Thanks for your suggestions! The molecular weight of the protein is 14kD. Another thing I want to make it clear is that though the mad data and the native data have the same/similar spacegroup C222 and packing, they are differed in cell parameters which were grown at different temperatures. With the mad data (62.6,98.4,40.1)1molecule/AU and 2molecules/AU in the native data(108.2, 118.6, 63.6).
I wonder that anything else have effect on the f' or f"? Another thing Edwin mentioned is that Se-Met crystal may have some defects. So what is the sign of defect of a crystal? I remember someone said that the R_merge of the low resolution shell (50-6.0)could be considered. For those R_merge of the low resolution shell >0.05, the crystal is somwhat non-optimal. Actually the data for my protein R=0.046 with native one and 0.056 with peak data. > You dont say the size of the protein but in fact errors in f' or f" will > have very little effect. You can correct the SE formator if you like, > but even so in most cases if you call the MET MSE then you get holes > over the Se in difference maps which probably indicate partial conversion. > > Frankly any model refined at 2.7A is not going to be optimal - it is > quite hard to model alternate conformations (which certainly will be > there for 5-10% of the residues) and the solvent is also hard to fix - > there are no very good geometric restraints to stabilise its refinement.. > > So if you have 2.2A data then unless there is something seriously wrong > with it, then that is the set to use.. > > If you transfer the model refined against one data set to another then > you need to preserve the Free R definitions. > > Easy if you have an mtzfile > Merge the data sets - then run uniqueify -f FreeRflag merged.mtz > > That will keep the existing flags and generate new ones for the new > reflections.. > > Then if you want to you can convert back to the CNS format. > Eleanor > > > Peng Zhang wrote: >> Hi, Peter, >> >> Does the radition damage have such a large effect? There are many >> positive >> peaks seeing from the difference map, seems abnormal. But I have seen >> such difference maps before which are OK for modeling and refining. >> >> Actually when I try to modelling the Se-met into the positive peaks, the >> R >> and free_R factor goes up about 2%. Does the occupancy of the Se affect >> the R factor, or should I change the occupancy? >> >> Another problem is f' andf f" prime, which I refine them when phasing >> with Solve. >> >> Actually, I did not use the same free set when refining with the native >> data and peak data.I try to compare them because I want to make it clear >> that the difference may be caused by the mad data while not the model >> itself. >> >> Thanks. >> >> >> >>> Hi Peng Zhang, >>> >>> The presence of radiation damage might cause some problems. >>> Do so see any obvious features in the difference map? >>> >>> Another problem (although I doubt it would cause such a big difference) >>> might be the fact that f' andf f" prime are incorrect. >>> Try and refine them (CNS or phenix.refine) maybe. >>> >>> Does your native data have the same free set as the peak data? if not, >>> you >>> are in trouble and have to start from scratch with your native data to >>> be >>> able to make a fair comparison. The 22/25 for 2.7 A seems awfully close >>> together. >>> >>> procheck has not very up to date standards of what is good and what is >>> not. Better use molprobity, available from: >>> >>> http://molprobity.biochem.duke.edu/ >>> >>> HTH >>> >>> Peter >>> >>> >>> >>> >>> >>> >>> >>> >>> >>> ----- Original Message ----- >>> From: Peng Zhang <[EMAIL PROTECTED]> >>> Date: Tuesday, March 20, 2007 6:04 pm >>> Subject: Re: [ccp4bb] modelling with sad/mad data >>> To: CCP4BB@JISCMAIL.AC.UK >>> >>> >>>> Maybe I did not make the questions clear, which leading to the >>>> misleadings.Firstly, I have collected the mad data and get the >>>> phase at synchrotron, >>>> the phased Se is quite good for modelling, and get over 70% of the >>>> molecule run with resolve autobuilding.The density seems also good for >>>> building. But when finally refining the model, the gap between the >>>> R(0.22)and free_R(0.32 )is big, even though modelled the Se- >>>> methionine. Before >>>> collecting the mad data at synchrotron, I already have another >>>> native data >>>> set collected at home diffractometer (rigku, with R-axis IV++). To my >>>> surprise, when I using the same model(first model) and run with >>>> this data >>>> set, it is quite good( with 2.7A resolution, R=0.24 and Rf=0.28 and >>>> further refine to R=0.22 and Free_R=0.25), and I got the final >>>> model.Thegeometry of the first model and final model(actually no >>>> big difference of >>>> the two models)is quite good with procheck.The omit map says good >>>> enoughwith both of the two models. >>>> >>>> So I wondering what happened with the peak data? Did the anomolous >>>> signalhave much effect on the data? and anyone have the similar >>>> experience? >>>> >>>> >>>>> First it is always best to refine your model against the highest >>>>> resolution good quality data that you have available. There was >>>>> correspondence about the geometric weighting - could you have >>>>> >>>> weighted> the Xray data too high and have bad geometry - see >>>> previous Emails! >>>> >>>>> And the Free R seems rather low for the Se data. >>>>> Did you transfer the same Free R set from the native to the Se data? >>>>> Eleanor >>>>> >>>>> >>>>> Peng Zhang wrote: >>>>> >>>>>> Dear friends, >>>>>> >>>>>> Recently, I have solved a structure using mad method. When >>>>>> >>>> using the >>>> >>>>>> peak >>>>>> data(2.3A) as the native for structure refinement, the gap >>>>>> >>>> between R >>>> >>>>>> factor and R free is big, about 0.1(0.22 and 0.32). I modelled the >>>>>> selenomethionine but the gap still exists. When I changed the >>>>>> >>>> data for a >>>> >>>>>> real native one(2.7A),it seems OK with R=0.24 and Rf=0.28. >>>>>> >>>>>> Does anyone have the similar experience? >>>>>> what should I pay attention to when using the sad/mad data as >>>>>> >>>> the native >>>> >>>>>> one for modelling and refinement? >>>>>> >>>>>> Thanks in advance. >>>>>> >>>>>> >>>>>> >>>>>> >>>>> >>>>> >>>> -- >>>> Peng Zhang, Ph.D. Student >>>> Institute of Biochemistry and Cell Biology >>>> Shanghai Institutes for Biological Sciences >>>> Chinese Academy of Sciences >>>> >>>> 320 Yue-Yang Road >>>> Shanghai 20031 >>>> P.R. China >>>> >>>> >>>> >>>> Tel: 021-5492-1117 >>>> Fax: 021-5492-1116 >>>> Email: [EMAIL PROTECTED] >>>> >>>> >>> >> >> >> > > -- Peng Zhang, Ph.D. Student Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 20031 P.R. China Tel: 021-5492-1117 Fax: 021-5492-1116 Email: [EMAIL PROTECTED]
