Re: [ccp4bb] Bicelle Crystallisation
Hi Rhys, I hope you are well. The JOVE article for bicelles is nice, plz check the video. There is a table in the text section showing some structures that originated from crystals grown at 4-7 % bicelle conc. using not only the DMPC/CHAPSO mixture... The other parameter that everyone tries is of course temperature which has effects on the crystallization process, viscosity phase diagram (because of detergents, and more complicated due to the additional components in this case), etc. Probably you know..: bicelles' term = bilayer + micelles... there's a thousand parameter one can adjust, and screening is the best way around it for now when used as a method. Once I read that a team obtained great crystals with it after six months at 4 °C... So many advices/logic in our field are time dependent :) Once you have the bicelle stock which can be stored for long time.. setting up the trials then is as easy as normal/classic methods. Not much experience, but for similar lipid-rich methods for example, minor amounts of detergent can strongly change the phase behavior... We might have heard of too many hypotheses, but some say that anything (more than one phase) that has a minimum and particular arrangement// i.e., is a bilayer etc, may result in crystals... Best wishes toufic el arnaout From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Friday, December 13, 2013 3:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Bicelle Crystallisation Hi All, I'm thinking of embarking on some crystallisation of a membrane protein in bicelles in the new year. In the methods I've read you simply take your protein in detergent and add the bicelle mxiture (Chapso+DMPC) to your protein allow the solution to equilibrate and set up your screens. What I was wondering is will the original concentration of detergent in your protein sample, effect the likelihood of crystallization (i.e. by changing the structure of the bicelle)? If so do people generally seek to minimize the detergent concentration of the sample before setting up the bicelles. Cheers, Rhys
[ccp4bb] Mr Ashley Bray
Dear CCP4 community, I am very stunned to hear about the death of Mr. Ashley Bray. He was a friend and colleague when I was in Dublin, where he was doing his PhD in membrane protein crystallography. He knew many techniques and was dedicated to his work, and to become a good scientist. Ashley finished his MSc last year at the University of Oxford with high scores. He lived mostly in the region between London and Southampton. He was a young guy in his early twenties. He was very polite, peaceful, and a human right activist. He was very skilled in playing piano. I still remember when I asked him why is he vegan, he then alerted me to the fact of how male animals are treated and killed since they are useless for the production of eggs and milk. To Ashley and his family, blessings during this time and always. toufic
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] Concentrating purified membrane protein
Hi, That is true, Michael.. can't remember the reference, but in some study they found that even if you use a higher MWCO (for example 100 kDa) with DDM (micelle 65-70 kDa), there is still 70-80 % retained detergent in the protein sample. Raji, another thing I would like to say is that the right MW cut-off may depend on the protein, the detergent micelle, but also the bound detergent molecules per protein. It could be 40-50 molecules of DM let's say for protein A, but 80-90 molecules for protein B. You can quickly evaluate the concentration step for your protein using SDS-PAGE or UV abs, and for the detergent there are many methods like colorimetric assays, TLC.. Best wishes t toufic el arnaout School of Medicine - 660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110, USA From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of R. M. Garavito [rmgarav...@gmail.com] Sent: Sunday, July 14, 2013 11:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Concentrating purified membrane protein Raji, One point the most people forget about is that whenever you concentrate any detergent-solubilized membrane protein is that you will ALWAYS concentrate the detergent. So regardless of the MWCO, if the protein-detergent complex concentrates, the overalldetergentconcentration also increases. What you want to shoot for is balancing protein loss with obtaining a sample having minimal EXCESS free detergent. While a concentration step with a Ni-column followed by dialysis will work, so will a wise choice of concentrator. One trick to moderate highexcess detergent is just to avoid the need for a many fold concentration where you will really concentrate the free detergent. Nonetheless, we have used concentrators effectively, anda Ni-column followed by dialysis as well, but if you still have too much free detergent, you can always use a spin desalting column with G-25 sephedex to bring remove detergent down to a nominal concentration. In all cases, you will lose some protein. Good luck, Michael R. Michael Garavito, Ph.D. Professor ofBiochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office:(517) 355-9724 Lab:(517) 353-9125 FAX:(517) 353-9334 Email:rmgarav...@gmail.com On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote: Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] off topic: good peak on gel filtration
Hello Peter, In addition to the great comments/details, please check the following points I have now in mind.. since you want to relate the size exclusion peak/profile to the crystallization: - occasionnaly, some perfect peaks that you might think are homogeneous actually correspond to a sample of hetergeneous protein (maybe the target protein will still crystallize, but problems happen during crystal optimization, or/and observing missing electronic density of the N- or C-terminus for example). It might also be reflected on the specific activity (btw if the protein you have is easy to assay, you could check the activity from different fractions if you think broad peak = problem). In some occasions, analyzing fractions from a perfect peak shows on SDS-PAGE a double band or sometimes far bands (I won't comment on oligomerization in this case). - not all proteins from good SE chromatograms crystallize... - some people only collect fractions from the centre of the peak (or for example they measure the A280 max, divide it by two, draw an horizontal line at that value, and collect the fractions/projection between where the line crosses the peak from both sides.. If you add one fraction before or after, the protein might not crystallize anymore. - from the literature, I have seen many shapes of chromatograms: perfect, bleeding, skewed (tail) to the right or left, little broad, etc.. which resulted in diffraction quality crystals. - re-running a protein sample (that crystallizes after the first SEC) for a second SEC might cause the protein not to crystallize anymore (for example membrane proteins might lose lipids etc). - for proteins that are subjected to SEC straight after Ni-NTA, and which have a perfect peak shape: a 4-16 hours delay before injection might show the aggregation effect. A peak shoulder will form, which you might not have seen if the sample was directly injected. The point is: maybe what you incubate for crystallization or work on after SEC might not be anymore that protein with the beautiful peak you had. Using different additives/chemicals/mutations may help in making the protein more stable/thermostable. You can check previous publications of Dr Tate CG, GPCRs for example. This is also a nice paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809. Regards toufic el arnaout School of Medicine - 660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110, USA From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry [ber...@upstate.edu] Sent: Saturday, June 29, 2013 9:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Thanks- guess I'm old-fashioned, using low-pressure columns. So apparently theoretical plates are still calculated, and have improved a lot- 25000/m is HETP .04 mm, way better than the figure I mentioned. (TP per dollar not so much.) No more sour grapes from me- eab Zhijie Li wrote: Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional to the
Re: [ccp4bb] Off-topic: NMR and crystallography
Hi Theresa, Here is a comparison between both methods (Table under 6-Summary):http://www.cryst.bbk.ac.uk/pps97/assignments/projects/ambrus/html.htm If you would like to have an idea about structures of membrane proteins (why was NMR used and what answers they got etc) solved by NMR to date please check:http://www.drorlist.com/nmr/MPNMR.html ; for solid-state NMR:http://www.drorlist.com/nmr/SPNMR.html I'm sure any lab would be happy to solve their structures using both methods (if they can) publish high impact papers. Even if structural differences exist, why not.. more to discuss :) Sometimes large differences are more related to the detergent/environment or many other factors that affect memb proteins. Regards toufic el arnaout School of Medicine -660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110 Date: Sun, 9 Jun 2013 16:36:15 0100 From: theresah...@live.com Subject: [ccp4bb] Off-topic: NMR and crystallography To: CCP4BB@JISCMAIL.AC.UK Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of off-board, to allow for a good discussion. Thank you. Theresa
