Dear Mohamed,
There are a few things to look at here
1) It may be worth processing your data with xia2 using DIALS as the integration program and see what you get. Different programs, different algorithms and hence different integration results
2) A solvent content of 8% and 3 molecules in the asu is extremely unlikely. Try to search for one molecule first and then look at the phasing solution in Coot and generate the symmetry molecules. If there are still gaps between the molecules look for a second one.
3) When running MR let the program look for all possible space groups of your point group found during data processing. Pointless usually gives a good indicator of what the correct solution may be but it if you have any issues like twinning or pseudo translation then Pointless can be fooled into the wrong conclusion. Did you check for twinning or pseudo translation? Did you check for radiation damage?
4) MR works best at lower resolution and programs normally use data to around 3.5A or so. If you want to make sure this is the case you can give the program this information. There is no need to cut back the resolution in data processing or scaling.
5) sequence identity of 35% makes your MR case a tricky one. Together with the low completeness you may struggle to find a solution. With such a weak homologue it is best to get experimental phases. If your protein happens to have an intrinsic metal cofactor this is easy. If not try soaking, Se-Met or sulfur-SAD.
6) In any case you will need to collect more data
HTH
Melanie
Dr Melanie Vollmar
Diamond Light Source Ltd.
Harwell Science & Innovation Campus
Didcot
Oxfordshire
OX11 0DE
United Kingdom
Tel: +44 (0)1235 778492
www.diamond.ac.uk
On 25 Jul 2016, at 12:57, Mohamed Noor <[log in to unmask]> wrote:
> Dear all
>
> I have a dataset with low completeness, processed with Xia2/XDS as shown below. As I am having difficulties in getting a good MR solution, I wonder whether it is worthwhile at all to solve the structure with this data. The closest homolog in the PDB has a sequence identity of 35 %.
>
> Secondly, the Pointless log has the following lines:
>
> Spacegroup TotProb SysAbsProb Reindex Conditions
>
> P 2 2 21 ( 17) 0.944 0.944 [l,h,k] 00l: l=2n (zone 2)
> P 21 21 2 ( 18) 0.944 0.944 [k,l,h] h00: h=2n, 0k0: k=2n (zones 2,3)
> ..........
> P 2 2 2 ( 16) 0.056 0.056
> P 2 2 21 ( 17) 0.056 0.056 00l: l=2n (zone 3)
>
> Does this mean I should focus on just SG 17/18? Why are there two lines for SG 17 with different probabilities? Is this related to alternative indexing?
>
> Using HHPred, I have prepared 10 search models. Based on Matthew's coefficient, there could be either one or two copies in the asu. With 3 copies, the solvent content is 8 %, so I guess I can safely exclude this. Although I could try different SG etc, would this make sense?
>
>
> High resolution limit 2.50 6.78 2.50
> Low resolution limit 38.97 38.97 2.54
> Completeness 84.1 75.6 84.9
> Multiplicity 3.8 3.7 3.7
> I/sigma 3.7 12.4 0.5
> Rmerge(I) 0.334 0.080 2.998
> Rmerge(I+/-) 0.312 0.073 2.775
> Rmeas(I) 0.380 0.091 3.433
> Rmeas(I+/-) 0.395 0.092 3.546
> Rpim(I) 0.177 0.043 1.629
> Rpim(I+/-) 0.239 0.055 2.178
> CC half 0.971 0.993 0.160
> Wilson B 0.00
> Total observations 45792 2282 2199
> Total unique 12038 617 595
>
>
> Thanks.
> Mohamed
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