If you have air in the packing that's worrysome. If symmetry mates don't make crystal contacts you are in trouble.
Have you checked a simple selfrotation function in your currently favored space group ? Do you have sufficient data collected to start out in P1 or C2 ? Then I would start there and systematically look at selfrotation functions in those space groups. Also check the native Patterson for translational NCS. 4 A is not great for stable refinement of cell parameters, which program did you use and which parameters did you fix? Did you use main.ncs=true in the SA approach ? Pointless or xtriage ? Why does it take a year to grow those crystals ? Out of curiosity, how did you collect on this crystal without overlapping reflections ? Good luck, Jürgen ...................... Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Dec 13, 2011, at 23:57, "Stefan Gajewski" <sgajew...@gmail.com> wrote: > I am looking at a highly unusual crystal lattice right now and can't figure > out what is going on, so I decided to ask the experts. > > I recently got data on a oligomeric protein with many highly correlated NCS > units (4.0A resolution, linear R-sym is 0.16-0.21 in I4, I422, F222, C2 and > 0.12 in P1) with severe anisotropic diffraction (according to diffraction > anisotropy server, the F/sigma drops below 3 at a=6.1 b=6.1 c=never, > suggested isotropic B-sharpening -125A^2) This lattice has a problem. The > apparent unit cell is rather huge (roughly 180 180 620 / 90 90 90) > > The unit cell dimensions are almost perfectly I4 and the presence of > systematic absent reflections >50 I/s in I41 and I4122 suggest no screw axis. > I used a very closely related structure solved at 4.2A as molecular > replacement model and got a solution from the anisotropy corrected data in > I422 space group with two oligomers in the asymmetric unit cell. > > Confidence of the MR "solution" is quite high since (a)the MR replacement put > one model one NCS raster off the "true" position resulting in a clash with > the second one in an empty region of the map and additional electron density > on the other side which corresponds perfectly to the wrongly positioned > monomer, and (b) after rotating the model in the "right" position I could > refine the structure to R-work=0.31. R-free=0.35 in one run of rigid body > refinement followed by NCS restrained simulated annealing refinement > (phenix.refine), which is in my opinion really good at such an early stage of > refinement given the low overall resolution and even lower completeness of > strong reflections in a and b due to high anisotropy (observables to atoms > ratio is about 3:1) . I can even see clear density for some of the bulky > sidechains which were not included in the model. > > Now here is the baffling thing. The unit cell is almost empty with an > apparent solvent content of >78%. The molecules cluster around the c-axis and > at the origin with an empty gap in a and b of at least 15A and up to 165A(!) > in the longest dimension. There is no sign of electron density that would > indicate a missing protein in that region and ~98% of my model is already > accounted for by the density in the 2Fo-Fc map, making a contact of > disordered protein regions across the ASUs unlikely. In fact, the protein > density is well defined at the closest gap and no mainchain atom is > unaccounted for in that region. The oligomer has a magnitude of ~105A x 70A. > I heavily doubt that a crystal lattice with such little contacts and holes as > huge as these can exist and therefore think that: > > (a) the R-factors are misleading me to think the solution is correct and > complete > (b) I must have been doing something really wrong > > Since proteins from this family have a well established history of producing > twinned crystals I had a look at that possibility. Analyzing the anisotropy > corrected I4 data for twinning (Padilla & Yeates method) revealed a 2-fold > twin law with a twin fraction of 0.42 which would make the discrimination > between an almost perfectly merohedral twin in I4 and a (non twinned ?) I422 > extremely difficult (to me). MR with anisotropy corrected I4 data gave the > same crystal packing and hence the same void solvent region. MR in lower > point groups was not successful so far although I haven't pursued that idea > vigorously. The same data in I422 has no indication for twinning and in C2 > three 2-fold twin laws. > > Anomalous data is not easily available since those crystals grow in about one > year and getting another crystal is also not very likely because this IS "the > other crystal". > > I am clueless now on how to proceed here and would appreciate advice from > experienced crystallographers on what to try first. > > Am I worrying too much about the packing? > Is it even possible to have such an enormously huge solvent region in a > protein crystal? > What is the recommended protocol when dealing with many and very strongly > correlated NCS units, putative twinning and severe anisotropy all at the same > time? > > Stefan Gajewski
