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Hi Zorg,
In these types of cases you normally want to ensure that all Rfree
reflections used in the original crystal form are also used for Rfree in
the new crystal form. In your case that would mean that you just keep
your original Rfree set. However, that way your Rfree set only samples
the strong h+k+l=2n subset of the lattice which will give a very
distorted comparison with Rwork which will include also all the
systematically weakened h+k+l=2n+1 reflections. So my suggestion would
be to keep half of your original Rfree set and select a new set of Rfree
reflections from the systematically weakened part of your data set.
I would do this in sftools. It clearly is a non-trivial use of the
program which illustrates how you can combine several of its flexible
commands to do complex and unusual tasks. I'm doing this by heart so
there may be a mistake but I hope you get the idea. Let me know if you
run into problems.
1 read P21212.mtz
2 read I222.mtz col rfree
3 calc r col random = ran_u
4 select col rfree = 0
5 select col random < 0.5
6 calc col rfree = 1
7 select all
8 set spacegroup I222
9 select sysabs
10 rfree 500
11 select all
12 delete col random
13 set spacegroup P21212
14 write P21212new.mtz
1 read the data for your P21212 set
2 read only the rfree column of the I222 mtz (assuming column label
for this is "rfree"
3 create a new column named "random" with uniform random numbers in the
range of 0 to 1
4 select reflections with a 0 in the rfree column (e.g. your rfree set)
5 select only the subset with random number less than 0.5, this should
give you a random selection of half your original rfree set
6 set the flag of this subset to 1 (e.g. put them in the working set)
7 reset the selection to include all reflections
8 change space group temporarily to I222
9 select all reflections that should be systematically absent in I222
10 select 500 rfree reflections in the h+k+l=2n+1 part of the data (set
500 to whatever number of reflections you want)
11 reset the selection to include all reflections
12 remove the temporary column
13 reset the space group to the correct setting
14 write out the new mtz with the desire rfree set
The alternative solution is to pick an entirely new Rfree set and shake
the bias out of your model. That would definately be easier to explain
in the methods section :)
Bart
Zorg Blue wrote:
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Hello,
We have solved the structure of a protein that crystallised in space
group I222 with 1 molecule per asymmetric unit. After refining the
structure to 3.5 A, we obtained a different crystal form that diffracts
to higher resolution; in this case, the space group appears to be
P21212, with the same unit cell as the first type of crystals and 2
molecules per asymmetric unit. The packing between the two molecules in
the P21212 asymmetric unit is identical to that of symmetry-related
molecules in the I222 crystals, suggesting that the space group of
crystal form 1 could also be P21212, with non-crystallographic symmetry
giving rise to strong pseudo-I222 symmetry at low resolution.
Since we have already refined our model against the low resolution data
processed in I222, what would be the best way to define a new free R set
in order to continue refinement against the higher resolution P21212
data? If our interpretation is correct, I would think that using a
completely new set would introduce some bias; on the other hand, is
there any way to somehow take into account the pseudo-symmetry we observe?
Thank you for any advice,
Zorg
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==============================================================================
Bart Hazes (Assistant Professor)
Dept. of Medical Microbiology & Immunology
University of Alberta
1-15 Medical Sciences Building
Edmonton, Alberta
Canada, T6G 2H7
phone: 1-780-492-0042
fax: 1-780-492-7521
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