Re: [ccp4bb] how to reduce protein solubility

Set up your trays with N:1 ratio of protein:well sol'n, where N > 1, up to, say 5:1? That will tend to concentrate the protein in the drop above the original concentration. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mattiroli,Francesca Sent: Monday, February 16,

Re: [ccp4bb] how to reduce protein solubility

Bringing the pH closer to the measured pI would definitely help. The pI can be measured on an IEF gel. Glycosylation, if applicable, could dramatically increase solubility. Roger Rowlett On Feb 16, 2015 11:33 PM, "Mattiroli,Francesca" < francesca.mattir...@colostate.edu> wrote: > Hi all, > > I a

[ccp4bb] how to reduce protein solubility

Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable

[ccp4bb] P321 space group problem

Dear all, I am trying to crystallize a small soluble protein. I have collected few diffraction data sets for the same. Every time I collect a data set I find that the crystal is diffracting at different angles only in one particular direction, the z direction. These are few examples for the nat

Re: [ccp4bb] Cis-peptide bond checking

To add to my rather spotty coverage of the literature on this subject: - M. S. Weiss, H. J. Metzner & R. Hilgenfeld (1998). Two non-proline cis peptide bonds may be important for factor XIII function. FEBS Lett. 423, 291-296. - M. S. Weiss, A. Jabs & R. Hilgenfeld (1998). Peptide bonds revisite

Re: [ccp4bb] Cis-peptide bond checking

Dear Wouter, That does sound like a useful tool indeed - finding the proverbial needle in a haystack! That's the challenge with such a rare event: rather like a "true" Ramachandran outlier, when they do occur they're usually a sign of an important motif in your protein that should be remarked

[ccp4bb] Application deadline for RapiData 2015 approaching

The 2015 RapiData Course on Data Collection and Structure Solution will take place at the Stanford Synchrotron Radiation Lightsource (SSRL) from May 3-8 2015. The aim of the course is to educate and train scientists in data collection and processing methods at synchrotron beamlines, using state-of-

Re: [ccp4bb] Cis-peptide bond checking

Great reminder. But there are real non proline cis peptide bonds, including highly conserved motifs in active sites, e.g. 3UAO and its homologs. I would hope these don't get "corrected". Roger Rowlett On Feb 16, 2015 5:09 AM, "Tristan Croll" wrote: > Dear all, > > > My apologies for the spam-l

Re: [ccp4bb] Cis-peptide bond checking

Hi Ed, That is exactly the point the validation tools in WHAT_CHECK, to detect cis-peptides if missed. I think these are more likely to have biological implications than the transpeptides modeled as cis. Most of those cases are part of poorly modeled regions with difficult to interpret density.

Re: [ccp4bb] Cis-peptide bond checking

However it is important to note that there are real non-proline cis peptides in high-resolution structures, and to not throw out these babies with the bathwater! In fact I think they are probably under-represented because people are hesitant to build a non-pro cis peptide even when the density

Re: [ccp4bb] Cis-peptide bond checking

Hi Tristan, Wouter, Gert and everyone else, I hope that the sudden surge of zeal for eradication of error, re wrongly assigned cis peptides that involve non-proline residues, would not become too great to accept that there are exceptions. I agree, 3% in one entry is probably unjustified, and ma

Re: [ccp4bb] Cis-peptide bond checking

Dear Tristan, Thank you for your post of earlier today regarding the problem of cis and trans peptide planes in the PDB. We also realised this problem a while ago and an article describing this problem and a solution is presently under review at Acta Cryst. D. After analysis of the PDB we can s

Re: [ccp4bb] Cis-peptide bond checking

Yes, but when the validation reports are as comprehensive as this, it's all too easy to assume that they've covered all possible issues and that the lack of a list of cis bonds means there are none. I understand that the next release of Mo

Re: [ccp4bb] Cis-peptide bond checking

Hmm..CISPEP records are provided. CISPEP 1 GLY A 63LEU A 64 0 1.59 CISPEP 2 LEU A 69PRO A 70 0 -14.05 CISPEP 3 PRO A 70PRO A 71 0-2.41 CISPEP 4 ASP A 162GLY A 163 0-8.79 Which can

[ccp4bb] Cis-peptide bond checking

Dear all, My apologies for the spam-like nature of my post, but I would like to draw your attention to an important issue (outlined in an upcoming short communication to Acta D, which will appear at doi:10.1107/S1399004715000826 once it's online). At present, neither the structural quality che