Re: [ccp4bb] protein degradation during concentration for crystallization trials

Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to near

Re: [ccp4bb] question about the zinc binding protein

Hi, I'm working with a possible zinc binding protein. Saw your post and was wondering, what is the proper pH range for zinc binding? Thanks, Peter

[ccp4bb] applying B_factor and scale correction to an MTZ

Hello, Is there a tool to apply both the scale and overall B-factor correction found by the wilson plot to a given MTZ file? After applying these corrections, is there another way than redoing wilson to check that the output MTZ is on absolute scale? Thanks a lot, Francois.

Re: [ccp4bb] Follow up to TLS, NCS and refinement

On Thu, Apr 8, 2010 at 3:26 PM, Daniel Bonsor wrote: > Following my previous question, there was something wrong with the staring > model for molecular replacement. Now that is sorted, I have 8 complexes in > the ASU. After a few rounds of refinement with NCS and isotropic Bfactors, > both the Rf

[ccp4bb] Follow up to TLS, NCS and refinement

Hello again! Following my previous question, there was something wrong with the staring model for molecular replacement. Now that is sorted, I have 8 complexes in the ASU. After a few rounds of refinement with NCS and isotropic Bfactors, both the Rfactor and Rfree get stuck at 30% and 36%, resp

Re: [ccp4bb] protein degradation during concentration for crystallization trials

Hi, MBP tag: 1.there might be a TEV cleavage site in your MBP variant. 2. your protein needs salt to stay in solution 3. your protein forms aggregates with MBP GST tag: you probably concentrate a protease together with your protein. You need a protease inhibitor kit to take care of different ty

[ccp4bb] protein degradation during concentration for crystallization trials

Dear all I am working on purification of 14 kd protein(pI  8.3, basic protein)  that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the

[ccp4bb] Phasing statistics

Dear CCP4ers, I've been asked by a referee to provide the phasing statistics for a SAD dataset that I used to solve a recent structure. Whilst I have been able to find a figure-of-merit for the data after phasing, I can't work out how to get any other statistics (e.g. phasing power or an equiva

Re: [ccp4bb] molprobity in coot: BL WARNING:: reduce didnt run ok, so stop here!

Hello Rongjin Guan, your problem most likely comes from different pdb version formats. The files which work are probably pre pdb v. 3.0. Although (Win)Coot is mainly v. 3 compliant here we are not (really (*)). A fix (at least for the files which are not working) may be to change two lines (1