Dear Dhirendra, Your problems probably stems from trying to solve your structure using tantalum clusters at too high resolution. You may also have high anisotropy in your data since you get that negative eigenvalue error message in CTRUNCATE.
First, I suggest you look at the Harker sections from a Patterson synthesis up to say 6-7 Å resolution. If they look reasonable, solve that substructure (manually or with RSPS if you are rusty) and generate some phases to 6-7 Å from the anomalous data with your favourite program. Use these phases to look for free heavy atoms (originating from split clusters) using difference fouriers. The pseudo-translational symmetry may help you in analysing and solving the heavy-atom substructure. When you have included all significant sites you should then hopefully be able to see the four separate protomers. You can then easily generate vectors for averaging by searching with a cut-out sphere of density (you can use PHASER). Make sure you see the pseudo-translational symmetry. Then, phase extend (with for example DM) using the fourfold averaging in ~300 steps starting from ~7-10 Å (try some variations to see what works best). I'd use 'O' to make and manually 3D-edit a good averaging mask (extend this mask to make a solvent mask, too). If the density isn't good enough after this you can try fourfold averaging in combination with multi-crystal averaging since your native was very non-isomorphous to your tantalum cluster data. Here, try phase extension using DMMULTI starting from ~7-10 Å in 300-500 steps (try some variations to see what works best). In parallel, it wouldn't hurt to try some other (smaller) heavy atoms. You can use the phases from the tantalum cluster data to find them easily. The CTRUNCATE problems you mention most probably stems from very high anisotropy in your data. We have found that the old truncate program works for some extremely anisotropic datasets (our typical datasets to be honest) when the new CTRUNCATE don't (giving the same error message as you got). Just select "old truncate" if you use the CCP4 interface. Later on, use the anisotropy server at NIH to analyse this. Using this server to generate an "anisotropically corrected" mtzfile will most probably enhance your results in the DM and DMMULTI sessions described above. Cheers, Martin On Mar 6, 2010, at 12:52 AM, Dhirendra K Simanshu wrote: > Dear all, > > I am trying to phase 172 aa protein using tantalum bromide (Ta6Br12) cluster. > Data belongs to space group C2221 with 4 molecules (2 dimers) in the > asymmetric unit. Phenix.xtriage indicates presence of pseudo-translation > (peak height= 39). I have collected data at Ta peak (1.2550 A) to a > resolution of 2.9A, Ta inflection (1.2553 A) at 2.8 A using one crystal. I > also collected one high energy remote (1.059 A) data at 2.6 A using another > soaked crystal. All three datasets shows presence of very good anomalous > signal till the highest resolution shell. > > Soaking with Tantalum bromide has changed the unit cell axes "a" and "b" by > 4-5 A so it is no longer isomorphous with the native data (2.7 A). But since > anomalous signal is very high in all these Ta soaked datasets, I have been > trying to find a solution using SAD/MAD but without any success. Processing > data in lower symmetry P21 and C2 also doesn't help. > > Another noticeable thing is..... "scalepack2mtz" fails when I try to run to > convert my sca file into mtz (using CTRUNCATE option ON) with error saying > "Anisotropic correction failed - negative eigen value". > > > > Since finding a solution has not been possible for reasons unclear to me. I > guess I am having problem because of presence of pseudo-translational > symmetry in my crystal. I would be grateful for any helpful advice or > suggestions in this regard. > > Thanks in advance! > Regards > Dhirendra > > . B. Martin Hallberg, PhD Junior research fellow Section for Cell Biology Department of Cell and Molecular Biology Karolinska Institutet 171 77 Stockholm Sweden http://tinyurl.com/yzn9y5j
