My first time posting to the CCP4BB board and I am very sure it will not be
my last. I have a general and specific question concerning molecular
replacements of protein-protein complexes. I have a good dataset (2.5A, 98%
complete), which has been integrated and scaled. Pointless suggested the
pointgroup P222, in agreement with imosflm and HKL2000. For the spacegroup
it could not decide between P2221 or P21221.
Spacegroup TotProb SysAbsProb Reindex Conditions
<P 2 2 21> ( 17) 0.889 0.900 00l: l=2n (zone 3)
<P 21 2 21> ( 18) 0.889 0.900 h00: h=2n, 00l:
l=2n (zones 1,3)
..........
<P 2 2 2> ( 16) 0.057 0.057
<P 21 2 2> ( 17) 0.057 0.057 h00: h=2n (zone 1)
..........
<P 2 21 21> ( 18) 0.039 0.040 0k0: k=2n, 00l:
l=2n (zones 2,3)
<P 21 21 21> ( 19) 0.039 0.040 h00: h=2n, 0k0:
k=2n, 00l: l=2n (zones 1,2,3)
I reindexed to both space groups and proceeded with Mathews Coeff. Assuming
a 1:1 complex had formed (MW = 39000) it predicts 1 complex per ASU. MolRep
using the larger protein (MW=27000) found the protein with a contrast of
3.33 (p2221) and 2.61 (p21221). However repeating MolRep for the smaller
protein in both spacegroups failed to locate it. Contrast was low, 1.11
(p2221) and 1.10 (p21221).
I reran Mathews Coeff in case the second protein was not their (though gels
of crystals show both proteins) and it predicted 2 proteins per ASU. MolRep
looking for 2 of the larger protein generated contrasts of 1.99 (p2221) and
2.27 (p21221).
Using Refmac5, I performed a rigid body refinement of all 4 cases with
details summarised below
p2221 1proteinper ASU - Proteins are arranged in layers separated by a
55Angstrom space. Some electron density is present but nothing that could be
generate a polypeptide. Perhaps two or three amino acids here and there.
Density fits the protein well. Rfac, 0.527, Rfree 0.523
p21221 1proteinper ASU - Proteins are again arranged in layers separated by
a 55Angstrom space. Again some electron density is present that could
accommodate two or three amino acids. Density also fits the protein well.
Rfac, 0.527, Rfree 0.512
p2221 2proteinper ASU - No layers are observed. Density fits the proteins
very poorly. Rfac, 0.541, Rfree 0.546
p21221 2proteinper ASU - No layers are observed. Density fits the proteins
very poorly. Rfac, 0.540, Rfree 0.547
My questions are this; In general what is the best procedure for molecular
replacement and refinement of protein-protein complex where the Kd is 1uM
and weaker and it is difficult to observe one of the proteins; In this case
I believe the second protein is their, though either the starting model is
poor (80% identical) or a gross conformational change of the protein has
taken place (which is unlikely based upon similar complexes that have been
solved). I am also concerned about Rfree being smaller than Rfac. Should I
continue with these two data set and try modeling alanine in the electron
density I can observe and see if further density is generated on further
refinement? Or should I go back and look at alternative space groups?
Thanks in advance for you opinions, suggestions and help
Daniel A. Bonsor,
Boston Biomedical Research Institute,
64 Grove Street,
Watertown,
MA 02472 USA
