Re: [ccp4bb] precipitation of protein in elution buffer

Hi Peter, we had a four similar cases recently. What really helped us was to add urea in amounts of 1-2 M. We tested the possible influence on secondary structure by limited proteolysis and CD/Trp-fluorescence and it seemed the protein maintained the same overall conformation as in the absence

[ccp4bb] degradation of protein durring freez thaw

Hello BB I apolozize an off topic query. I am working with small proetin-protein complex of 24kDa. I purify this N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, 0.3M NaCl . After purification this protein complex are dialysed in 20mM tris pH=8.0.I am able to purify enou

[ccp4bb] precipitation of protein in elution buffer

Hello all I apolozize for a off topic question on BB. I am working with small proetin-protein complex, each have molecular weight 10kDa. I elute this N-terminal His-tagged complex through Ni-NTA resin in 50mM of NaH2Po4, 0.3M of NaCl, 5% glycerol and 250mM of Imidazole.Similarly, Lysis buffer and

Re: [ccp4bb] sigma cutoff for fitting waters in model

Like so many rules of thumb, the 3-sigma fofc and 1-sigma 2fofc is a reasonable guideline that works very well in most cases despite being based on a flawed assumption. The "0.3% chance" of a peak being above 3 "sigmas" assumes that the histogram of electron density values is Gaussian. It is

[ccp4bb] Takeda San Diego-Membrane Protein Chemistry

If someone has an interest in the following positions listed below, please feel free to forward your resume or pass along to your network, if you know someone who may be a good fit! We are looking for individuals who have industry experience. Thanks! Josie Tatone Takeda Pharmaceutic

Re: [ccp4bb] geometry problems with sugars

JED's example is very illustrative and it shows that chirality may need to be added to this link definition. then sugar validation may be easier (at least ASN-NAG with only one sugar). If chirality is wrong then rotate around ND2-C1bond as a rigid group. Just like you do with rotamers. Here

Re: [ccp4bb] geometry problems with sugars

Garib Murshudov wrote: As I see there is no chirality definition for NAG-ASN link (perhaps there should be but then people will be unhappy even more). Only reason i can see for this flattening is conflict between geometry and electron density. Your example shows that even if electron density

Re: [ccp4bb] sigma cutoff for fitting waters in model

Dear Tassos, Am 19.04.10 17:31, schrieb Anastassis Perrakis: The sigma issue a bit more complicated. What we call usually sigma is the root mean square deviation (rmsd) of the map. Lets first recall, that the variation within the protein region is quite large, while the solvent is rather f

Re: [ccp4bb] geometry problems with sugars

As I see there is no chirality definition for NAG-ASN link (perhaps there should be but then people will be unhappy even more). Only reason i can see for this flattening is conflict between geometry and electron density. Your example shows that even if electron density is weak it may play a r

Re: [ccp4bb] geometry problems with sugars

I've seen flattening of the C1 atom recently in Coot - in that case the reason was that, as the density wasn't really clear around the sugar, I'd fitted it 180 degrees rotated around the ASN-ND2---C1-NAG bond. In such cases Coot happily flattens the sugar ring such that C1 C2 C3 C5 O5 are in a p