Its possible you are in a lower space group, perhaps with some twinning, but your search
model is different enough to only find a "solution" when things are over-merged.
Try refining your P6522 model against data merged in P65. If the other copy (symmetry mate in
P6522) does not show up, you may be in trouble (wrong MR solution). I'd also try
refinement/building in the other triogonal/hexagonal space groups, but again, start with the PDB
file that you got for P6522. Just change the space group in the header, and switch out the MTZ
file. You will need to merge your data in each space group and also check the a-b "flip"
re-indexing for most of them. Have a look at the CCP4 "reindexing" list for the h,k,l
operators to try:
http://www.ccp4.ac.uk/html/reindexing.html
note how similar they are to the "twinning" operators:
http://www.ccp4.ac.uk/html/twinning.html
If I have counted right, that means you have 36 jobs to run.
I'd also recommend turning the "TWIN" option in refmac off and on for each of these cases. This will always give you a lower R factor, because of the dynamic range compression you get with twinning, but if one particular combination of twinning with a particular space group and axis reindexing is markedly better than all the others, then you have just found your right space group. So, now we are up to 72 jobs, but hardly a lot of work compared to growing the crystals in the first place.
You might also want to try being "clever" and generating the symmetry mates of
your P6522 model and refine these partners as separate molecules as you reduce the
symmetry of the data. It's tricky, but think of it as an exercise. Which real-space
operator becomes what reciprocal-space operator? You can check your answer by loading it
up in coot and seeing if symmetry mates clash with the input coordinates.
Yes, its a lot of work to try all these combinations, but that's the annoying
thing about twinning, it opens up a lot of ambiguities.
Good luck!
-James Holton
MAD Scientist
On 12/14/2013 6:44 AM, D Bonsor wrote:
Dear all,
I have collected ~160 degrees of data on a new crystal form of a protein which
has already been solved. Data was processed with XDS and reindex, scaled and
truncated with Aimless. Both XDS and Pointless suggested a Laue group of
P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall
Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a
completeness of 99.1% and resolution of 2.8Ang.
With cell dimensions of 63.1 63.1 243, only one protein chain can be found in
the asymmetric unit (two copies would leave a solvent content of 8%). I ran
phaser with all alternative space groups and a single solution in P6522 with a
TFZ of 10.0.
I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I
open the structure and map in Coot and could see that there was a large
conformational change of helix-turn-helix actually becoming just a long helix
(https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then
dimerizing through the long helix with one of the symmetry mates.
This section was rebuilt
(https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through
Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of
the structure I see nothing else really to be modeled. Nothing that could bring
the Rfactors down to a reasonable range.
I have therefore tried several things. I ran the structure through Zanuda server to look
at other space group possibilities. The server suggested I was in the correct space
group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and
reran phaser in "search all alternative space groups" using the original search
model but found no solutions. I did reprocess the data in P1, though I did not collect
enough data.
Twinning tests show no twinning. Although that does not mean there is no
twinning, I can see that P6522 has no twin laws. Does that mean no twinning can
occur in P6522 or that it can occur but there is no law to be able to separate
the amplitudes?
I also collected data on a single point mutation of the protein. Although this
diffracted to a slightly weaker resolution (3.2Ang), I also observe the same
problem of good maps in P6522 but no solution in the groups described above, a
clear indication that this helix has elongated but terrible Rfactors.
Based upon that the maps look good in P6522 do you believe that I have solved
the structure in the correct space group but my data collection is at fault or
in fact that I have some form of pseudosymmetry or something else going on and
that the space group has lower symmetry but not in the space groups I have
checked. Or is it something else.
Any suggestions, criticisms or you need further information please contact me
and enjoy your weekend.
