Hi,
Sometimes automated model building needs more manual intervention than one
might expect, although it sounds like you've already carefully inspected the
"good" regions.
Could you use a model of the N-ter (from a homolog) simply to create a
solvent envelope, then see if solvent flattening (or more sophisticated modern
solvent-massaging tricks) improves the density in that region?
Phoebe
________________________________
From: CCP4 bulletin board [[email protected]] on behalf of Pravinkumar
Jagtap [[email protected]]
Sent: Tuesday, April 11, 2017 1:54 PM
To: [email protected]
Subject: [ccp4bb] High Rfree: Phasing issue or partial crystal disorder
Dear All,
I am stuck with this problem for 2 months and hope you could help.
We have a 2.1 A dataset for a 380 amino acid long protein. The space group is
I4 (single molecule in asymmetric unit, 48% solvent content) and the dataset is
quite perfect (no obvious pathologies). The protein itself is organised in 2
lobes (N and C terminal lobes). The sequence identity to nearest homologue
structure is 17%.
We could get the phases by SeMet SAD phasing (3A resolution dataset, 5 SeMet
(excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe and 2
partial occupancy (~0.5 each; present on surface) SeMet in N-terminal lobe).
Automated model building (at 2.1 A) yielded nice model for the C-terminal lobe
(215 residues) and manually I could build parts (around 80 residues) of
N-terminal lobe with high confidence. In addition we could also build a ligand
which is sandwiched between C and N terminal lobe.
However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some patchy
density left at the N terminal lobe but as it is discontinuous, I cannot build
anything in it (except lots of water molecules). In total I am missing around
85 residues. These residues are predicted to be present in secondary structure
(and not flexible).
As I have around 75-80% model built, I would expect that I would have all the
phases and should get nice density for the remaining part. But as I dont see
it, could the rest part be flexible? But again, this is not reflected in the R
factors (I would then expect low Rfree).
Could it be that I still lack phases (due to partial occupancy of SeMeth in
N-terminal lobe ) and have to try to get them by heavy metal soaking, or there
is disorder in the N-terminal lobe? I have also tried solving different
datasets for same crystal but this has not been useful.
Regards,
Pravin.
