Re: [ccp4bb] protein dodecamer

Check with PISA@EBI, it has database searches where you can fetch all dodecamers in the PDB Eugene On 20 Feb 2013, at 06:29, Hui Wang wrote: Dear all, I am looking for proteins that form a dodecameric ring structure (Not two rings of hexamer Nor tetrahedral distribution of subunit trimers).

Re: [ccp4bb] protein dodecamer

Hi You can do this type of selection from this service at PDBe. It provides you with many options that you can combine into a query to make selections of PDB-ID. http://www.ebi.ac.uk/pdbe-as/pdbeselect/PDBeSelect.jsp From this page : select from the drop down [Assembly] - Assembly Name Click

[ccp4bb] Problems in running refine_den in cns_solve

Dear all, I have a problem while running CNS DEN refinement. After running the CNS minimize refinement, the program was aborted and an error message was displayed as: ** Error: test set array test does not exist

[ccp4bb] off topic question BIAcore problem

Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I

[ccp4bb] Thrombin cleavage of membrane protein with fusion tag

Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon

Re: [ccp4bb] off topic question BIAcore problem

I assume you use CM5 chips ? I further assume you run at pH 7.5 perhaps ? What's the pI of your analyte ? 7.5 ? Do you get significant binding to your reference cell under the conditions you are running ? You might get rebinding to your negatively charged surface and the dissociation you are

Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag

Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions

Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag

Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times.

Re: [ccp4bb] off topic question BIAcore problem

Dear Xianchi, unfortunately, dissociation rate constant kd 10^-6 s^-1 was just beyond the limit of Biacore in 1999 (e.g. see Fig. 1 in http://www.ncbi.nlm.nih.gov/pubmed/10556876). I am not sure about these days. As Juergen noted, you may have a problem with rebinding (you could try to reduce

Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag

Dear Pascal and Toufic, Many thanks to both of you for the pointers. Toufic, I actually poked around online and bought exactly the same kit that you are suggesting, especially because the beads are reusable. So your experience is reassuring. So I'll find out soon. I also plan to play around with

Re: [ccp4bb] off topic question BIAcore problem

Hi Xianchi, First of all: a very slow dissociation rate can also be an artifact: the analyte can be simply precipitating on the surface. You do need to rule this out by proper controls. But there is no rule saying that 10e-6 s-1 off rate is not realistic. Even with protein-small molecule

[ccp4bb] Dell linux box and stereo monitor

Hi All, I am about to order some linux boxes for protein crystallography use from DELL. Could you guys share the experience which model would be the best for this purpose? Also I know the stereo monitor topic has been discussed previously, but I would like to get the opinion for the current best

[ccp4bb] Opening for a Post Doctoral Fellow in Crystallography at Genentech

Dear CCP4bb readers, We have an opening for a talented Post Doc in X-ray crystallography in the Genentech Department of Structural Biology. If interested, please apply online at http://www.gene.com/careers/detail/00411397/Postdoctoral-Research-Fellow-Xray-Crystallography . Postdoctoral

[ccp4bb] Improving Homology Models

Dear Crystallographers, it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would put in a sort of polishing step, but they don't seem to. Is

Re: [ccp4bb] Dell linux box and stereo monitor

My 0.02 on this as always. For monitors, I was worried about this at first. I replaced all my SGI units (with what was good graphics and CRT displays), and I worried about getting a new monitor and losing information with different stereo options. I bought zalman monitors, and they work

Re: [ccp4bb] Improving Homology Models

On Wed, Feb 20, 2013 at 12:39 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: it has been my experience that homology modelling programs get folds pretty well, but sometimes the details are pretty obviously bad, like too-close contacts. One might think that the modelling software would

[ccp4bb] Off Topic GST-tag Protein Purification

I am attempting to purify a protein complex of two proteins. The Protein A and Protein B have been co-expressed in media containing N15-Cys. Protein A has a 6-histadine tag. I am able to purify this complex via a Ni-NTA column with good results. I now have the Protein A-labled/B-labled

Re: [ccp4bb] Off Topic GST-tag Protein Purification

Protein A must be co-expressed as it does not express well alone. Have you tried separating A+B on the Nickel column by flowing 2M urea to help tickle off protein B. The amount will vary depending on the affinity. In my case (~50nM) requires about 1000-1700ml of 20mM Tris, 2M urea pH 7.5. Then

Re: [ccp4bb] off topic question BIAcore problem

Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM.

Re: [ccp4bb] Improving Homology Models

Hi Jacob, In our experience using MODELLERhttp://salilab.org/modeller/ for the homology modeling part, followed by refinement with CHARMMhttp://www.charmm.org/ leads to structures with very reasonable high quality statistics as reported by MolProbity. Both programs are integrated in our

Re: [ccp4bb] Improving Homology Models

I used proteinmodelportal.org to generate homology models. It worked well for us. Huiming Sent from my iPhone On Feb 20, 2013, at 12:39 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, it has been my experience that homology modelling programs get folds

Re: [ccp4bb] off topic question BIAcore problem

Well that looks pretty real then. You might have wrong concentrations in one or the other experiment perhaps hence the difference. Jürgen On Feb 20, 2013, at 5:45 PM, xianchi dong wrote: Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein

Re: [ccp4bb] off topic question BIAcore problem

Hi Xianchi, How did you treat the control flow cell? And what is the composition of the control buffer that you use to disrupt binding? Is it denaturing or a mild condition? If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 1RU from 150RU (assuming you can reach

Re: [ccp4bb] Problems in running refine_den in cns_solve

many thanks. I'll try again. best, On Wed, Feb 20, 2013 at 10:11 PM, Mecy Shi mecy...@gmail.com wrote: Dear all, I have a problem while running CNS DEN refinement. After running the CNS minimize refinement, the program was aborted and an error message was displayed as: