HOW sure are you of the spacegroup? The only difference between I4 ans I41 absences is that l=2n is absent for I4, l=4n for I 41.
If you have the wrong choice half your symmetry equiv molecules will be corret but not the others.. Getting the screw axis wrong is a good way to get a reasonable but not acceptable R factor Eleanor On 12 April 2017 at 16:26, Phoebe A. Rice <pr...@uchicago.edu> wrote: > 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 [CCP4BB@JISCMAIL.AC.UK] on behalf of > Pravinkumar Jagtap [pravinja...@gmail.com] > *Sent:* Tuesday, April 11, 2017 1:54 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *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. > >
