Dear Mark, Thanks for your reply. I have already tried P1 SG (we do have 360 degree data) and Zanuda server without success. I do now have crystals in different conditions (and visually they are of different shape). In parallel I am trying heavy metal soaking and hope one of the strategy will yield better model. But its good to know that other people had such cases as there is not much literature about this on the internet.
Thanks and regards, Pravin. On Wed, Apr 12, 2017 at 9:56 AM, Mark J van Raaij <mjvanra...@cnb.csic.es> wrote: > Dear Pravin, > > we've had a couple of these unfortunately, in our case the only solution > has been to find alternative crystal forms. Or several crystal forms in > which different domains are disordered, but allowing to make a composite, > complete structure for interpretation. > Your N-terminal domain is probably is several alternative positions in > different copies of the protein in the crystals. This would give rise to > disordered density, but not complete flexibility, and thus highish Rs. > However, before being sure, you should probably try integrating in lower > symmetry space-groups, going down to P1 if necessary. If you don't have a > dataset of 180 or even better 360 degrees perhaps collect it from a fresh > crystal. And check diffraction images carefully for missed weak spots and a > possible larger cell. > In this case, I don't think better phases would help, but you never know. > I would try placing the nearest structural homologue of the N-terminal > domain as well as you can and see if some density appears, that way you > have corrected the solvent envelope more or less and this could maybe help > a bit. > > Greetings, > > Mark > > Mark J van Raaij > Dpto de Estructura de Macromoleculas > Centro Nacional de Biotecnologia - CSIC > calle Darwin 3 > E-28049 Madrid, Spain > tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016> > http://wwwuser.cnb.csic.es/~mjvanraaij > > On 11 Apr 2017, at 20:54, Pravinkumar Jagtap <pravinja...@gmail.com> > wrote: > > 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. > > > -- Pravin Kumar Jagtap, Department Chemie Lehrstuhl für Biomolekulare NMR-Spektroskopie Technische Universität München Lichtenbergstr. 4 85747 Garching / Munich Germany. Helmholtz Zentrum München Office 110. Building 43b Institute for Structural Biology (STB) Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Ingolstädter Landstr. 1 85764 Neuherberg
