Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu
On 24 March 2013 02:38, Raji Edayathumangalam <r...@brandeis.edu> wrote: > Dear Appu, > > I am not sure that I have a complete sense of the issue at hand since some > of the information needed to think your issue through is missing in your > email. For example, to what high resolution cut-off were the data measured? > What resolution limits were used for the MR search? How do the unit cell > dimensions and space group in the two cases compare? > > I am guessing the ligand binding domain in your protein has the identical > sequence to that of the published ligand binding domain that you use as a > template in your MR search. In any case, here are a couple of my thoughts: > > (1) It might be worth setting up different runs of MR with different > numbers for expected copies (not just two copies but also one copy and > three copies just in case you have one of the extreme cases of solvent > content)? > > (2) If the MR solution is correct and there is physical room for a DNA > binding domain in your lattice (check by displaying symmetry mates), > perhaps the DNA binding domain is disordered. In that case (and if all > attempts with current data fail), you may have to crystallize the protein > in presence of DNA. > > > Good luck! > Raji > > > > > On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar <appu.kum...@gmail.com> wrote: > >> Dear members, >> >> I am doing a molecular replacement of a >> transcription factor whose ligand binding structure(24000 Da) is available >> in PDB but not for the DNA binding(13000 Da). When i am searching for the >> two copies from ligand binding domain as a template model, i am getting >> very good solution but i am not getting any density for the DNA binding >> domain to build up in density. The space gorup is P 1 21 1 (4) and unit >> cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 >> 90.00. Please guide me how to get the complete model structure. Table below >> show the matthews statistics >> >> For estimated molecular weight 37000. >> Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) >> _____________________________________________________________ >> 1 5.71 78.46 0.00 0.01 >> 2 2.85 56.91 0.62 0.70 >> 3 1.90 35.37 0.37 0.29 >> 4 1.43 13.82 0.00 0.00 >> _____________________________________________________________ >> >> >> The phaser molecular replacement gives the following table. >> istogram of relative frequencies of VM values >> ---------------------------------------------- >> Frequency of most common VM value normalized to 1 >> VM values plotted in increments of 1/VM (0.02) >> >> <--- relative frequency ---> >> 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 >> | | | | | | | | | | | >> 10.00 - >> 8.33 - >> 7.14 - >> 6.25 - >> 5.56 - >> 5.00 - >> 4.55 - >> 4.17 - >> 3.85 -- >> 3.57 --- >> 3.33 ------ >> 3.12 ---------- >> 2.94 **************** (COMPOSITION*1) >> 2.78 ----------------------- >> 2.63 -------------------------------- >> 2.50 ----------------------------------------- >> 2.38 ------------------------------------------------ >> 2.27 -------------------------------------------------- >> 2.17 ----------------------------------------------- >> 2.08 -------------------------------------- >> 2.00 -------------------------- >> 1.92 --------------- >> 1.85 ------- >> 1.79 --- >> 1.72 - >> 1.67 - >> 1.61 - >> 1.56 - >> 1.52 - >> 1.47 * (COMPOSITION*2) >> 1.43 - >> 1.39 - >> 1.35 - >> 1.32 - >> 1.28 - >> 1.25 - >> >> $TABLE : Cell Content Analysis: >> $SCATTER >> :N*Composition vs Probability:0|3x0|1:1,2: >> $$ >> N*Composition Probability >> $$ loggraph $$ >> 1 0.306066 >> 2 0.00141804 >> $$ >> >> Most probable VM for resolution = 2.27817 >> Most probable MW of protein in asu for resolution = 92664.2 >> >> Thank a lot in advance >> >> >> >> > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > >
