Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?
What is the solution to this? Hi Meisam, you have it, it is just three molecules in the asu. Look at the overall crystal lattice packing and see if you have contacts supporting each molecule. Generate a large representation of your symmetry mates, I suspect you have a channel in your crystal lattice, explaining why your Matthews coefficient leads you in a wrong direction. If you are not convinced, calculate a selfrotation function with Molrep and look at the Chi=120 section and compare it to the Chi=90. At least judging from your R-factors, if I understand you correctly you have not done much of a refinement yet. Have you performed a real space refinement and adjusted side chains etc. ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Nov 21, 2013, at 12:35 PM, Meisam meisam.nosr...@gmail.commailto:meisam.nosr...@gmail.com wrote: Dear CCP4ers I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% linear Rfactor. The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric unit, 53% for 4 molecules, and 36% for 5 molecules. Molecular replacement just finds 3 molecules in the asymmetric unit. Running Phaser also gives a partial solution with 3 molecules. When I refine just the back bone of the protein for the 3 molecules the Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular replacement on the refined structure again, and I fix it as a model to search for another molecule, it still does not find it. I have attached a photo to show the density for the fourth molecule in the asymmetric unit. What is the solution to this? Thanks in advance for your help Meisam An Extra Molecule.png
Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?
half-seriously...old-school method: - apart from the excellent suggestions by Juergen and Phil, you could experimentally determine the density of the crystals and calculate their protein content from that. This looks like a fun method to try: http://journals.iucr.org/j/issues/1999/05/00/wb0070/wb0070.pdf (some work, but would be an independent way to estimate the solvent content) Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 21 Nov 2013, at 18:35, Meisam wrote: Dear CCP4ers I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% linear Rfactor. The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric unit, 53% for 4 molecules, and 36% for 5 molecules. Molecular replacement just finds 3 molecules in the asymmetric unit. Running Phaser also gives a partial solution with 3 molecules. When I refine just the back bone of the protein for the 3 molecules the Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular replacement on the refined structure again, and I fix it as a model to search for another molecule, it still does not find it. I have attached a photo to show the density for the fourth molecule in the asymmetric unit. What is the solution to this? Thanks in advance for your help Meisam An Extra Molecule.png
Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?
Meisam: Probabilities are just that: many of us have had structures with large solvent contents that are statistically unlikely. Pedantic quibble: It scales in P21 Space group with 7% linear Rfactor. really means that it scales in primitive monoclinic with a reasonable Rsymm, and I hope you also checked P2 as well as P21 when doing molecular replacement. P2 is rare, but not unprecedented. When you say refine just the back bone do you mean you're refining just a poly-ALA model or a non-mutated one ? Because if so, absent the side-chains and any waters, an R-factor of 31% quite good. If so, add side-chains and then waters and continue refining and see how things look. 3-fold averaging across the current monomers plus your decent resolution should make the sequence interpretation straightforward. But: * if you can see interpretable density for the fourth molecule, build in secondary structure elements, refine those, repeat until you can see a substructure you recognize, then place the monomer manually. * use Arp/wArp to autobuild your structure - this has the benefit that often the map you'll get out will be a very good one even if you build the remaining monomer manually. If you're lucky it'll build it all for you. * Autobuild in Phenix can also do much the same (I would do the first two, and perhaps all three, in parallel until one emerges a clear winner) * if the above still doesn't resolve things, consider the possibility that the fourth molecule is not what you think it is, or may be statistically disordered Phil Jeffrey Princeton On 11/21/13 12:35 PM, Meisam wrote: Dear CCP4ers I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% linear Rfactor. The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric unit, 53% for 4 molecules, and 36% for 5 molecules. Molecular replacement just finds 3 molecules in the asymmetric unit. Running Phaser also gives a partial solution with 3 molecules. When I refine just the back bone of the protein for the 3 molecules the Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular replacement on the refined structure again, and I fix it as a model to search for another molecule, it still does not find it. I have attached a photo to show the density for the fourth molecule in the asymmetric unit. What is the solution to this? Thanks in advance for your help Meisam
