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
