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hi all i m very thankful for such a quick response and valueable suggestions. As pointed out by Rizkallah, PJ the 2 trimers present in the asu belong to the different molecules n while making the packaging dig the molecule appears as hexamer. i am especially thankful to Gerard DVD Kleywegt for providing me literature on NCS. Vineet On 12/8/06, Rizkallah, PJ (Pierre) <[EMAIL PROTECTED]> wrote:
Hello Vineet, There is something more fundamental about this arrangement than simple NCS. If the protein is genuinely hexameric, then you cannot generate a hexamer from a trimer in P41. What happened to the 2-fold? Two trimers stacked on top of each other do not make a hexamer, one half is the wrong way up! I would check the space group first, it ought to have a 2-fold, like P4122 or P41212. Maybe you should lower the symmetry instead, to P2/P21. Then you can worry about NCS. In my opinion, you should apply loose restraints to the side chains in REFMAC so that the wayward side chains can enjoy their freedom from the NCS straitjacket, while the main chain weights need to be medium. I believe these are the defaults in the REFMAC script generated by CCP4i, but you can verify that. Good Luck. Pierre Rizkallah ************************************************************************ ******* Pierre Rizkallah, Daresbury Laboratory, Warrington, Cheshire WA4 4AD, U.K. Phone: (+)44 1925 603808 Fax: (+)44 1925 603124 e-mail: [EMAIL PROTECTED] html: http://www.srs.ac.uk/px/pjr/ ________________________________ From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Vineet Gaur Sent: 07 December 2006 17:07 To: [email protected] Subject: [ccp4bb]: when to remove NCS during refinement Hi all i m trying to solve the structure of a protein crystallized in p41 space group by molecular replacement. it is a hexameric protein. each asymmetric unit have two trimers. i m solving the structure at 3.2A, by applying NCS between the six protomers. present status of refinement is Rfree: 30.60% and Rcryst: 30.30%, with a fairly good steriochemistry. Among the noncrystallographically related molecules i can see no difference in the main chain but considerable differences in the sidechains. At this stage of refinement with strict NCS i m not able to refine the structure further. i m not able to judge weather i should remove the strict NCS and start refining the individual subunit. so, how should one decide when to remove the NCS and start refining the individual subunits. My second problem related to the NCS is, whenever i remove NCS n start minimizing the pdb obtained after removing NCS, the Rfree start shooting up very rapidly. is it bcoz of larger differences among the NCS related molecule or bcoz of poorly refined structure. pls suggest me what should i do. I am using CNS for refinement. Thanx in advance Vineet Gaur
