Well - first Solvent content.. 1) there could be 4 molecules, or 3, or 2.. Solvent content varies a lot as you know. Is there reasonable density for parts of all the chains?
2) Does the MR search clearly show 4 molecules? ie improvement in scores with extra molecules? 3) Have you checked for sensible NC symmetry - submit your model to the PISA server and see if it suggests a teramer, or 4 fold symmetry or maybe 2 dimers? PISA will also suggest buried surface area of each chain - maybe parts of the C & D chains have nothing to fix them in place Eleanor On 26 January 2017 at 14:11, Pooja Kesari <pkesar...@gmail.com> wrote: > We have a 2.6 A structure showing four chains in an asymmetric unit. Our > protein is 360 residues around 40 kDa . Mattews shows four chain in an > assymetric unit (solvent 49% mattews coeff 2.44). The template has about > 60% homologous with our protein. The molecular replacement against this > template gave an initial free R of 38. We did chain tracing and found that > we have good density (2Fo-Fc) for chain A and B but poor density for C and > D. > > 1. The density for a particular stretch of 10 amino acids (disordered loop > region) is absent in all the chains. We could not found density for this > flexible loop region in any of the already known structures. Any suggestion > on how can we build this region? > > 2. We did not find density for most of the loop regions in chain C and D > which were well traced in chain A and B. How can we improving the density > for these two chains based on chain A and B (Density modification)? > > 3. We analysed the data using phenix xtriage and found that our data shows > severe anisotropy. Any suggestion of anisotropy correction? > > Pointless and Ctruncate analyses didn't show twinning or NCS. I have > checked the space group using Zanuda. We are stuck at a free value of 32. > > On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson <eleanor.dod...@york.ac.uk > > wrote: > >> This is a bit too vague to help much. >> How did you solve the structure? >> Eleanor >> >> On 26 January 2017 at 03:50, Pooja Kesari <pkesar...@gmail.com> wrote: >> >>> Dear All, >>> Thank you all for reply. >>> >>> We have checked the data for twinning. >>> Our protein is 360 residues around 40 kDa protein. >>> We have tried TLS refinement. >>> chain A and B don't superimpose well with chain C and D. (A and B chains >>> also share slight difference ) >>> Since we don't have proper density for *some regions* chain C and D, >>> we are not sure whether these chain have similar or different >>> conformations. >>> We tried anisotropy correction and the model refined a bit. >>> >>> >>> On Wed, Jan 25, 2017 at 10:32 AM, Debanu <debanu....@gmail.com> wrote: >>> >>>> Hi Pooja, >>>> >>>> Are you positive you have the correct space group and there are no >>>> other issues like twinning, etc? >>>> >>>> If sure, did you define NCS groups in refinement? TLS refinement? Try >>>> different refinement programs? >>>> >>>> How big is the molecule? Was it solved by MR or experimental phasing? >>>> >>>> You can try superimposing A/B on C/D and refinement with tight NCS then >>>> adjust NCS restraints during model adjustments based on local differences >>>> or also see if phenix autobuild helps. >>>> >>>> Best, >>>> Debanu >>>> -- >>>> Debanu Das >>>> Accelero Biostructures >>>> >>>> >>>> On Jan 24, 2017, at 8:42 PM, Pooja Kesari <pkesar...@gmail.com> wrote: >>>> >>>> Dear All, >>>> >>>> I have a 2.6 A resolution structure having four chains in an asymmetric >>>> unit. >>>> The chain A and B have density for almost all residues however we don't >>>> have proper residue density in chain C and D.What can be tried to build >>>> chain C and D ? >>>> >>>> >>>> >>>> Many Thanks >>>> Pooja >>>> >>>> >>> >>> >>> -- >>> Thanks & Regards, >>> Pooja Kesari >>> Research Scholar >>> Department Of Biotechnology >>> Indian Institute of Technology Roorkee >>> INDIA >>> >>> >> > > > -- > Thanks & Regards, > Pooja Kesari > Research Scholar > Department Of Biotechnology > Indian Institute of Technology Roorkee > INDIA > >
