Hi, Everyone, Thank you all very much for the nice suggestions. I am trying to reply within this email.
I agree that the problem may be rooted from the crystal itself, we noticed during data collection that a wedge of the rotation was very mosaic, HKL2000 was able to pick up the right spots, but the scaling gives high chi^2, and when I used the rejection files, HKL2000 complained "more than 50000 rejections". Colleagues suggested tweaking the error model, the complaint of "more than 50000 rejections' went away and rejection dropped to below 300 spots. The new error model reduced the chi^2 as well as the I/sigI in the low resolution shells. I run the P222 data set with Xtriage, the report says no twining, but the symmetry was too low. However, HKL2000 won't even pick up higher symmetry groups during indexing. I also rescaled the data omitting the bad wedge, xtriage gives "normal" report. Refinement was done with combination of simulated annealing, TLS, ADP, individual sites in Phenix. The molecular replacement was done with CDR-loop-trimmed antibody Fab and antigen structures. The map quality was good and I was able to rebuild the new loops without any problem. I will have beam time later this week, I think it will be better to put a better crystal on. Best regards, Tongqing Tongqing Zhou, Ph.D. Staff Scientist Structural Biology Section Vaccine Research Center, NIAID/NIH Building 40, Room 4607B 40 Convent Drive, MSC3027 Bethesda, MD 20892 (301) 594-8710 (Tel) (301) 793-0794 (Cell) (301) 480-2658 (Fax) ****************************************************************** The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. ****************************************************************** -----Original Message----- From: Eleanor Dodson [mailto:c...@ysbl.york.ac.uk] Sent: Tuesday, June 15, 2010 4:46 AM To: Zhou, Tongqing (NIH/VRC) [E] Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Stuck refinement When this happens, I firstly suspect that the spacegroup may be wrong. We had a case where the symmetry was pseudo I4212 but was really I222 (or was it really I212121) Anyway most of the structure obeyed the I41212 symmetry but there was a tail which did not..) Feed the unmerged reflections into pointless and see what it suggests Eleanor Zhou, Tongqing (NIH/VRC) [E] wrote: > Hi Everyone, > > I have some problem in refining a structure. The data goes to 2.4A (with some > 30% completeness at 2.15A), the structure was solved by MR with Phaser, > refinement was done with Phenix, but the r and r-free are now staying at 26% > and 32%, even with all possible waters and missing fragments added. Data was > collected at APS at cryo condition. One thing I noticed during HKL2000 data > processing was that the chi^2 were way too high at lower resolutions shells, > I had to adjust the default error model in HKL2000 to get the chi^2 to around > 1, but this adjustment reduced the overall I/sigI ratio a lot (from around 20 > to 5). > > The quality of electron density maps looks fine to me for a 2.4 A data set > and I was able to build all the missing CDR loops for the antibody in the > complex. I am lost now, should I just re-collect a new data set? > > Thanks, > > > Tongqing > > Tongqing Zhou, Ph.D. > Staff Scientist > Structural Biology Section > Vaccine Research Center, NIAID/NIH > Building 40, Room 4607B > 40 Convent Drive, MSC3027 > Bethesda, MD 20892 > (301) 594-8710 (Tel) > (301) 793-0794 (Cell) > (301) 480-2658 (Fax) > ****************************************************************** > The information in this e-mail and any of its attachments is confidential and > may contain sensitive information. It should not be used by anyone who is not > the original intended recipient. If you have received this e-mail in error > please inform the sender and delete it from your mailbox or any other storage > devices. National Institute of Allergy and Infectious Diseases shall not > accept liability for any statements made that are sender's own and not > expressly made on behalf of the NIAID by one of its representatives. > ****************************************************************** > >
