Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 zhangqia...@gmail.com Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 wdh0...@gmail.com 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 zhangqia...@gmail.com Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 yet - try a wet mount. Ezra On 8/30/2010 9:24 PM, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 yet - try a wet mount. Ezra On 8/30/2010 9:24 PM, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 crystallization. These are two very different things. If you are lucky you may not need to change, but once you have extracted your rmembrane protein in one detergent, you should try to characterize its homogeneity by size exclusion chromatography in different detergents ( with shorter or longer chains and/or belonging to a different class (change from a choline or a phospho-glycero-lipid to an alkyloside or from a charged to an uncharged detergent etc). This scouting is tedious but is extremely informative and it can be done on analytical scales (so it does not require too much protein). If you like statistics about detergent use you can look there. *http://www.mpdb.tcd.ie/* depending on the class of membrane protein beta-barrel versus all-alpha helical etc etc you can initially concentrate your efforts on a subset of detergents. 2- The diffraction. As mentioned, starting with very poorly diffracting crystals is not uncommon (as it is for RNA crystals). My own personal experience is that you can get from 40 A resolution to the dreadful 6-4.0 A resolution barrier by tweaking the purification/extraction conditions (1/ changing detergent (shorter chain) and 2/ carefully controlling the amount of detergent present in the sample used for crystallization (to avoid or at least minimize the phase separation problem)). 3- The cryo conditions. Crystallization drops in presence of detergent are actually not as homogenous at it seems. Within the same drop you may have crystals of identical size and morphology and freeze them in the same condition and still get very different diffraction limits. When you freeze your crystals matching the detergent concentration in your cryo-condition with the 'expected' concentration in the drop can be extremely important especially with alkylosides (personal experience). Good luck, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 by a) dehydration (chances are slim since you have no diffraction at all) b) microseeding c) additive screen (SilverBullets?) d) screening a lot of crystals (how small are the small ones? They may be good enough for microfocus beamlines and yes, smaller crystals generally tend to produce better diffraction quality) e) this one sounds silly, but make sure you are hitting the crystal with the beam (just shift it up and down a notch and take single shot - I've seen this trick actually working several times) The list is not comprehensive. But frankly, having non-diffracting crystals is a poor place to start, these guys waste a lot of time and often still don't diffract no matter what you try. Good luck, Ed. On Mon, 2010-08-30 at 21:24 -0400, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏 -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 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? -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 get misled into perhaps wasting months/years on something not particularly promising. But, as I said, I am happy to be contested/refuted... Jacob On Tue, Aug 31, 2010 at 11:39 AM, Ed Pozharski epozh...@umaryland.eduwrote: 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 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? -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 crystals, and yet they have to be checked in the beam just in case. I think you touch on a quite difficult question, which is when one has to give up on a crystallization lead as hopeless. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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-Tips-for-Membrane-Protein-Crystallization http://mcl1.ncifcrf.gov/nihxray/Tips-and-Tricks_Crystallization_Membrane_Protein.pdf good luck, Eric __ Eric Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Mon, 30 Aug 2010, qiangm zhang wrote: Hi all, I got a crystal of one membrane protein (~60kD) from Na/K phosphate condition (see getit_4), then I got the improved crystal like getit_5 after trying seeding, different detergents, lipids and additives. But this crystal does not diffract at all, I already tried Izit staining which shows it is protein crystal (detergent crystal?). Does anyone have any good suggestions for the optimization of this membrane protein crystallization? Thank you in advance. Best regards Qiangmin Zhang Biomedical Science Tower 3 Room1034 3501 Fifth Avenue Pittsburgh, PA 15260 -- 张强敏
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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
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 suggested, but I found this reference most helpful by far: A general protocol for the crystallization of membrane proteins for X-ray structural investigation. http://www.ncbi.nlm.nih.gov/pubmed/19360018 (this is actually the paper in the link that Eric posted: http://kb.psi-structuralgenomics.org/update/2009/06/full/th_psisgkb.2009.25.html) Best, Matt On Tue, Aug 31, 2010 at 12:53 PM, Parveen Goyal parveen...@gmail.comwrote: Hi, The picture looks like detergent crystals, specially DDM crystals. I have same crystals in many conditions. With regards, Parveen -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
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 a few ideas there that have already been put forward. My personal opinion is that (assuming your crystals are protein) detergent is the deciding factor. I second the suggestion for shorter chain detergents, but also suggest that you carefully consider your detergent concentration. While you want it to be above cmc, I have found excessive concentration to be bad for crystallisation. If you are going back to the drawing board, I can highly recommend the MemGold and MemStart/Sys screens developed by the lab of So Iwata at the Imperial College, and sold by Molecular Dimensions (in the UK). It has given me a lot of success in getting initial hits for various membrane proteins. MemGold has been designed to specifically target alpha-helical membrane proteins, as described in this paper; http://onlinelibrary.wiley.com/doi/10.1110/ps.073263108/full Hope you find something useful here. Good luck. Damon.
