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
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