would like to ask what may be some procedures I can try for checking and removing these biases" Can you state what type of maps these are? The reason is the following. Taking PHENIX twinning maps as an example we have, in the manual - "By default, data is detwinned using algebraic techniques, unless the twin fraction is above 45%, in which case detwinning is performed using proportionality of twin related Icalc values. Detwinning using the proportionality option, results in maps that are more biased towards the model, resulting in seemingly cleaner, but in the end less informative maps. The 2mFo-dFc map coefficients can be chosen to have sigmaA weighting (two_m_dtfo_d_fc) or not (two_dtfo_fc). IN both cases, the map coefficients correspond to the 'untwinned' data. A difference map can be constructed using either sigmaA weighted detwinned data (m_dtfo_d_fc), a sigmaA weighted gradient map (m_gradient) or a plain gradient map (gradient). The default is m_dtfo_d_fc but can be changed to gradient or m_gradient if desired. " For the high twin fraction which you have (near 50%). my view is that 2Fo-Fc type maps are not optimal for this as they don't take account of the increased bias resulting from the extra degrees of freedom (i.e. splitting the intensity between the two reflections based on Icalc). For the reflections related by twinning higher order coefficients (e.g. 3Fo-2Fc or perhaps 4Fo-3Fc) would give noiser but less biased maps. Fibre diffraction folk do similar things when their Bessel function terms overlap. Cheers Colin From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Peter Chan Sent: 27 October 2010 02:00 To: [email protected] Subject: [ccp4bb] Help with model bias in merihedral twin Hello All, Not long ago I posted for some help with my twinned dataset at 1.95 A, and have confirmed the twinning of P6(5) into P6(5)22. Molecular replacement was successful and the twin refinement in Refmac yieled R/Rfree of 21%/26%, with a twin fraction of 0.46. Although the electron density map looks good, I am not sure if I should have too much confidence in it because I was not able to obtain 'strong electron densities' from omitted sections of the model in a refinement. I don't know if this is an indicator for bias introduced somewhere. I would like to ask what may be some procedures I can try for checking and removing these biases, and a few additional related questions. As suggested to me previously, I have generated a total omit map with sfcheck in ccp4i, using the refined pdb and unrefined data in P6(5). The .map file look a little worse in quality (is this because of the twinning?) but is still reasonable, with a few breaks in the main chain and side chains. Interestingly, when I do a real space refinement against the total omit map, I get slightly better Rfree at the earlier rounds of Refmac which diverges into the numbers above. Why is this the case? Cycle R fact R free 0 0.2301 0.2523 1 0.2205 0.2534 2 0.2164 0.2545 3 0.2140 0.2554 4 0.2123 0.2559 5 0.2117 0.2570 6 0.2116 0.2575 7 0.2112 0.2582 8 0.2112 0.2584 9 0.2109 0.2587 10 0.2106 0.2597 Secondly, I read that I should make sure the Free R flags should be consistent throughout the twin-related indices. What may be the adverse outcome if this isn't enforced? Is Refmac aware of this in a twin-refinement? If not, which tool could I use for this? I would very much appreciate any comments and suggestions. Best, Peter Chan -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
