Thanks to all that responded. I sorted this out.

It all appears to stem from the C2->I2 conversion. Forcing everything in processing to stick with C2 fixes all the issues!


Thanks again,

--paul

On 11/03/2016 12:39 PM, Paul Paukstelis wrote:

CCP4BB,

I posted some time back about a DNA oligonucleotide structure we were working on. I had difficulty phasing it despite strong signal from bromines, but finally managed to get reasonable enough maps from a few datasets to build, only to find that despite the density looking quite good, it simply wouldn't refine past R/Rfree of around 28/32. With help from ccp4bb it began to become clear that this might be a candidate for a lattice with translocation defects.

I had my student make a variant in which two 3' nucleotides that weren't involved in base pairing contacts were removed. This crystallized under the same conditions in a different space group and was now diffracting to ~1.0 A (from about 2.2 in the previous space group). Images overall looked good, though we collected on some crystals that clearly had more than one lattice that made indexing more difficult. The best looking data still had some tails on spots, but was easily indexed in C2, which Pointless quite happily changed to I2 to minimize the beta angle. There are no clear alternating strong/weak intensities. Phaser finds a strong solution using the previously built dimer, but notes a 25% over origin peak in native Patterson. Maps look very good, though after the first round of refinement it is apparent that there is another dimer in the ASU, but it is clearly overlapping the first. If I'm not mistaken, all these clues suggest lattice translocation defects. Question 1: any thoughts on how likely it would be for a molecule to intrinsically pack in such a way that it results in lattice translocation defects?

I thought it would be worthwhile pressing on given the high resolution it would be possible to do grouped occupancy refinement of the dimers without taking too huge a hit in observation/parameters. Refinement with refmac using occupancy groups leads to a best R/Rfree of 18/24, though geometry could be better in some spots. Curiously, refmac (or phenix.refine) in the PDB header reports only 50% completeness in the resolution range, though all the data reduction and analysis (aimless, xtriage) report 99% completeness. Question 2: Why is this? Phenix logfile says something about removing about half the reflections as systematic absences. I have been working with everything in I2 after Pointless switched it over.

Question 3: Any other suggestions on how to proceed with refinement in a case like this? My gut instinct tells me that it would be better to not do intensity correction due to the high resolution, but perhaps that's something to pursue?

Thank you in advance.

--paul