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