Re: [ccp4bb] Rpim and how its related to anomalous signal

2012-11-04 Thread James Foadi
I also think Rpim, as commonly defined, is not directly useful for measuring the anomalous signal. Commenting on its use, though, I find it quite useful to judge whether one is adding useful data to already existing ones. In a multiple crystals context I always look at both Rmeas and Rpim.

[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation,  I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures  with different

Re: [ccp4bb] protein cleavage

2012-11-04 Thread D Bonsor
You do not mention what buffer you are trying to do your cleavage in. You need a reducing agent for TEV to work (e.g. reduced gluthionine, DTT, mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease and the presence of divalent metal ions will/eventually inhibit TEV. TEV

Re: [ccp4bb] protein cleavage

2012-11-04 Thread Cynthia Kinsland
Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. It may be that your protein is not stable/soluble without the MBP fusion. Different buffer conditions may help if your protein is

Re: [ccp4bb] low-resolution data and SG

2012-11-04 Thread Tim Gruene
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear SDY, if you can see extra density after MR into which you can even build or correct the model it is a good sign your chose the correct space group. Check the geometry of your model. I suppose it is very distorted - a matrix weight of 0.03

Re: [ccp4bb] protein cleavage

2012-11-04 Thread SD Y
Rana, I understood that these proteases are very efficient in cleavage. One time, I had a construct MBP-3C-protein, I never able to cleave this particular construct while I could do well with other truncations.The MBP you are seeing might be co-purified with DHBx and unless gel band intensity

[ccp4bb] protein cleavage

2012-11-04 Thread rana ibd
Dear All    Thank you for all your replies, the buffer for the TEV protease that I have used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I have tried using this buffer without NaCl but the TEV protease precipitates when dialyzing over night, as for using glutathione

Re: [ccp4bb] low-resolution data and SG

2012-11-04 Thread Jim Pflugrath
This looks like an output from SCALEPACK. Unfortunately, one has no way to know from the output if 21 and 90 are strong intensities or not. One cannot go by the I/sigmaI alone. For example, suppose there is thermal diffuse scatter at these positions or perhaps there is a cosmic ray or

Re: [ccp4bb] protein cleavage

2012-11-04 Thread Bosch, Juergen
@Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the

Re: [ccp4bb] protein cleavage

2012-11-04 Thread Cynthia Kinsland
I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage as the existence of the MBP band post-cleavage would not have been noteworthy if it had also been there pre-cleavage. A time course or at least a time zero sample is always a good idea. Truncation products prior to

Re: [ccp4bb] protein cleavage

2012-11-04 Thread VAN RAAIJ , MARK JOHAN
We once had a more-or-less MBP-sized fragment before cleavage, but this turned to be a spontaneous mutation. This expression experiment had been started from a glycerol stock with an unknown number of growth cycles prior to expression. Starting from a fresh transformation with the purified

Re: [ccp4bb] Extra electron density

2012-11-04 Thread Sangeetha Vedula
Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with a (charged?) head. Partially occupied perhaps, as they're so close together. On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar yogesh.khando...@gmail.com wrote: Dear All I am working on an enzyme which involved in fatty

Re: [ccp4bb] protein cleavage

2012-11-04 Thread Sangeetha Vedula
Do you see the same MBP band (or corresponding to the same MW, in case it isn't MBP) before cleavage, at the same total concentration of protein? If not, your protein could be crashing out. Even so, if you use SDS and heat up the sample, I am surprised that your protein just disappeared. TEV

Re: [ccp4bb] Unusually low B factors with phenix

2012-11-04 Thread mingzhu wang
Pavel, 1.8.1-1168 also has this problem. I found this problem existed with some dataset, but not all dataset. 1.8.1 give better map, so I built model with 1.8.1 map and go back to 1.7.3 before I deposit the structure to PDB. When I compared the B factor from 1.8.x and 1.7.3, I found