Re: [ccp4bb] membrane protein optimization
Dear Frank, It is difficult to say something without the diffraction images but probably they are detergent ( or lipid) crystals. These crystals are in general soft and don't diffract well. My suggestion is to know well what your crystals really are before any optimization. Best, Isabel Dr Isabel De Moraes, MRSC Membrane Protein Laboratory Diamond Light Source Ltd, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 ODE, UK On 23 Oct 2013, at 17:22, crystalboy wrote: Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank [cid:7839c9ff-b567-4b4b-b191-07dc6c335f34@fed.cclrc.ac.uk] -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] membrane protein optimization
The problem you're having is a very common one, unfortunately. There are about 10,000 things you can try to improve diffractiongenerally going from low to solvable resolution is very, very hard. I would say the two most important ones are (i) to vary the detergent and (ii) to go to another homolog if (i) doesn't work. You don't mention which detergent you're using (DDM?), but the best chance of success would be to try harsher detergents, ie those that form small micelles (shorter chain maltosides, b-OG, LDAO etc). Give bicelles a try too, and LCP if you know someone with an LCP robot. If your protein is stable only in DDM you're out of luck and the best thing would be to look for an ortholog that is more stable. Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 5:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] membrane protein optimization
Hi Frank, unfortunately it is very common with membrane protein crystals to get stuck with diffraction quality around 20A. From what you describe you could consider the following: a) revise the detergent you solubilize your MP with (e.g. use OG instead of DDM), and consider to change to another (shorter) detergent that might allow tighter crystal packing b) engineer your MP,mutate charged residues to alanine (check function!) c) try homolog protein d) try to crystallize in lipid cubic phase Best of luck! Susanne On Oct 23, 2013, at 9:22 AM, crystalboy wrote: Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank 20130917020056875.jpg Dr. Susanne Ressl Otto Hahn Fellow of the Max-Planck Society Stanford University School of Medicine James H. Clark Center 318 Campus Drive, Room E300 Stanford, CA 94305-5432 Phone: +1-650-736-1715
Re: [ccp4bb] membrane protein optimization
Hi Frank, Do not forget that membrane protein crystals are often fragile and difficult to manipulate. Finding good cryo condition can be difficult and small temperature variation can destroy crystals within minutes, this makes room temperature diffraction tests not always obvious. The time of harvest may also be critical. In addition to the previous suggestions, it is often usefull to check if there is a large amount of lipids in the sample. This can often easily be done by thin layer chromatography. HTH Daniel Le 23/10/2013 18:22, crystalboy a écrit : Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] membrane protein optimization
Hi Frank, The previous suggestions are great. In my case, I had soft crystals (bend from 180° to ~130-140°) in the loop using the LCP but they still diffracted. There are a lot of heroic stories on how some people solved a structure, you should just try as many ways as possible (do not only rely for a long time on only one condition or construct), and remember methods are just methods (i.e., there are hundreds of LCP robots in the world..). Protein engineering, homologs (maybe hyperthermophiles).. stabilization.. are great ways. Recently I had tested various crystals grown by vapor diffusion for a protein purified in different ways/detergents, so obtaining crystals is very common, but it's just the start. I also had crystals using bicelles but saw huge spots to 2 A (no patterns.. rather looked like detergent/lipid). Good luck toufic el arnaout From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank
Re: [ccp4bb] membrane protein optimization
Dear Frank, Toufic is right. The trick is no trick, you just have to try everything. In my experience if u have a protein crystals in a mild detergent and it is very fragile no point going to a harsh or shorter chain detergent like OG. I got crystals in NM (9 C) and they were fragile and dissolved after 1 day. I could only get stable crystals when I moved to DM (10 C) and DDM (12 C). Once you get a stable crystal you may consider dehydrating them in higher PEG so as to improve protein protein contact. Again the trick is no trick, you just try everything suggested. Good luck with your trials. Emmanuel Nji. On 23 October 2013 19:46, El Arnaout, Toufic elarnao...@biochem.wustl.eduwrote: Hi Frank, The previous suggestions are great. In my case, I had soft crystals (bend from 180° to ~130-140°) in the loop using the LCP but they still diffracted. There are a lot of heroic stories on how some people solved a structure, you should just try as many ways as possible (do not only rely for a long time on only one condition or construct), and remember methods are just methods (i.e., there are hundreds of LCP robots in the world..). Protein engineering, homologs (maybe hyperthermophiles).. stabilization.. are great ways. Recently I had tested various crystals grown by vapor diffusion for a protein purified in different ways/detergents, so obtaining crystals is very common, but it's just the start. I also had crystals using bicelles but saw huge spots to 2 A (no patterns.. rather looked like detergent/lipid). Good luck toufic el arnaout From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy [yyb...@gmail.com] Sent: Wednesday, October 23, 2013 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] membrane protein optimization Hi CCP4BB Forks, In recently I got a membrane protein crystal in the quite normal membrane protein crystallization conditions as other persons reported, like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using sitting drop method. These crystals are around 50-100 uM. They look like trapezoid crystal. My problem is all of my crystals have not diffraction in home source X-ray and just poor diffraction at Synchrotron (lower than 20 A). My crystals like to appear on the surface of the drop. Look like my crystals are quite light. I had tried to use a needle to touch them. Unlike other protein crystal, my crystal looks like quite soft. When I touch it, it didn't crack, but was bend or mashed. I had tried to do additive screen and detergent screen. It seems they are not useful. Do anyone have good ideas to optimization these crystals? Thanks for your suggestions. Frank -- Dr. Emmanuel Nji Department of Biochemistry and Biophysics Centre for Biomembrane Research Stockholm University 106 91 Stockholm Sweden Cell: 0046736457727
