Dear CCP4 community,
I've been having trouble with a crystal dataset for the past couple of
weeks and am completely stumped about how to best proceed.
I have a small 12 kDa beta-sheet sandwich protein that crystallised in 0.2
M ZnAc, 0.1 M NaCac pH 6.5 and 10% 2-propanol. Through a stepwise increase
of glycerol 10%-15%-20% and decreasing conc of 2-propanol, I was able to
get diffraction to 2.2 Å at the MX1 beamline (Aust synchrotron).
I have processed the data in mosflm, with a unit cell of 50.4, 71.4, 126.1,
90, 90, 90 and sg of P222.
Running through aimless and pointless suggests cutting the data to 2.4 Å
and all other stats look reasonable (can supply if requested). Pointless
guesses the sg to be P22121.
Matthews suggests 4 mols in the ASU, and phaser identifies a translational
NCS vector.
Peak Distance Vector
22.3% 45.7Ã…: FRAC +0.000 +0.417 +0.275 (ORTH 0.0 29.7 34.8)
Searching for 4 mols in the ASU using a chainsawed structure (4U3H), where
there are a handful of mutations only in the loop regions gives a single
solution.
SOLU SET RFZ=4.9 TFZ=9.5 +TNCS PAK=1 LLG=157 RFZ=3.1 TFZ=9.5 +TNCS PAK=2
LLG=113
LLG=497
SOLU SPAC P 2 21 21
The solution looks like a reasonable tetrameric arrangement, however,
problems arise during refinement.
Here, I am unable to have my R-free drop below 0.39. If I delete large
sections of a single monomer and refine, I find that the electron density
disappears.
To me, this indicates that I do not have a correct solution.
I have tried reprocessing in P1, but am unable to find a solution of the
estimated 16 mols in the ASU.
Going back to my diffraction images (attached), it seems that there are
streaky spots throughout my dataset (even on multiple crystals) - so
perhaps mosflm is having trouble correctly indexing.
Any help or suggestions would be greatly appreciated.
Regards, Ben
--
Ben Porebski,
PhD Candidate (A/Prof. Buckle Lab)
Department of Biochemistry and Molecular Biology,
Faculty of Medicine, Nursing and Health Sciences,
Building 77, Monash University, Clayton, Vic 3800, Australia
