Thank you for the reply. On Thu, Jan 26, 2017 at 9:07 PM, <[email protected]> wrote:
> Dear Pooja, > > > > A few remarks: > > > > - Matthews does not show 4 chains in the asymmetric unit, it > suggests 4 chains. However, in reality it can be more or less chains, > although rare, 75% solvent (2 chains) is not unheard of. > > - An initial Rfree of 38% is ok, 32% after refinement is a bit > high > > - Disordered loops do exist and you may have to live with them > (not be able to build them). > > - To correct for anisotropy, I suggest the staraniso server from > global phasing. > > - Unless you ran your molecular replacement in P1, Zanuda will > confirm the space group you choose for MR. So run MR in P1 (if your > symmetry is not too high) and run Zanuda again. (Have someone) look > critical at the assigned space group and assess whether another choice > might also be possible. > > > > Good luck! > > Herman > > > > *Von:* CCP4 bulletin board [mailto:[email protected]] *Im Auftrag von > *Pooja Kesari > *Gesendet:* Donnerstag, 26. Januar 2017 15:12 > *An:* [email protected] > *Betreff:* Re: [ccp4bb] Bad density for chains > > > > 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 <[email protected]> > 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 <[email protected]> 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 <[email protected]> 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 <[email protected]> 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 > > > -- Thanks & Regards, Pooja Kesari Research Scholar Department Of Biotechnology Indian Institute of Technology Roorkee INDIA
