Re: [ccp4bb] offtopic: effect of compound impurities on ITC?

2010-08-26 Thread Savvas Savvides
Hi Francis I guess it depends on how much residual high-affinity binder you have in the mixture and what the difference in affinity is between Y and deriv-Y. Another issue is of course whether Y and derY compete for the same binding site and have the same stoichiometry. A well designed

Re: [ccp4bb] homology modeling

2010-08-26 Thread Christian Rausch
Dear Paul! you might want to have a look at http://salilab.org/modeller/ . The stand alone version is free for academia. Christian ___ Dr. Christian Rausch Lehrstuhl für Biologische Chemie Technische Universität München, Germany On Thu, Aug 26, 2010 at 12:03 AM, Paul Kraft haresea...@yahoo.com

Re: [ccp4bb] Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified?

2010-08-26 Thread Paul Emsley
On 25/08/10 23:24, Garib Murshudov wrote: However 1) coot uses 2mFo-DFc maps Typically yes, but it uses whatever Refmac (or other programs) provide (of course) 2) you should be able to feed any map you want to coot Yes. so it is nice place for experimenting this kind of calculation

Re: [ccp4bb] Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified?

2010-08-26 Thread Eleanor Dodson
Well - that is exactly what COOT does isnt it? Eleanor Hailiang Zhang wrote: Hi, Can some utilities of CCP4 do the real-space refinement locally with the residue range explicitly specified? By the way, I have registered phenix bb. Just didn't realize this before, sorry again. Best Regards,

[ccp4bb] Problems in purification

2010-08-26 Thread ganesh pathare
Dear all, I have problems in purifying a protein. The protein is 38,000 daltons and has a N-ter His-Tag. The protein expression levels are low and as a result I have a limit for the purification steps. Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300 mM NaCl, 20 to 250

Re: [ccp4bb] offtopic: effect of compound impurities on ITC?

2010-08-26 Thread Justin Hall
Hi Francis, I might save you some time by telling you up front you should just go back and purify your compound to remove the impurity, you dont even need to read the rest of this, just go. Along the lines of what Savvas was saying, with any equilibrium binding assay between two direct

[ccp4bb] CALL FOR PROPOSALS FOR ESRF BEAMTIME WITH ONLINE MICROSPEC (22nd to 25th of October 2010]

2010-08-26 Thread David Flot
- CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC Proposal Deadline *9th September 2010* There will be beam time available at the ESRF for MX data collection

[ccp4bb] Protein X-Ray Crystallographers, Oxford

2010-08-26 Thread Richenburg, Alexandra
Senior Scientist X-Ray Crystallographers within our Structural Biology Department Salary: £26,000 - £39,000 + benefits Location: Oxfordshire, UK Full time; Permanent and Temporary (12 month contract) Evotec (UK) Ltd is currently seeking X-Ray Crystallographers for our

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Roger Rowlett
I generally find it is possible to purify most overexpressed proteins, even those at low levels of _expression_, with a combination of IEX and HIC methods. We use step-gradients to do our routine purifications, but may use gradients for polishing. If running

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Ed Pozharski
On Thu, 2010-08-26 at 11:35 -0400, Roger Rowlett wrote: We routinely polish protein preps on Q-sepharose (Mono-Q should be even better) with at least 10 CV gradients over a narrower range of NaCl concentrations, maybe 0-0.5 M or even smaller. Just wanted to add that in my experience the

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Matthew Bratkowski
Hi. What size are the impurities? If they are smaller than your protein, then they could actually be truncation products, which will be difficult to purify away since they maintain some of the same characteristics as the full length protein. You can check for C-terminal truncations using a

Re: [ccp4bb] LIGPLOT or similar (quick summary)

2010-08-26 Thread Mark J van Raaij
Dear All, people have pointed me to: LigPlot+ (http://www.ebi.ac.uk/thornton-srv/software/LigPlus/) MOE from ChemComp (not free) PDBSUM (http://www.ebi.ac.uk/pdbsum/): prerun LIGPLOT results, only for released pdb coordinates. looks like I am stuck to Windows for now - at least it runs.

Re: [ccp4bb] LIGPLOT or similar (quick summary)

2010-08-26 Thread Acharya, Priyamvada (NIH/VRC) [F]
Hi, PDBsum can be used for not released co-ordinates as well- by using the generate option (found on the left panel on the webpage). Priyamvada -Original Message- From: Mark J van Raaij [mailto:mjvanra...@cnb.csic.es] Sent: Thursday, August 26, 2010 12:46 PM To: CCP4BB@JISCMAIL.AC.UK

[ccp4bb] Heme Proteins

2010-08-26 Thread Hari Namboodiri
Hi CCPers Can anyone provide insights about expressing heme containing proteins in E.col? Does E.coli need any porphyrin precursor during expression or you need special E.coli strains. I have references mentioning delta-aminolevulinic acid for one ortholog but none for another. The enzyme is

Re: [ccp4bb] Heme Proteins

2010-08-26 Thread R. M. Garavito
Dear Hari, You might look at Lucy Waskell's experiences with full length mammalian Cytochrome P450 2B4. While she got 50 mg of purified protein per liter of culture, many other heme proteins are much harder to express in E. coli with heme assembly proteins, chaperones, etc. You just

Re: [ccp4bb] Heme Proteins

2010-08-26 Thread Edward A. Berry
I think you need a special trick to express C-type heme proteins. For B hemes (protoheme, protoporphryn 9), which I think is what the P450's have, E.coli is always expressing several proteins which contain it. But I guess there may be some way to up the production in case of an overexpresser.

[ccp4bb] Can CCP4 refine B factors for several residues only?

2010-08-26 Thread Hailiang Zhang
Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Is there anyway CCP4 can do this? Thanks for any suggestions! Best Regards, Hailiang

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Phoebe Rice
Have you tried expression tricks like Rosetta cells? Testing different colonies and/or starting from fresh transformants? Sometimes that matters. If your protein is an oligomer and your contaminants are degradation products, you might try adding some urea. If desparate, you could spike the

Re: [ccp4bb] Heme Proteins

2010-08-26 Thread James Qiu
Hari, I have expressed sulfite oxidase (with a N-terminal heme) in E. coli strain TP1000 which resulted in red protein. I did not supplement the media with iron or a porphyrin precursor. Sincerely, James On Aug 26, 2010, at 12:56 PM, Hari Namboodiri wrote: Hi CCPers Can anyone provide

Re: [ccp4bb] Can CCP4 refine B factors for several residues only?

2010-08-26 Thread Ethan Merritt
On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote: Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try that). Maybe you could tell us what this reason is? Is there

Re: [ccp4bb] Can CCP4 refine B factors for several residues only?

2010-08-26 Thread Hailiang Zhang
Thanks a lot Ethan, I will give it a try. Best Regards, Hailiang On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote: Hi, I want to refine B factors for several residues only (all the other B factors and all coordinates fixed, I know it sounds weird but there is a reason to try

Re: [ccp4bb] Can CCP4 refine B factors for several residues only?

2010-08-26 Thread Garib N Murshudov
One more additional way is to apply harmonic restraint on all coordinates and all B values apart from those you want to refine. If harmonic restraints are strong enough then B values will not move much. But I do not like this option. Regards Garib On 26 Aug 2010, at 19:56, Hailiang Zhang

[ccp4bb] AW: [ccp4bb] Heme Proteins

2010-08-26 Thread Jan Schoepe
Hary, Delta-aminolevulinic acid is the classical heme (heme b) precursor for your experiments. As far as I know, the synthesis of porphyrin goes very quickly in E. coli, since I tried it once with a bacterial P450 and got a fat overexpression band after ~2hrs in the lysate. Also the

Re: [ccp4bb] turn granular to crystal

2010-08-26 Thread yybbll
Hi, A little danger this condition. I suggest you should firstly make sure they are protein crystals. I know it is very difficult. I suggest you can add proteases to the drops. If crystals dissolve, no problem, they are protein crystals. Best Yibin 2010-08-27 yybbll 发件人: rui 发送时间:

Re: [ccp4bb] Problems in purification

2010-08-26 Thread Ho Leung Ng
I assume you are trying to do a non-denaturing prep. Have you run your sample on a size exclusion column to see if it is aggregated? If it is aggregated, it can stick to a lot of contaminating proteins which will be difficult if not impossible to separate. ho On Thu, Aug 26, 2010 at 8:24 AM,