Dear Yarrow your toroidal structure suggests that the protein may actually have the propensity to assemble as such in solution, hinting a connection to a biologically relevant state. Do you have any experimental information that it does so? e.g. via SAXS, MALS, native PAGE etc.?
best regards Savvas ---- Savvas Savvides Unit for Structural Biology, L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html On 19 Mar 2014, at 16:58, Yarrow Madrona <amadr...@uci.edu> wrote: > Thank you to everyone for their input. I am posting a picture to some of the > symmetry related molecules shortly. There are six dimers related by symmetry > (60 degrees) with a "donut" hole in the middle. This was troubling to me as I > have solved mostly tighter packing structures (monoclinic or orthorhombic) in > the past. If expanded further there are a bunch of tightly packed donut holes > (though I didn't show these). > > I want to know if this is really a viable solution. The crystals are huge > (300microns X 300microns) and this would maybe explain why they are only > diffracting to 3.2 angstroms. Thank you! > > https://www.dropbox.com/s/r01u37owbkz9pon/donut.png > > -Yarrow > > > > On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona <amadr...@uci.edu> wrote: > Yes in the first couple of rounds of refinement it refines very well for a > 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs > contiguously except for a "donut" hole in between six dimers that are related > by symmetry. Trying to put a molecule there disrupts the symmetry and leads > to clashes. I have a synchrotron trip next week, hopefully this should help > clear things up a bit. > > > On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson <eleanor.dod...@york.ac.uk> > wrote: > I think you have solved it! That is an excellent LLG and if you can't see > anything else in the map, then there s prob. not another molecule. > Does it refine? If you look at the maps following refinement any missing > features should become more obvious. > Solvent content of 65% is not uncommon. > Eleanor > > > On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: > >> Hello CCP4 Users, >> >> I recently collected data in-house on an Raxis IV and am trying to solve a >> 3.2 angstrom structure. >> I have obtained only "partial solutions" using Phaser and would like some >> help. I believe I only have two molecules in the ASU instead of three as >> suggested by the mathew's calculation. I believe I have two molecules in the >> ASU with a space group of P312 despite a high solvent content. I have >> outlined by line of reasoning below. >> >> 1. Indexes as primitive hexagonal >> >> 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm >> assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis >> in the P312 space group. See this link, >> https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf >> >> 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. >> Phaser gives solutions for only 4 molecules. >> >> 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. >> Phaser gives solutions for only 2 molecules. >> Mathews calculation for data scaled in P312: >> >> For estimated molecular weight 44000. >> >> Nmol/asym Matthews Coeff %solvent P(3.20) P(tot) >> >> ____________________________________________________________ >> >> 1 6.84 82.03 0.00 0.00 >> >> 2 3.42 64.07 0.18 0.13 >> >> 3 2.28 46.10 0.81 0.86 >> >> 4 1.71 28.13 0.01 0.01 >> >> 5 1.37 10.17 0.00 0.00 >> >> ____________________________________________________________ >> >> Phaser Stats: >> >> >> Partial Solution for data scaled in P312: >> >> RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 >> TFZ==30.3 >> >> 6. No peaks in patterson map (No translational symmetry). >> >> 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 >> bromo-phenyl Immidazole) despite not including them in the search model. >> 6. There is only one "black hole" where it would be possible place another >> subunit but there is not much interpretable density and the symmetry of the >> space group would be broken if this was done. Six Dimers are arranged around >> this hole. I can post a picture if anyone wants to see it. >> 6. Early refinement of the "partial solution" gives an Rwork/Rfee ~ 24%/31% >> for a 3.2 angstrom data set. Probably over parameterized judging by the gap >> in R/Rfree but still better than I would guess if I had only 2/3 of the ASU >> composition. >> My belief is that there really is only two molecules in the ASU and that >> there just happens to be a very large solvent channel giving a 65% solvent >> content. >> >> I would like help in determining whether this is likely or if I have missed >> something. Thank you for your help in advance! >> >> -Yarrow >> >> >> >> Post Doctoral Scholar >> UCSF >> >> Genentech Hall, Rm N551 >> >> 600 16th St., San Francisco, CA 94158-2517 >> > > >
