Radiation Damage? Why don't you try scaling back on the time of each frame, get better completeness and redundancy, while taking a hit on resolution? That could get you your heavy atoms, and then you could apply those phases to a "roasted" crystal. The heavy atoms are the first to go, it seems.
Jacob ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [email protected] ******************************************* ----- Original Message ----- From: Kumar To: [email protected] Sent: Friday, March 20, 2009 2:26 PM Subject: [ccp4bb] Problems with phasing a protein (1300aa) Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. 'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very quickly on most of the beamlines. We have scanned at Se wavelength and it gives very strong signal as it contain ~45 Se in AU (1300 aa). It is difficult to collect a complete dataset (maximally we get 50-60 % completion with Rmerge ~15) out of one crystal on regular beamline. At microfocus beamline (APS), we were able to collect data in 3-4 batches and merge them to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data collected on microfocus beamline (at peak wavelength) for locating heavy atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no difference in original and inverted (contrast and connectivity). Our phasing attempts with datasets obtained after merging two incomplete dataset from two different crystal has also been disappointing. My another worry is absolute value of average intensity, which seems to be quite low in most of the datasets. Below I have pasted last table of scale.log (HKL2000). Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 7.53 45.4 1.6 1.3 1.295 0.055 0.047 7.53 5.98 11.4 1.3 1.3 0.672 0.135 0.114 5.98 5.23 11.2 1.6 1.6 0.643 0.171 0.152 5.23 4.75 16.8 2.0 1.9 0.736 0.148 0.118 4.75 4.41 18.8 2.2 2.2 0.739 0.143 0.132 4.41 4.15 14.6 2.4 2.4 0.653 0.190 0.175 4.15 3.94 11.3 2.5 2.5 0.582 0.247 0.226 3.94 3.77 10.1 2.8 2.8 0.511 0.280 0.191 3.77 3.63 8.0 3.1 3.1 0.450 0.315 0.285 3.63 3.50 7.6 3.3 3.2 0.483 0.311 0.270 All reflections 15.5 2.3 2.2 0.694 0.153 0.106 Now, I want you to help me by answering some of my queries: 1. Is it possible to get MAD/SAD phasing done from a dataset having more than 15% Rmerge and resolution in the range of 4 - 4.5 Ang? 2. Will a complete data set obtained from merging various batches(30-40 frames each) from one or more than one crystal will have proper anomalous signal for phasing? I am worried as weak anomalous signal may get lost while merging. 3. Will such a low value of average Intensities (as shown above from HKL scale log file) will be good enough for MAD/SAD phasing or I really need to improve crystal quality for stronger diffraction. 4. For MAD/SAD phasing, till what resolution we need to have anomalous signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A (calculated using Phenix.xtriage). 5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge (14-15%), lower value of average intensity, anomalous signal up to 6 A or so..... which programs will be more useful for heavy atom location and to prevent false positives from being selected? We have been also trying our luck with heavy atom soak but that also has not been very encouraging. I would appreciate any suggestions in this regard. Thanks in advance and sorry for such a long mail. Kumar
