Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Ho Leung Ng
Some other things to try: 1. Co-crystallize with a ligand 2. crystallization with lipid cubic phase or bicelles 3. limited proteolysis to define a rigid core ho - Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Pascal Egea
Hi Qiangmin, All the comments and references that were already mentioned to you are excellent, I would stress 3 points. 1- The detergent. A clear distinction should be made between the detergent used for extraction/solubilization and the detergent (or cocktail of detergents/lipids) used for cryst

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Ezra's gmail
I cannot say what you have - running a gel, MS, etc of a washed crystal could confirm what you have. What you did not indicate is if your lack of diffraction was of frozen or unfrozen crystals. I have seen too many cases where it is the cryo condition killing diffraction. So if you have not tried

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Ezra's gmail
I cannot say what you have - running a gel, MS, etc of a washed crystal could confirm what you have. What you did not indicate is if your lack of diffraction was of frozen or unfrozen crystals. I have seen too many cases where it is the cryo condition killing diffraction. So if you have not tried

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Joseph Lyons
When diffraction fails - one can always look at the crystals with a UV microscope (assuming you have Trp) - they should light up. Or if its possible - harvest them, wash and run a gel. (or if its detergent a TLC) Joe 2010/9/1 wu donghui > It does not look like protein crystal and IZIT stainin

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread wu donghui
It does not look like protein crystal and IZIT staining is not reliable in determining protein or other. Mostly it is like detergent or PEG crystal or quasi-crystal. Good luck Wu 2010/8/31 qiangm zhang > Hi all, > > I got a crystal of one membrane protein (~60kD) from Na/K phosphate > co

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Damon Colbert
I have had a lot of problems of my own with poorly diffracting (or not at all) membrane protein crystals. After a previous discussion here, I summarised the suggestions I got in this ccp4 user wiki; http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality There are

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Matthew Chu
Yea, Parveen has actually asked about this here a year ago and I found this discussion quite useful indeed: www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg11717.html and I think we all get tons of these crystals especially in conditions with PEGs. There are lots of things you can try and have been

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Parveen Goyal
Hi, The picture looks like detergent crystals, specially DDM crystals. I have same crystals in many conditions. With regards, Parveen

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Eric Larson
Hello Qiangmin, Here are links to a few web resources that have tips for membrane protein crystallization that may help with your optimization strategy: http://kb.psi-structuralgenomics.org/update/2009/06/full/th_psisgkb.2009.25.html http://web.emeraldbiosystems.com/blog/bid/33916/8-Practical-

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Ed Pozharski
On Tue, 2010-08-31 at 11:57 -0500, Jacob Keller wrote: > I just don't want this guy to get misled into perhaps wasting > months/years on something not particularly promising. Trouble is, of course, that one never knows if a particular trick will work this time. We routinely get PEG/fluoride salt

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Jacob Keller
I have to say that in my limited experience with membrane protein crystallization, these liquid crystal / spherulite type things are very common, and seldom turn into anything useful. Perhaps others on the list more experienced than I can corroborate or refute this. I just don't want this guy to ge

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Ed Pozharski
Well said. I've seen three cases by now when switching to a homologue from a different organism led to solving a structure (and way too many cases when crystals just did not diffract, either at all or well enough :). On Tue, 2010-08-31 at 18:48 +0300, Tommi Kajander wrote: > Or might be worth goi

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Tommi Kajander
Or might be worth going back to the drawing board to design more constructs (and check them around the same conditions), thermostable homologs etc.. what about reductive methylation, anyone had luck with membrane proteins? tommi Quoting "Ed Pozharski" : Unfortunately, some crystals don't

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-08-31 Thread Ed Pozharski
Unfortunately, some crystals don't diffract at all. You may want to try to 100% verify that it's protein either by SDS-PAGE or mass-spec (100x100x100 micron crystal could contain ~0.5mcg of protein, so you my need to use silver staining). If it is, I'd consider trying to get diffracting crystals