How about plotting the solvent content along with resolution limits of the structures?
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel Afonine Sent: Wednesday, December 14, 2011 12:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] very strange lattice: high anisotropy, 78% solvent content and maybe merohedral twinning Hi Stefan, 1) just out of curiosity I wrote a tiny script using CCTBX that estimates solvent content via bulk-solvent mask, and quickly run this script for all PDB structures for which I could re-calculate the R-work within 5% from published value. Also, this script extracted the solvent content values reported in PDB file header. Here is what I get: Histogram of solvent contents (estimated via mask): Solvent content Number of structures 5.980 - 14.482 : 11 14.482 - 22.984 : 109 22.984 - 31.486 : 396 31.486 - 39.988 : 3590 39.988 - 48.490 : 11442 48.490 - 56.992 : 11707 56.992 - 65.494 : 6524 65.494 - 73.996 : 2561 73.996 - 82.498 : 510 82.498 - 91.000 : 19 Histogram of solvent contents (extracted from REMARK records): Solvent content Number of structures 6.000 - 14.300 : 91 14.300 - 22.600 : 550 22.600 - 30.900 : 2046 30.900 - 39.200 : 6487 39.200 - 47.500 : 9566 47.500 - 55.800 : 9050 55.800 - 64.100 : 5853 64.100 - 72.400 : 2420 72.400 - 80.700 : 720 80.700 - 89.000 : 86 So, your 78% is not that uncommon although it is at the high(ish) end. 2) Does Xtriage suggest twinning? If so what happens if you refine with the twin law? 3) Make sure you look at both, 2mFo-DFc with and without missing Fobs filled with DFc (depending on completeness of your data that may make a big difference). Pavel On Tue, Dec 13, 2011 at 8:47 PM, Stefan Gajewski <sgajew...@gmail.com<mailto: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
