Hi,
assuming you've exhausted modeling choices and applied proper refinement
strategies, after all Rree=32% is not unheard of at 2.6A resolution (which
actually may be even worse than 2.6A if data set is not 100% complete).
Have a look at R-factors of models in PDB with data resolution around 2.6A:
Histogram of Rwork for models in PDB at resolution 2.50-2.70 A:
0.119 - 0.145 : 10
0.145 - 0.171 : 147
0.171 - 0.197 : 814
0.197 - 0.223 : 1454
0.223 - 0.248 : 951
0.248 - 0.274 : 183
0.274 - 0.300 : 31
0.300 - 0.326 : 3
0.326 - 0.352 : 0
0.352 - 0.378 : 1
Histogram of Rfree for models in PDB at resolution 2.50-2.70 A:
0.175 - 0.204 : 51
0.204 - 0.233 : 420
0.233 - 0.262 : 1328
0.262 - 0.291 : 1344
0.291 - 0.320 : 386
0.320 - 0.349 : 58
0.349 - 0.378 : 6
0.378 - 0.407 : 0
0.407 - 0.436 : 0
0.436 - 0.465 : 1
Histogram of Rfree-Rwork for all model in PDB at resolution 2.50-2.70 A:
0.002 - 0.012 : 27
0.012 - 0.022 : 137
0.022 - 0.031 : 360
0.031 - 0.041 : 645
0.041 - 0.051 : 750
0.051 - 0.061 : 754
0.061 - 0.071 : 498
0.071 - 0.080 : 251
0.080 - 0.090 : 114
0.090 - 0.100 : 58
Number of structures considered: 3594
Sounds like you are not alone...
Pavel
On Thu, Jan 26, 2017 at 6:11 AM, Pooja Kesari <[email protected]> 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 <[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
>
>
