Hi, I would like to seek some advices on experimental phasing.
I have datasets of the following: 1. Around 4-6 datasets per crystal collected at sulfur edge. There's no anomalous signal but I used one of the best dataset collected as the native dataset (nat2). 2. Peak (aupeak) and inflexion (auinf) for a gold derivative 3. Peak for a hg derivative (hgE3pk) 4. One native dataset Here's the scaleit summary for the different datasets Table: The Rfactor Riso |F_nat2 FP_hgE3pk F_aupeak F_auinf ---------------------------------------------------------------- F_nat2 | 0.380 0.239 0.239 FP_hgE3pk |0.380 0.388 0.379 F_aupeak |0.239 0.388 0.126 F_auinf |0.239 0.379 0.126 Table: Normal Probability for acentric data Normal Prob. |F_nat2 FP_hgE3pk F_aupeak F_auinf ---------------------------------------------------------------- F_nat2 | 3.112 4.661 5.057 FP_hgE3pk |3.112 2.803 2.777 F_aupeak |4.661 2.803 2.120 F_auinf |5.057 2.777 2.120 Table: Normal Probability for Centric data Normal Prob. |F_nat2 FP_hgE3pk F_aupeak F_auinf ---------------------------------------------------------------- F_nat2 | 2.688 3.127 3.091 FP_hgE3pk |2.688 1.966 2.017 F_aupeak |3.127 1.966 1.288 F_auinf |3.091 2.017 1.288 Table: Anomalous Differences ( FPHi+ v. FPHi-) Anom difference |nref_cent Prob_cent nref_acent Prob_acent Rfactor -------------------------------------------------------------------------------------- F(+)_hgE3pk v F(-)_hgE3pk|361 0.346 1538 0.732 0.209 F_aupeak(+) v F_aupeak(-)|353 0.020 1552 1.094 0.104 F_auinf(+) v F_auinf(-) |557 0.024 2949 1.200 0.105 Sulfur SAD with the datasets collected at the sulfur edges was not successful. - There are 14 S in the 309 amino acids long protein. - These datasets range from 2.5A to 3A. - No anomalous signal can be detected even (all the CCanom << 0.1 across all the resolution shells) SAD with the Hg peak datasets wasn't successful too - Unable to find the positions of the Hg atoms with shelxd/phenix hyss But! I was able to find 3 au atoms using shelxD using the SIRAS (SAD/MAD did not work) protocol with aupeak (resolution to 4A) and nat2 (resolution to 2.5A). - shelxE built about 2/3 of the model with nice (but disconnected) helices. - After density modification, the map improved with obvious solvent edges. However, after rounds of refinement using refmac, the Rfree remains >0.5. - Autobuilding using arp/warp and phenix autobuild did not complete the model. In fact, only 1/3 of protein was build as loops. - I attempted to build the rest of the model and connect the helices manually using coot but there's a lot of ambiguous electron densities extending in two different directions. I can't really comprehend it. It is so near yet so far. Is the structure really solved? If it's solved, is there anything that I could do to build the model? Can I make use of the other datasets that I have to help improve the phases? Really hope to hear from you soon! Many thanks in advance!! Cheers, Kelly
