Dear all,
I have determined a structure with the resolution 2.0A and the spacegroup P3121 from crystals grown at 4 degree. This apo-form structure is composed of an N-terminal (a/b)8 TIM barrel (~300 AA) and two C-terminal domains (~100 AA) which are named as Sub1 and Sub2. Meanwhile, I have also got crystals at 20 degree. Although the diffraction of these crystals is not good, I could confirm that the spacegroup has changed to C2. Recently, I have collected several datasets from crystals with different substrates at 20 degree. All of them have relative low resolution from 3.5A to 2.8A and completeness from 80% to 90% and have the same spacegroup and almost the same cell parameters. Considering that the biological unit is dimer and the C-terminal domains have high flexibility, I use both monomer and dimer forms of the TIM barrel as the search models when I do MR in the best dataset (2.8A resolution and 85% completeness). I use three programs CNS, Molrep, and Phaser and they all could find the same optimal solution which contains a dimer and about 68% solvent in one asymmetric unit. After several rounds of refinement, the R and freeR have dropped to 35.6 and 41.6, respectively and the density fit well at the TIM barrel region. Between the symmetric molecules exists a lot of discontinuous positive peaks which have no obvious secondary structure patterns. All the information above show that something unknown is not determined in this structure. I superpose the full-length dimer to the determined TIM barrel dimer but find that the C-terminal domains have severe conflict with the symmetric molecules at this packing form. I continue the MR using the TIM barrel, Sub1, Sub2, or Sub1+Sub2 as search models with fixed TIM barrel dimer, however no right solutions are obtained. I also do the MR with the full-length monomer and dimer forms but there are still no right solutions. Then I have tried the other datasets, however I get the same results. Now I am trying to build the model manually. Because of the discontinuous densities, it is hard to finish the building work at a relatively short time and I think it is not a right way to cut the knot. I don't know how and what to do next. Could anyone give me a reasonable method to solve this tough problem? Any suggestion is appreciated. Jian Wu -- Jian Wu Ph.D. Student Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS) Tel: 0086-21-54921117 Email: [email protected]
