Hi Vikram, lots of questions for you: - what do the images look like? Were there “contaminating” lattices? Did the spot shape look ok? Do they get worse later in the data collection, i.e. do you have radiation damage? Is the image quality regular or worse at certain angles? - how good is the data near 2.6Å? Is the data anisotropic? If the data is not so great, perhaps Rfree 41% is as good as you can get. Nevertheless: - are you sure the molecular replacement solution is correct? Did you see density differences for residues you had to “mutate” in COOT that support the correctness of the molecular replacement solution? - what does model validation say? Any regions that still need improving? Any major differences between the two monomers that are not explained by the density? - did you try refining with local NCS in REFMAC? If not, this should get the Rfree down a bit more. - residues 1-49 and 370-377 may be genuinely disordered and not modellable, how hard did you try?
Hope this helps, Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser.cnb.csic.es/~mjvanraaij > On 25 Oct 2016, at 14:16, Vikram Dalal <vikram.dalal...@gmail.com> wrote: > > Hi everyone, > > We are trying to solve a protein structure of 2.6 A. We have processed it > with HKL2000. We have even tried processing with mosflm and xia2. It is in > C2221 space group (checked by pointless) and data is not twinned. It has 31% > identical with a search model and has 57% sequence coverage. There are 2 > subunits in asu. > > I did not get proper phases with MOLREP and phaser. I have tried Balbes (R > free 50) and Mr BUMP (R free 54).But, density from balbes look some > reasonable. > So i have refined it in ccp4i and phenix differently and then model build it > by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and now i > have amino acid 50 to 369 in both chains, still 49 amino acid at N terminal > and 8 amino acid at C terminal are missing and some loops are missing in > between too. > > I have tried the ARP/wARP, but it does not work for it. > > I have even tried phase and build of phenix but condition remain same. i got > stuck at R free at 42 and same around 49 amino acids at N terminal and 8 > amino acids at C terminal and some internal loops still absent. > > Thank you in advance. > > > > > > > > > > >
