Thank you very much to all those who suggested a way out for our situation. We have so far refined the occupancies on phenix and the ligand shows an uniform occupancy of 0.8 post the refinement. The B-factors of the ligand are around 20 (atleast for the regions with a well defined electron density), which is slightly lower than the average B-factors of the whole structure which is 24.
We have a few poorly defined regions in our electron density. This was the starting point of our problem and it remains to be a problem. @ Robbie ---> we would like to run EDSTAT on CCP4 but we dont find the program in both 6.3.0 and 6.3.1 versions. It would be kind to know if we are doing something wrong to not find it on the ensuite. @ Herman ----> We did add the cryo from the crystallisation condition as another strategy but that also doesnt look too convincing. There is just that enough more to the density to think its our substrate. The density also does not compare well with the apo structures. @ Eleanor----> We set the occupancies to zero and refined the structure but we did not get any conclusive answers from it. We have a continious density for the best part of the ligand; but as you mentioned a few carbon atoms which are wobbly are poorly defined. We will look carefully into the geometrical restraintsas you suggested. Thank you all again forthe suggestions! Srinivasan ________________________________ From: "Bosch, Juergen" <[email protected]> To: [email protected] Sent: Tuesday, 12 March 2013 4:32 PM Subject: Re: [ccp4bb] [ccp4bb] validating ligand density Going back to the initial question. I would recommend looking at AFITT http://www.eyesopen.com/afitt Works like a dream (in certain cases). Jürgen P.S. I wish I had some stocks from them but I don't ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Mar 12, 2013, at 11:25 AM, <[email protected]> <[email protected]> wrote: You are the one who should judge your statement, but it looks plausible to me. > >Now that I think of it: why do we need referees if every scientist should judge their own hypothesis? Publication will be a lot faster if we no longer need to heed the remarks of some grumpy referees and send in revision after revision. Also the number of publications will increase significantly if every scientist is allowed to judge their own papers! > >HS > > >>________________________________ >> From: Jacob Keller [mailto:[email protected]] >>Sent: Tuesday, March 12, 2013 4:14 PM >>To: Schreuder, Herman R&D/DE >>Cc: [email protected] >>Subject: Re: [ccp4bb] validating ligand density >> >>Dear Jacob, >> >>You are overinterpreting, the statement is about judging, not proving a >>hypothesis. I am sure Mr. Edwards judged his statement to be ok. >> >> >>I guess there is a good likelihood that you are right, but who am I to judge? >> >> >>JPK >> >> >> >> >> >> >> >> >>>Herman >>> >>> >>>>________________________________ >>>> From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of >>>> Jacob Keller >>>>Sent: Tuesday, March 12, 2013 3:44 PM >>>> >>>>To: [email protected] >>>>Subject: Re: [ccp4bb] validating ligand density >>>> >>>> >>>>One final quote that is not in the twilight paper summarizes it nicely: >>>>> >>>>>"The scientist must be the judge of his own hypotheses, not the >>>>>statistician." >>>>> A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept >>>>>of likelihood and its application to scientific inference , p. 34. >>>>> >>>> >>>> >>>>There must be a lot of thinking behind this statement--while it seems >>>>plausible, it seems far from proven prima facie. Also, it assumes that the >>>>scientist is not a statistician. >>>> >>>> >>>>Jacob >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>> >>>>Btw, the book is good reading. >>>>> >>>>>Best, BR >>>>> >>>>> >>>>>-----Original Message----- >>>>>From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Robbie >>>>>Joosten >>>>>Sent: Tuesday, March 12, 2013 10:03 AM >>>>>To: [email protected] >>>>>Subject: Re: [ccp4bb] validating ligand density >>>>> >>>>>Dear Srinivasan, >>>>> >>>>>Although the Twilight program can only look at deposited PDB entries, the >>>>>tips about ligand validation in the paper are very useful. I suggest you >>>>>start from there. >>>>>You can use EDSTATS in CCP4 to get real-space validation scores. Also look >>>>>at the difference map metrics it gives (and the maps themselves of course), >>>>>they will tell you whether you misidentified your ligand. Occupancy >>>>>refinement in Refmac can also help you: if the occupancy drops a lot >>>>>something is wrong. That can be partial binding (not that much of a problem) >>>>>or worse, a ligand that isn't there. By the way, I've been playing with >>>>>that recently and some ligands/hetero compounds in the PDB were so >>>>>incredibly 'not there' that Refmac would crash (that bug seems to be fixed >>>>>in the latest version). >>>>> >>>>>HTH, >>>>>Robbie >>>>> >>>>>> -----Original Message----- >>>>>> From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of >>>>>> R.Srinivasan >>>>>> Sent: Monday, March 11, 2013 23:03 >>>>>> To: [email protected] >>>>>> Subject: [ccp4bb] validating ligand density >>>>>> >>>>>> Hello all, >>>>>> >>>>>> We co-crystallized an inactive variant of our enzyme in >>>>>> the >>>>>presence of >>>>>> substrate and have determined the structure at 1.85A. >>>>>> >>>>>> Now, we want to validate the fitting of the ligand into >>>>>> the >>>>>electron >>>>>> density. We tried validating using the difference map (2Fo-Fc) after >>>>>refining >>>>>> the structure without the ligand. But, it is still a bit inconclusive >>>>>> if >>>>>the density >>>>>> fits the ligand. >>>>>> >>>>>> It would be very kind to know if there are tools for >>>>>validating this >>>>>> electron density. We were excited about twilight but turns out it can >>>>>> only >>>>>be >>>>>> used with deposited structure. >>>>>> >>>>>> >>>>>> We will appreciate your help and suggestions. >>>>>> >>>>>> >>>>>> Many thanks, >>>>>> Srinivasan >>>>> >>>> >>>> >>>> >>>>-- >>>>******************************************* >>>> >>>>Jacob Pearson Keller, PhD >>>> >>>>Looger Lab/HHMI Janelia Farms Research Campus >>>> >>>>19700 Helix Dr, Ashburn, VA 20147 >>>> >>>>email: [email protected] >>>> >>>>******************************************* >> >> >> >>-- >>******************************************* >> >>Jacob Pearson Keller, PhD >> >>Looger Lab/HHMI Janelia Farms Research Campus >> >>19700 Helix Dr, Ashburn, VA 20147 >> >>email: [email protected] >> >>*******************************************
