Re: [ccp4bb] Improve diffraction ...any ideas?
Use random microseeding to pick up new conditions, and work with those. See http://www.douglas.co.uk/mms.htm and http://www.douglas.co.uk/MMS_proc.htm for theory, references and practical details On 24 May 2013 03:20, Urmi Dhagat udha...@svi.edu.au wrote: Hi, I am working on a protein antibody complex which readily crystallizes (crystals form overnight and grow over 2-3 days) in 0.1M Bicine pH 9, 10 % PEG8000. The crystals are chunky - shaped like a parallelogram but they diffract poorly to about 8 Å. I have tried the following to improve diffraction: 1. Screen different temperatures 4°C - crystals have bad form and 10°C crystals grow slower but diffraction does not improve. 2. I have done an additive screen – A few hits came up like Yttrium Chloride and Acetonitrile but they don’t improve diffraction either 3. I have tried streak seeding this does not help either 4. Tested different cryo protectants – MPD, PEG400, Ethylene glycol and glycerol - 10 - 15% glycerol seems to work best 5. Not sure if cryo protectant affects diffraction in this case – I will look at room temp diffraction soon to rule this out. 6. Typical diffraction images attached Does anyone have suggestions on what I could try to improve diffraction of my crystals? Urmi Dhagat St Vincent's Institute -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Improve diffraction ...any ideas?
Rajiv, I don't quite get your idea. Once the crystals of the single proteins have grown, you can't soak the other protein in, can you? Or do you mean something else? Umri, if you do get crystals of one of the components it's well worth trying cross-seeding into the complex, again using random screens. There are a few examples where this has worked, particularly with antibodies. Patrick On 24 May 2013 06:43, Rajiv K Bedi rkn...@essex.ac.uk wrote: Dear Umri, I think the main problem is co-crystallization. What I would do is crystallize protein and antibody separately and then soak protein crystals into reservoir solution containing antibody or vice versa. And do try to get crystals from different conditions which may alter the space group and thereby improve diffraction quality, hopefully. All the best, Rajiv -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Improve diffraction ...any ideas?
Hello Scott Setting up rMMS by hand works fine, but it's a bit slow and uses more protein and (sometimes much more important!) more seed stock. We recommend using a Hamilton syringe, preferably with a rounded needle, to set up by hand. 1.0 protein + 0.7 reservoir solution + 0.3 seed stock works well. Dispense the protein and reservoir solution first, then add the seed stock You can clean the needle by passing it through the reservoir just before you dispense seed stock. It's surprising how accurately you can dispense the seed like that with a syringe and your fingers. I can't say whether dispensing by robot or by hand gives better results. The most productive group that I know that used this method had great success by hand before started using a robot, so I'm sure that both work well. Bear in mind that you're likely to get showers of small crystals at first when you use this method. So you will probably need to dilute the seed stock. What works extremely well for this is our new Combinatorial script. Here you arrange the hits in columns, and add a different seed stock to each row. Make a dilution series from neat seed stock to say a 1:100,000 dilution. Add the most dilute seed stock to the first row, the next most dilute to the second and so on. Its a really quick way to get 1-5 crystals per well, and you can easily set up a whole plate with almost the same number of crystals per well. We use the robot to do this but I'm sure it would be easy to do the same thing by hand. Best wishes Patrick On 24 May 2013 13:46, Scott wrote: Dear Patrick, Have you had better optimization using MMS by setting up trays with a robot or into larger sitting drops by hand? Thank you for your suggestions. Cheers, Scott On May 24, 2013, at 8:01 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Use random microseeding to pick up new conditions, and work with those. See http://www.douglas.co.uk/mms.htm and http://www.douglas.co.uk/MMS_proc.htm for theory, references and practical details On 24 May 2013 03:20, Urmi Dhagat udha...@svi.edu.au wrote: Hi, I am working on a protein antibody complex which readily crystallizes (crystals form overnight and grow over 2-3 days) in 0.1M Bicine pH 9, 10 % PEG8000. The crystals are chunky - shaped like a parallelogram but they diffract poorly to about 8 Å. I have tried the following to improve diffraction: 1. Screen different temperatures 4°C - crystals have bad form and 10°C crystals grow slower but diffraction does not improve. 2. I have done an additive screen – A few hits came up like Yttrium Chloride and Acetonitrile but they don’t improve diffraction either 3. I have tried streak seeding this does not help either 4. Tested different cryo protectants – MPD, PEG400, Ethylene glycol and glycerol - 10 - 15% glycerol seems to work best 5. Not sure if cryo protectant affects diffraction in this case – I will look at room temp diffraction soon to rule this out. 6. Typical diffraction images attached Does anyone have suggestions on what I could try to improve diffraction of my crystals? Urmi Dhagat St Vincent's Institute -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein concentration for crystallization
attachments) is strictly prohibited. If you have received this message in error, please contact the sender by reply e-mail message and destroy all copies of the original message (including attachments). -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] structural homologs as cross seeds
There are many examples where cross-seeding with homologous proteins has worked, both in classical and recent work. You should add seed-stocks from any crystals with significant homology to your routine screening experiments in the first place. The important thing is to use *random *screens at first. This has the effect of giving you new leads and also optimizing the ones that you have (plus you can control the number of crystals per drop). See http://hamptonresearch.com/documents/ramc/RAMC2011_T11_Obmolova.pdf (Omolova and colleagues have examples where complexes were seeded with crystals of one of the components and vice versa.) The excellent review by Stura and co covers most of the theoretical points. (1999). Epitaxial jumps. *J. crystal growth*, *196*(2), 250-260. For practical details see the original paper by D'Arcy (Acta Cryst D: 63.4 (2007): 550-554) and also our paper Random microseeding: a theoretical and practical exploration *Crystal Growth Design* 11.8 (2011): 3432-3441. also http://www.douglas.co.uk/MMS_proc.htm Acoot, this is also the approach to use with your cubic crystals. Don't think about it too much, just try it! The only thing that I can't explain is why this random seeding method, including cross-seeding, is not more well-known. Hope it works Patrick On 27 December 2013 21:03, Mark van Raaij mjvanra...@cnb.csic.es wrote: the differences are likely to be on the protein surface, and these would make the crystal contacts, i.e. it would be unlikely the protein could crystallise in the same crystal habit. Never say never in crystallisation (i.e. try anything), but I would go for other things first like additives, different crystallisation techniques, temperatures, concentrations etc. On 27 Dec 2013, at 20:29, Mahesh Lingaraju wrote: Dear all, I was wondering if it sounds logical to use the crystals from a possible structural homolog as seeds to induce nucleation ? (in terms of overall sequence, the proteins are considerably different but based on sequence alignment and structures from other related proteins, it is highly likely the protein would have the same structure.) Please comment if any of you had experience with this. Thank you Happy holidays :) Mahesh -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] seeding, reproducibility
Mahesh, this is a very interesting and slightly controversial question. One approach is to mix together all of the crystals that you have in the initial screen. The idea at the beginning of the project is to get as many diverse hits as possible - you can worry about crystal size, space group and quality later on. If you can collect data from crystals and determine the unit cell then you can be more rational. You can construct a library of polymorphs having different unit cells. This *may *allow you to push the space group in a given condition to the unit cell or space group that you want - maybe you find e.g. that your ligand can only diffuse into the active site with a certain unit cell. There is a very interesting paper with several examples of how to use polymorphs by the Stura group, see below. However, seeding can give rise to crystals with different but related space groups. A very nice example is shown in the wikipedia article about epitaxy - titanium oxide growing on iron oxide. Also Stura's paper on epitaxial jumps is interesting and relevant. Make sure you dilute your seed stock in a systematic way as part of your final optimization - one great advantage of microseeding is that oyu can control the number of crystals per drop by diluting the seed stock. Also, make sure that you use fresh crystals to make the seed stock. For some strange reason old crystals sometimes fail to act as seeds, even though they can be crushed and still diffract. Maybe the unit cell shrinks, or maybe the crystals become cross-linked. Make the seed stock as soon as your crystals stop growing, then freeze them. Seed stocks can almost always be frozen. Best wishes, Patrick *Library of polymorphs:* Vera, Laura, Claudia Antoni, Laurent Devel, Bertrand Czarny, Evelyn Cassar-Lajeunesse, Armando Rossello, Vincent Dive, and Enrico A. Stura. Screening Using Polymorphs for the Crystallization of Protein–Ligand Complexes. Crystal Growth Design 13, no. 5 (2013): 1878-1888. Stura, Enrico A., Jean-Baptiste Charbonnier, and Michael J. Taussig. Epitaxial jumps. Journal of crystal growth 196, no. 2 (1999): 250-260. *Cross-seeding:* Obmolova, Galina, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet, and Gary L. Gilliland. Promoting crystallization of antibody-antigen complexes via microseed matrix screening. Acta Crystallographica Section D: Biological Crystallography 66, no. 8 (2010): 927-933. Abuhammad, Areej, Edward D. Lowe, Michael A. McDonough, P. D. Shaw Stewart, Stefan A. Kolek, Edith Sim, and Elspeth F. Garman. Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by cross-seeding with the homologous protein from M. marinum: triumph over adversity. Acta Crystallographica Section D: Biological Crystallography 69, no. 8 (2013): 1433-1446. *Seed stability etc* Shaw Stewart, Patrick D., Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen, and Peter FM Baldock. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization. Crystal Growth Design 11, no. 8 (2011): 3432-3441. *Original description of the random microseeding method*D'Arcy, Allan, Frederic Villard, and May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica Section D: Biological Crystallography 63, no. 4 (2007): 550-554. On 17 January 2014 22:24, Mahesh Lingaraju mxl1...@psu.edu wrote: Hi Folks The protein that I am working on gives several initial hits which are needles. And at random, I picked the needles from a condition (0.2 M cacl2, 0.1 M HEPES pH 7.5 28% PEG 400) and seeded into another screen where I added 1µl protein + 1µl reservoir solution + 0.3 µl seed stock. I prepared the seed stock fresh by adding 36 µl of the reservoir solution to preexisting 2µl drop. One of the conditions from the seeded screen gave me a hit that looks really promising ( see attached). I am sort of positive that these crystals are protein as they are UV active. I tried to reproduce these but with needles from another condition which actually look much nicer than the seeds i used to produce these crystals. However, I failed to do so. While i understand that seed quality is important, I find it interesting that the crystals do not reproduce with similar or better looking seeds. Is it common that the seeds absolutely need to be from the same condition to reproduce hits ? thanks for any suggestions Mahesh -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] seeding, reproducibility
One or more ingredients in the original hit (which you use to suspend the seed crystals in) can indeed be essential, and may be all you need to get nice crystals. In D'Arcy's original paper you will see that he needed Ca2+ to get nice crystals of one of his proteins. The same is true in Stoddards paper, which inspired D'Arcy, ref below. However in the majority of cases it is just the seed that you need, i.e. there was a nucleation problem in the new hits that appear with seed stock. You can see this very clearly because (in most cases) diluting the seed stock in the same solution gives a corresponding reduction in the number of crystals. Ireton, Gregory C., and Barry L. Stoddard. Microseed matrix screening to improve crystals of yeast cytosine deaminase. *Acta Crystallographica Section D: Biological Crystallography* 60, no. 3 (2004): 601-605. On 18 January 2014 00:18, Mahesh Lingaraju mxl1...@psu.edu wrote: Thanks you for the suggestions, Patrick and Matthias. I was actually wondering if any of the components from the seeding solution actually were important but your explanations sound more logical. I apologize for the large attachment. I did not realize that it was so big. Have a nice weekend ! Mahesh On Fri, Jan 17, 2014 at 6:18 PM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Mahesh, this is a very interesting and slightly controversial question. One approach is to mix together all of the crystals that you have in the initial screen. The idea at the beginning of the project is to get as many diverse hits as possible - you can worry about crystal size, space group and quality later on. If you can collect data from crystals and determine the unit cell then you can be more rational. You can construct a library of polymorphs having different unit cells. This *may *allow you to push the space group in a given condition to the unit cell or space group that you want - maybe you find e.g. that your ligand can only diffuse into the active site with a certain unit cell. There is a very interesting paper with several examples of how to use polymorphs by the Stura group, see below. However, seeding can give rise to crystals with different but related space groups. A very nice example is shown in the wikipedia article about epitaxy - titanium oxide growing on iron oxide. Also Stura's paper on epitaxial jumps is interesting and relevant. Make sure you dilute your seed stock in a systematic way as part of your final optimization - one great advantage of microseeding is that oyu can control the number of crystals per drop by diluting the seed stock. Also, make sure that you use fresh crystals to make the seed stock. For some strange reason old crystals sometimes fail to act as seeds, even though they can be crushed and still diffract. Maybe the unit cell shrinks, or maybe the crystals become cross-linked. Make the seed stock as soon as your crystals stop growing, then freeze them. Seed stocks can almost always be frozen. Best wishes, Patrick *Library of polymorphs:* Vera, Laura, Claudia Antoni, Laurent Devel, Bertrand Czarny, Evelyn Cassar-Lajeunesse, Armando Rossello, Vincent Dive, and Enrico A. Stura. Screening Using Polymorphs for the Crystallization of Protein–Ligand Complexes. Crystal Growth Design 13, no. 5 (2013): 1878-1888. Stura, Enrico A., Jean-Baptiste Charbonnier, and Michael J. Taussig. Epitaxial jumps. Journal of crystal growth 196, no. 2 (1999): 250-260. *Cross-seeding:* Obmolova, Galina, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet, and Gary L. Gilliland. Promoting crystallization of antibody-antigen complexes via microseed matrix screening. Acta Crystallographica Section D: Biological Crystallography 66, no. 8 (2010): 927-933. Abuhammad, Areej, Edward D. Lowe, Michael A. McDonough, P. D. Shaw Stewart, Stefan A. Kolek, Edith Sim, and Elspeth F. Garman. Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by cross-seeding with the homologous protein from M. marinum: triumph over adversity. Acta Crystallographica Section D: Biological Crystallography 69, no. 8 (2013): 1433-1446. *Seed stability etc* Shaw Stewart, Patrick D., Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen, and Peter FM Baldock. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization. Crystal Growth Design 11, no. 8 (2011): 3432-3441. *Original description of the random microseeding method*D'Arcy, Allan, Frederic Villard, and May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica Section D: Biological Crystallography 63, no. 4 (2007): 550-554. On 17 January 2014 22:24, Mahesh Lingaraju mxl1...@psu.edu wrote: Hi Folks The protein that I am working on gives several initial hits which are needles. And at random, I picked the needles
Re: [ccp4bb] First stucture of FCFV
Those who use lysozyme as a model protein should note that fungal spores of an unknown strain can have a dramatic effect on lysozyme crystal nucleation, as noticed by my student many years ago (I wasn't completely happy with the report because we never knew what we were working with!). Chayen, N. E., Radcliffe, J. W., Blow, D. M. (1993). Control of nucleation in the crystallization of lysozyme. *Protein Science*, *2*(1), 113-118. http://onlinelibrary.wiley.com/doi/10.1002/pro.5560020112/full I've often wondered why crystallization experiments at room temp don't become a sea of bacteria and fungi within a week. Is it because the high precipitant concs used stop growth? After all, medical saline is only 150 mM - much less than the average NaCl conc used for crystallization, which is about 1.7M. I guess not many bugs can grow in 20% PEG either. Patrick On 3 April 2014 15:25, Reza Khayat rkha...@ccny.cuny.edu wrote: I think fungus dependent crystallization has occurred for some labs. A paper that pops into mind is from my graduate laboratory (not my work though): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225192/ Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org Original message Date: Thu, 3 Apr 2014 06:36:37 -0700 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Chad Brautigam cabrautc...@yahoo.com) Subject: Re: [ccp4bb] First stucture of FCFV To: CCP4BB@JISCMAIL.AC.UK I once encountered mold-dependent crystallization of a protein. Wouldn't that have made for a lively Methods section? Luckily, we determined the structure from crystals derived from a different, non-moldy condition. Whew. Chad From: Artem Evdokimov artem.evdoki...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 3, 2014 7:55 AM Subject: Re: [ccp4bb] First stucture of FCFV Common molds like aspergillus or penicillium. After a while you sometimes get sporangia, then you can tell with more certainty. .. A. On Apr 3, 2014 3:50 AM, Bernhard Rupp hofkristall...@gmail.com wrote: Several people were asking what this FCFV tentacles actually might be. I think it is some fungus/yeast growing out of nutritious drops. Does resemble fungus/mushroom mycelium. I have also some that look like huge bacteriophages with nice heads on them, probably yeast buds. There is also a yeast lab next to the Xtallization facility :-/ *** feel free to speculate. Best, BR -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] PDB passes 100,000 structure milestone
@JISCMAIL.AC.UK *Sent:* Wednesday, 14 May 2014, 19:22 *Subject:* Re: [ccp4bb] PDB passes 100,000 structure milestone On Wednesday, 14 May, 2014 13:52:02 Phil Jeffrey wrote: As long as it's just a Technical Comments section - an obvious concern would be the signal/noise in the comments themselves. I'm sure PDB would not relish having to moderate that lot. Alternatively PDB can overtly link to papers that discuss technical issues that reference the particular structure - wrong or fraudulent structures are often associated with refereed publications that point that out, and structures with significant errors often show up in that way too. I once did a journal club on Muller (2013) Acta Cryst F69:1071-1076 and wish that could be associated with the relevant PDB file(s). Perhaps some combination of those two ideas? The PDB could associate with each deposited structure a crowd-sourced list of published articles citing it.They already make an effort to attach the primary citation, but so far as I know there is currently no effort to track subsequent citations. While spam comments in a free-format forum are probably inevitable, spam submission of citing papers seems less likely to be a problem. - Ethan On Wed, May 14, 2014 at 12:32 PM, Zachary Wood z...@bmb.uga.edu mailto:z...@bmb.uga.edu wrote: Hello All, Instead of placing the additional burden of policing on the good people at the PDB, perhaps the entry page for each structure could contain a comments section. Then the community could point out serious concerns for the less informed users. At least that will give users some warning in the case of particularly worrisome structures. The authors of course could still reply to defend their structure, and it may encourage some people to even correct their errors. -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg MS 357742, University of Washington, Seattle 98195-7742 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] PDB passes 100,000 structure milestone
I may be missing something here, but I don't think you have to rebut anything. You simply report that someone else has rebutted it. Along the lines of Many scientists regard this published structure as unreliable since a misconduct investigation by the University of Alabama at Birmingham has concluded that it was, more likely than not, faked [1] [1] http://www.nature.com/news/2009/091222/full/462970a.html On 15 May 2014 18:00, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, May 15, 2014 at 9:53 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: It seems to me that the Wikipedia mechanism works wonderfully well. One rule is that you can't make assertions yourself, only report pre-existing material that is attributable to a reliable published source. This rule would be a little problematic for annotating the PDB. It requires a significant amount of effort to publish a peer-reviewed article or even just a letter to the editor, and none of us are being paid to write rebuttals to dodgy structures. -Nat -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] PDB passes 100,000 structure milestone
Hi Joel and Jaime - very nice to hear from you. I hope everything is going well in Rehovot. Proteopedia is the natural place to put comments etc. However it might look more natural if there was more info there in the first place - ie if people gave more explanation about the significance of their and other people's structures, then comments like the one I suggested could be added. I don't know how you get people to become more active at Proteopedia. Maybe your students could post occasional messages to eg the CCP4bb with comments that show how useful Proteopedia can be. Tricky though! Best wishes, Patrick On 16 May 2014 14:35, Joel Sussman joel.suss...@weizmann.ac.il wrote: 16-May-2014 Dear Patrick, *Proteopedia* [*http://proteopedia.org] http://proteopedia.org%5D* uses exactly the same style for referencing published material. *Proteopedia* allows for the easy insertion of Pubmed and DOI references by only requesting from the user to enter the Pubmed or DOI ids. We have extended the same software used in Wikipedia for the internal *Proteopedia* engine to, based on this reference ID, retrieve, format and insert the correctly formatted reference at the bottom of the page. For example, *type refPMID 18673581/ref or refdoi 10.1093/nar/gku213/ref* in the wikitext box and save the page. If you type the reference in this manner, the properly formatted reference will be created automatically at the bottom of the page (or wherever you place the necessary wikitext references/). See *http://proteopedia.org/w/Help:Editing#Citing_Literature_References http://proteopedia.org/w/Help:Editing#Citing_Literature_References* and Proteopedia pages for actual examples. best regards, Jaime Joel On 15May, 2014, at 13:48, Patrick Shaw Stewart patr...@douglas.co.uk wrote: I may be missing something here, but I don't think you have to rebut anything. You simply report that someone else has rebutted it. Along the lines of Many scientists regard this published structure as unreliable since a misconduct investigation by the University of Alabama at Birmingham has concluded that it was, more likely than not, faked [1] [1] http://www.nature.com/news/2009/091222/full/462970a.html On 15 May 2014 18:00, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, May 15, 2014 at 9:53 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: It seems to me that the Wikipedia mechanism works wonderfully well. One rule is that you can't make assertions yourself, only report pre-existing material that is attributable to a reliable published source. This rule would be a little problematic for annotating the PDB. It requires a significant amount of effort to publish a peer-reviewed article or even just a letter to the editor, and none of us are being paid to write rebuttals to dodgy structures. -Nat -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers
When I hear of a reviewer holding up a publication and then publishing something similar, my first reaction is fury and I feel the case should be investigated and this immoral individual should be exposed. However I can see that there are many shades of gray here. We're all biased in that we tend to ignore information that conflicts with our previous cherished beliefs and focus on things that confirm them. So it can take a long time to change your mind - sometimes months. This can lead to indecision and delays, but in retrospect we tend to think that we would have come to those conclusions in any case so there's no harm in using the info. People with a strong sense of duty will get the review done quickly and make sure that they don't take advantage of the data, but I can see that it can be tempting. I think the idea of getting reviewers to sign a piece of paper saying that there is no immediate conflict of interest i.e. they are not about to publish something similar, is a good one. The author could prepare simple statement describing the topics covered (not the abstract which gives, or should give, the conclusions). Then it's not a matter of proving that the reviewer cheated, only that they had the opportunity to cheat. I always communicate freely with the editors, e.g. telling them why I don't want such-and-such to review the paper. Wouldn't it be possible simply to ask the editor to check that the reviewer asking for co-ordinates etc is not close to publishing something that could benefit from the data? I don't think it's a good idea for reviewers' names to be visible because that would mean that we would all have to do a far more professional job of the review. (I'm not a career scientist but I've been asked to review a few papers.) I also agree with those who say that this competitive focus on high impact journals etc. stifles creativity, is inefficient and gives poor value for money. Just some thoughts - probably stating the obvious Patrick On 20 April 2012 01:18, Edward A. Berry ber...@upstate.edu wrote: Bosch, Juergen wrote: To pick a bit on George's point with MR citation. Here's how you can read it in the paper from your favourite competitor: A homology model was generated using [fill in any program for ab initio prediction] and subsequently used for molecular replacement with Molrep. The structure was refined to an Rwork of 21% and Rfree of 24 %. Or maybe the structure was solved by MIR, using a lot of heavy atom data that they had been unable to solve until a fortuitous MR result gave phases which located the heavy atoms -- No, No, it was that new improved version of autosol! Anyway, who cares how the heavy atoms were located- the structure was solved entirely using their data and they have the raw data (image files even) to prove it. It was just bad luck with the derivatives that kept them from solving it 6 months earlier. They really deserve to have the first publication! -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] zinc with HEPES/seeding
It sounds as though microseeding worked very well, but the crystals are still growing far too quickly. Try diluting the protein and/or the reservoir solution to half the concs you are using or lower. To have enough protein you need larger drops, say 500 + 500 + 200 by hand if you can't do it with the Phoenix (wrong robot of course ;) Dispense the seed with a Hamilton syringe, rinsing the needle in the reservoirs before adding to the drops. You could also try using large volumes, say 4 ul total, with dilute ingredients, and make a small hole in the tape with a pin to let the wells slowly dry out. On 11 May 2012 18:35, Rajesh Kumar ccp4...@hotmail.com wrote: Dear Patrick, You along with others had made some suggestions last time. May be its a good time to update. With classical screening, I got a crystal like appearances/shower with HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and with different conc of Lithium I could only get very very thin needles which shower and difficult to pick even with 0.05 loop. changing conc of protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, glycerol, ethylene glycol, didnt reduce shower. I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) 100nl+100nL+50nL seeds using Phoenix. Got several nice hits which very good size individual crystals in conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after plates were set and there was no luck. Crystals started to appear from 20 min onwards and keep growing in next couple of hours. I thought of trying dehydration but they were already in dehydrating conditions them selves. I wanted to ask if anyone ever failed with MMS but thought not waste others time on this. Cross seeding to full length protein and Se met protein also gave beautiful crystal but again no diffraction. I have not checked SeMet as they were bit small. I am still open to ideas if you have any thing on seeding. I did try in microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul seeds, same as sitting drop which gave me very good looking crystals) but I didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but it didn't give me anything. Currently, I am making entropy mutations, new constructs of different lengths to solve the above problem. I still want to improve this condition with needles, because I collected a 4.5A data on one of the needle (just one). I need phases. I have sent some Iodide soaks to synchotron (yet to collect data) but manipulating these crystal drops with more than 100 tiny needles with a tough membrane on it has been frustrating as I end up loosing several drops to just to fish out 1-2 needles. I am ready to try if there any trick left. Thanks for lots and lots of help. Regards, Rajesh -- Date: Fri, 11 May 2012 18:09:54 +0100 Subject: Re: [ccp4bb] zinc with HEPES From: patr...@douglas.co.uk To: ccp4...@hotmail.com Rajesh How did you do the MMS? By hand or with a robot, and what screens did you use? and why did you change to HEPES out of interest? Patrick On 11 May 2012 18:05, Rajesh Kumar ccp4...@hotmail.com wrote: The rationale was to see if Zn could make differences in crystal morphology. This is because the protein has CxxC and CxxH similar to a zinc finger motif. All my efforts, additive screening, MMS, streaking, micro batch, hanging drop, changing drop ratio, drop shape, did not help me to either increase thickness or change the shape of very very thin needle crystals. Yes, I will try very less, 50uM. Thanks for helping me to understand. Rajesh Date: Fri, 11 May 2012 12:53:53 -0400 Subject: Re: [ccp4bb] zinc with HEPES From: liehy...@gmail.com To: ccp4...@hotmail.com Rajesh, 10mM zinc seems a bit too high. I normally used it at 50uM conc. ray On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar ccp4...@hotmail.com wrote: Dear All, This question sounds simple but I dont know the answer. I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES. Could you please tell me why is this? I appreciate your help. Thanks Rajesh -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd
Re: [ccp4bb] Crystallization with antibody
Try seeding the complex with the crystals that you have. If you have crystals of both proteins, crush them and mix them together. Suspend the seeds in whichever reservoir solution has the least salt in it* (or suspend in 50% PEG**) . Use random Microseed Matrix Screening (rMMS) i.e. microseeding into random screens***. rMMS with crystals of one component of a complex has successfully given crystals of the complex in the past. It's very quick and easy to try with a robot or by hand. *Radaev and Sun, J. Appl. Cryst. (2002). 35, 674-676 ** Cryst. Growth Design (2011) 11(8), 3432-3441 *** D’Arcy et al. Acta Cryst. (2007). D63 550-554 http://www.douglas.co.uk/MMS_proc.htm On 28 June 2012 13:25, Theresa Hsu theresah...@live.com wrote: Dear crystallographers Trying to crystallize a membrane protein complex of 100 kDa with a soluble protein of 20 kDa which is interact with the membrane protein. So far, no co-crystals in 200 conditions. Some conditions gave crystals but mass spec of crystals show only either one protein present. I am thinking of antibody but don't know where to start. Can I use the anti-His tag antibody? Thank you. -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Punctuation etc.
Could I make a request? Could people who ask questions to the bb possibly lay out their questions clearly, capitalize the word I and use the correct punctuation such as question marks? It makes it so much easier to read, and - this is more important - you will get more helpful responses if you help people to understand what you're asking. Giving your message a meaningful title is also very helpful! Best wishes to all, Patrick -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Improvement in crystal quality
Niks Seeding often works very well with needles. But start with seeding in RANDOM SCREENS, i.e. rMMS References - Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007) 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 **Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 *Chatty article that I wrote for SerCat newsletter in 2009*: http://www.douglas.co.uk/SER-CAT09_1.html *A bit of theory*: http://www.douglas.co.uk/mms.htm *Procedure*: http://www.douglas.co.uk/MMS_proc.htm A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D66 (2010) 568-576. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444910005512 Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf On 24 July 2012 14:40, Niks nik...@gmail.com wrote: Dear All, I am trying to crystallize a recombinant dehydrogenase protein. Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M sodium formate and 0.2M Potassium Chloride) after two days. Crystals looks like needles most of time, sometime broader needles (Pictures attached). UV crystal scanner says those are protein crystals, but when we tried to pick up one and shoot at room temperature, diffraction patterns looks like similar like of powder diffraction (picture attached). I have tried 50 of the 96 additives(whichever I can arrange of) mentioned in the additive screen from Hamptons . I have tried detergent screen from Hamptons (this time original screen solutions). I have tried incubating the plate at 28degrees as well as 10degrees, Though waiting for 10degree results but one drop showed needles again after normal two days of growth period. I tried to slow down the supersaturation by adding 100ul of 1:1 ratio of silicon oil and paraffin oil over the 1ml of well solution. This time no crystals but some precipitation. If anyone spare any word of wisdom to improve these crystal quality, I will be very grateful. If seeding is the only obvious thing to try, any reference for the seeding procedure will be highly appreciated. Thanks very much Nishant Varshney PhD student, National Chemical Laboratory,Pune,India -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Improving microcrystals
Samuel Clearly you should try the rMMS random microseeding approach where you add a seed stock made from the spherulites to a random screen. See refs: Acta Crystallographica section D63 (2007), 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442. http://www.douglas.co.uk/SER-CAT09_1.html On 25 August 2012 02:11, Samuel Johnson samueljohnson...@yahoo.in wrote: Hi everyone, I have been working on a protein for the past year. After a number of trials at crystallizing the protein i have identified conditions for getting spherulites/micro-crystaline material under micro batch method. I have confirmed that the crystalline material is protein, by using Izit-dye test. The condition is 50mM CaCl2, Mes pH 6.5 and 40% PEG 400. I will be happy to get suggestions on improving conditions to obtain single crystals. I have already tried varying a number of parameters like salt, precipitant concentration and buffer pH but that didn't help. Thanks. -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Off-topic: Best Scripting Language
Like most computer users and many scientists I don't write scripts to organize or analyse my data unless I get desperate. I've used both Python and Perl a few years ago, but it would take quite a lot of time and effort and staring at on-line tutorials to get back into either of them right now. So I end up using massive Excel files that kind of work, but are a pain. I've noticed that quite a few structural biologists have the same problem. I've never understood why there can't be a simple programming language that is completely self-explanatory bercause it uses English sentences. Our robot scripting language uses syntax like Dispense 0.5 * DropVol ul to TargetWells using ProteinSyringe That is pretty obvious. So why can't I have a language where I can write Carry_out_a_sequence_where x is 1 to 10 with_step_size 1 : if age of person(x) is_greater_than 50 then print name of person(x) is an old man (or woman) . Repeat_for_next x . ? I don't care if it's efficient (anything is efficient compared to Excel) or if it's easy to write big programs in. All I care about is that it's easy to get going. Later on I can learn to write simply Sequence instead of Carry_out_a_sequence_where. I could click a button that would make the replacement to make my code more compact and readable to a trained eye. And of course is_greater_than could be written as . Any intelligent school-child could understand it too, which would be fantastic here in the UK where kids aren't taught to program any more. Does such a language exist? On 13 September 2012 17:08, James Stroud xtald...@gmail.com wrote: On Sep 13, 2012, at 3:24 AM, Tim Gruene wrote: I have the impression that python programmers spend a lot of effort in trying to convince others that python is a good choice. Why bother rather than let people make their own decision? Someone asked. Plus, python programmers put no more effort than any other programmer. It's just that python has more advocates (for good reason) so the apparent effort is amplified. Don't hate us because our preferred programming language is beautiful. James -- James Stroud http://www.jamesstroud.com -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] off topic: reproducing hits from organic solvents
Peter We make a 96-well plate that was originally made for microbatch-under-oil, which has a large moat or well around the whole plate. My old friend Lesley Haire had a lot of success with this plate for a condition that contained isopropanol. She set up the drops without isoprop, covered them with Al's Oils (silicone mixed with paraffin oil from Hampton Research), then put water containing the appropriate concentration of isoprop in the moat (in her case 10 - 20%). THe isoprop diffused through the oil and into the drops. Since the oil is also saturated with isoprop it makes a very good barrier and stops the isoprop from evaporating from the drops when you harvest your crystals. As Lesley pointed out you can even set up a random screen without isopropanol, then diffuse the isopropanol in afterwards. Let me know if you - or anyone else - would like some samples of the plate. Unfortunately it won't fit on a Mosquito, but you can use it by hand, see ref below. Regarding your question below, bear in mind that water and oil can both go through plastic tubes (slowly). I hope it works for you Patrick Refs and info: See http://www.douglas.co.uk/winner1.htm http://www.douglas.co.uk/vb.htm *Nature* *431* (2004), pp 481-485. On 3 October 2012 17:37, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I recently got some hits from a Mosquito grown at 18C with PEG4000 and 10% propanol. I've tried reproducing these hits using the standard 24 well format with no success, even with playing with PEG and propanol concentrations. The initial screening solution was from a kit that's been sitting in the cold room for the better part of 2 years. Does anyone here have any tips or tricks in reproducing crystals grown from organic solvents? Many thanks in advance, Peter -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Plate crystals
Jahan It sounds as though the protein crystallizes well, so microseeding (done the right way) is very likely to solve the problem. Make a strong seed stock with as much crystalline material as possible from several wells, including different hits if possible. Just mix them all together, but keep PEG conditions separate from salt conditions (or you will get two layers). Make a set of serial dilutions from neat up to 1 in 100,000 and freeze them at -80. You need to seed into *random screens*, so that you can pick up new conditions. Then you should optimize two or three new conditions by using the diluted seed stock. For example, if you estimate that there are 1000 crystals in the drop, you use the 1:1000 dilution. For info and references see http://www.douglas.co.uk/mms.htm On 15 October 2012 23:01, Jahan Alikhajeh ja...@graduate.org wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Stabilization of crystals and ligand exchange
I say (of course I would!) why not try co-crystallization with random microseeding using the crystals with the original ligand? It usually allows you to control the number of crystals per drop too On 18 October 2012 15:59, Dmitry Rodionov d.rodio...@gmail.com wrote: Hi Sabine, Glutaraldehyde crosslinking worked pretty good for various soaks in my experience. J. Appl. Cryst. (1999). 32, 106-112[ doi:10.1107/S002188989801053X ] A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography C. J. Lusty Best regards, Dmitry On 2012-10-17, at 12:26 PM, Sabine Schneider wrote: Hi everyone, I am trying to get the structure of a protein-ligand complex were I need to exchange the ligand which it co-crystallises nicely with. Problem: either they crack, disolve, turn brown,... OR they still look very nice, well shaped but do not show a single reflection at the synchrotron!!! Here is what I tried so far: 1) initially stabilising with higher precipitant (here PEG1500) before slowly transferring (*) it to the ligand-removal solution (= artifical mother liquor with higher PEG, ethylen glycol or glucose, but without initial ligand) (*) by slow exchange I mean : initially mixing drop solution with stabilising/ligand-removal solution and adding it back to the drop stepwise before fully transferring it. Or calculation wise I have fully exchange the solution to the new solution 2) here I let them ist over night (if they did not disolve, crack or whatever) 3) slow exchange transfer to the artificial ML with the new ligand (10mM), left them over night and directly froze them 'Best' so far (crystals still looking nice but no reflection...) was slow exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also adding ethylenglycol to the reservoir), let them sit for over night, before again slow exchange to the solution with the new ligand in higher PEG and 30% ethylen glycol. As I said here the crystals keep shape, but don't diffract at all anymore. Just freezing them with 30% ethylen glycol they diffract nicely to 2.5A on a home source. But already after step one they are sometimes not happy anymore. Co-crystallisation failed since when I add the ligand, which is not that soluble to the purified protein, everything crashed out of solution. I am thinking about to test adding the ligand to the diluted protein and concentrate it together. But I don't have that much ligand, since the synthesis is quite tedious The ligand can be dissolved in 30% ethylenglycol to ~50mM Thus I was wondering if someone has done successfully ligand exchange with glutaraldehyd stabilised xtals? Or any ideas how to stabilise them? I appreciate any ideas or comments! Sorry for the lengthy email! Best, Sabine -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] inflluence of pH for crystallization on protein 3-D structure
Acoot You may be interested in some data-mining that we did a few years ago http://www.douglas.co.uk/PDB_data.htm#Acid-base We didn't see any obvious correlations On 28 October 2012 03:00, Acoot Brett acootbr...@yahoo.com wrote: Dear All, A protein crystal can be got at pH 5 or 8, or a pH with much extreme value. What will be the relatively extreme pH value to get the crystal on the protein structure solved based on the crystal got? I mean usually we regard the physiological pH as 7. If a crystal was got at pH 5, the structure solved may be different from the protein structure at pH 7. But it seems there is rarely analysis on the discrepancy of the protein structures when publishing 3-D structure with the protein crystal got at relatively extreme pH. I am looking forward to getting your comment on it. Cheers, Acoot -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Problems in crystallization
Eva Yes batch under oil will solve the problem. We regularly used to crystallize conditions with high levels of MPD under oil without difficulty. I agree that the protein might be reducing the surface tension, but I've never heard of such a strong effect. Maybe the protein is unstable and wants to denature onto the surface of the plate. Is it possible that you have any low-molecular-weight organics in your protein sample? You could try the UV-compatible versions of crystallization plates because they're more hydrophobic than the regular PS ones. The polypropylene ones are even more hydrophobic, similar to siliconized glass. Salt precipitants will tend to reduce drop spreading, PEG to increase it. It's partly a matter of surface tensions, partly of the affinity of the ingredients for the plastic/glass. The air-liquid interface energy competes with the liquid-solid interface energy. Patrick On 7 November 2012 13:41, anna anna marmottalb...@gmail.com wrote: Why don't you try batch under oil? 2012/11/7 Eva Bligt-Lindén eva.bl...@abo.fi Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva __**__ Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Glass Capillaries
The Triana range of capillaries are very easy to use for screening etc On 12 November 2012 16:13, Michael Roberts mrobert...@talktalk.net wrote: Dear All, I would be interested to learn of other crystallographers' experience in their use of glass capillaries for protein crystal growth and X-ray diffraction clarity. There are many types of glass available - quartz, soda glass, borosilicate, etc. Are there specific types which people prefer for best results overall? Best wishes, Michael Roberts -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Equilibrium Relative Humidity matching
Matt, how well does it work in practice? Did you check the colligative predictions against the measurements that you originally made? I agree that the PEG has virtually no effect on the drop equilibration. This can be seen in comparisons of batch and v.d. e.g. http://www.douglas.co.uk/convert.htm. Therefore I always thought that equilibration in high-PEG experiments was driven by any salts that may be present - including the salt in the protein, which is easy to overlook. I remember a paper by Luft and De Titta about chaperone salts. However, unless you know the relative effects of different salts, the result is hard to predict! On 23 November 2012 16:05, Boaz Shaanan bshaa...@exchange.bgu.ac.il wrote: *Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710* ** ** * * -- This leads to a counter-intuitive observation - it is only the number of molecules in solution that affect the RH and not the type of molecule/ion - therefore one molecule of glycerol has the same contribution as a chloride ion or anything else. This means that there is no effect for charge etc. What does matter is how many species the salt dissociates into - this means that a given concentration of sodium malonate (3 species) will have a lower RH than ammonium sulphate (2 species (NH4+ and (NH4SO4)-) and not 3 as might be expected). This means that relatvie humidity is a colligative property: http://en.wikipedia.org/wiki/Colligative_properties Doesn't it? So it should not be too surprising. Boaz On 22/11/2012 18:32, Patrick Shaw Stewart wrote: Matt My old Rubber Book (Handbook of Physics and Chemistry, 1976) has a table (attached) showing the lowering of vapor pressure by salts in aqueous solution, taken from the Smithsonian Tables. I've never been able to make any sense of it in terms of cations, anions, valency, or charge density (position in Hofmeister series), whether concentrations are expressed as M or N solutions. For example, of the salts mentioned on your website, MgSO4 seems to be anomalous. My spreadsheet also has a little converter that my colleague wrote many years ago to convert vapor diffusion conditions to batch. This might be of interest to people who have to work in batch, e.g. people making crystals for X-FEL data collection. You can download it as a program from http://www.douglas.co.uk/vdtomb/vdtomb.htm - it seems to work pretty well. Best wishes Patrick On 22 November 2012 12:11, Matthew Bowler mbow...@embl.fr wrote: Dear All, after a few requests, I have now added equations to the online calculator that uses Raoult's law to calculate the relative humidity equilibria for precipitant solutions (see http://go.esrf.eu/RH). The new equations (4 and 5) allow the calculation of salt concentrations that will be in equilibrium with a certain PEG or other molecule solution - this will allow slow and controlled dehydration experiments to be designed in vapour diffusion plates, by slowly increasing the salt concentration in the reservoir above the equilibrium and thereby reducing the amount of water in the crystallisation drop by a controlled amount. Hope it is useful, cheers, Matt. -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ === -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- Matthew Bowler Synchrotron Science Group European Molecular Biology Laboratory BP 181, 6 rue Jules Horowitz 38042 Grenoble Cedex 9 France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.88.29.04 http://www.embl.fr/ === -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] To cryo or not to cryo...
Yuri Whatever happens, keep the remains of the crystal to make a seed-stock after data collection. You can also try making a seed stock from the rest of the drop, even if you can't see crystals, and even if it has dried up. Use part of your seed-stock to seed into one or more *random screens* You can make the seed stock by suspending crushed crystals in the reservoir solution that gave the original hit (preferably taken from the original plate as it may have dried out a bi9t). Patrick Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007), 550–554. http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 On 1 December 2012 09:22, Yuri Pompeu yuri.pom...@ufl.edu wrote: Dear community, I have what seems to be a pretty decent single crystal that grew from a screen set up 2 weeks ago. I am trying to reproduce it but so far I have not succeeded. I am however afraid the crystal that did form will start to deteriorate. So this brings me to dilemma, I feel like I should try and mount this crystal and shoot it. But since I only have 1 sample, I do not want to mess this up... I am inclined to try cryo conditions, but I am afraid the addition of a cryo such as glycerol could destroy the little guy. The crystal formed in 30% PEG 4000, 0.1M NaCitrate pH5.6 and 0.2M NH4AcO, I wonder if this is a cryo condition already? Any suggestions would be appreciated. best, -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] protein crystals or salt crystals
Good morning Frank On a related idea, do you typically use a limited number of buffers (buffer plus salt) for the final purification step of your proteins? If so, do you have a chart of where salt crystals may appear in the screens that you use most often? Could you put that chart on your web site to help the community? People could pick one of your standard buffer mixes to make their lives easier later on. Best wishes Patrick On 8 February 2013 07:18, Frank von Delft frank.vonde...@sgc.ox.ac.ukwrote: Test the diffraction - that's the only way. But given the other junk in the drop, chances are they're salt. (And don't post 5Mb attachments, please.) On 07/02/2013 22:24, amro selem wrote: Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] protein crystals or salt crystals
Amro Here is an extract from our paper, describing a method that is almost infallible, and not too hard to do if you're organized. It can never give false positives and (in the 3 cases we looked at it) only gave false negatives when there was heavy precipitate in the drop. Best wishes, Patrick Ref: Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line athttp://pubs.acs.org/doi/abs/10.1021/cg2001442. In the cases of crystals of the proteins concanavalin A, trypsin, and thaumatin, we used an interesting novel method of making the distinction, which is a modification of the method of Pusey et al.17 We covalently labeled 50 μL aliquots of the proteins with the fluorescent dye DyLight 350 NHS Ester (from Thermo), following the manufacturer’s instructions except that we used higher protein concentrations (30 mg/mL for trypsin and concanavalin A, 36 mg/mL for xylanase). We added 20 nL samples of labeled protein to wells containing putative protein crystals after the crystals had grown. We photographed crystals in a darkroom by illuminating with the UV Pen-280 or with an FL4BLB UV lamp (Luxina), which has a peak wavelength of 370 nm. As shown in Figure 2, crystals fluoresced brightly and were unambiguously identified as protein rather than salt. (The DyLight kits are very easy to use because all resins, columns, etc. are provided. We chose the label that is excited at 350 nm because it is not necessary to use a filter since most cameras have built-in UV filters.) The advantages of the method are (1) since it allows protein to be seen directly, it does not give false positives or negatives (except when the drop contains a lot of precipitate, see below). (2) It cannot interfere with the crystallization process. (3) Labeled protein need only be prepared if crystal identification by other methods fails; (4) even needles and small crystals can be identified. The method does not work well when the drop contains a lot of protein precipitate, which may absorb the labeled protein before it can reach the crystals. Note also that protein sometimes coats salt crystals in crystallization experiments, giving a superficially similar appearance. Such cases can, however, easily be distinguished by comparing UV images with visible light images because the protein coating is outside the salt crystal. (17) Pusey, M.; Forsythe, E.; Achari, A. Methods Mol. Biol. 2008, 426, 377–385. On 11 February 2013 09:37, Ganesh Natrajan ganesh.natra...@ibs.fr wrote: Dear Amro, What you could try is this. Make a solution of 0.5 % (w/v) commassie brilliant blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your drop and close the cover slip. If the crystals are protein, they should turn blue after some time (typically 30 mins). Salt crystals will not turn blue as they are not stained by commassie. You could also try using Hampton's Izit crystal dye for this, but the problem I have faced with it is that the izit itself crystallizes (gives lovely blue crystals) under certain buffer conditions. cheers Ganesh Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] salt or not?
Careina, One thing to try if other ideas don't work or are too difficult, is covalently (therefore unambiguously) labelling a little of your protein with a fluorescent dye. If you add 20 nL of this to the drop *after the crystals have grown*, protein crystals will light up, but salt crystals will not. Thermo make some very easy-to-use kits for labelling. See methods section of our paper *Cryst. Growth Des.*, 2011, *11* (8), pp 3432–3441. Could you also label the DNA . . . ? Hope it helps, best wishes, Patrick On 15 April 2013 11:18, Careina Edgooms careinaedgo...@yahoo.com wrote: Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
I've just remembered another approach that I've heard suggested, although I don't know anyone who has tried it. Dissolve plenty of the small molecule in paraffin or silicone oil, and use this mixture to cover the drop. This can be used with regular vapor diffusion or (probably better) microbatch-under-oil. The oil acts as a reservoir that contains excess small molecule that (you hope) will be fed into the crystals. Patrick Shaw Stewart -- [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk http://douglas.co.uk/ or http://www.douglasinstruments.com http://www.douglasinstruments.com/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Green, Todd Sent: 22 January 2007 20:40 To: CCP4BB@JISCMAIL.AC.UK Subject: crystal friendly solvents that are useful for dissolving hydrophobic small molecules? Hello All, I am trying to soak some crystals with a small molecule that is quite hydrophobic. I am having trouble with solubilty of the small molecule. It will dissolve up to about 1 mM in 100 % DMSO, but precipitates at concentrations of less than 15 micromolar when the DMSO concentration is below 20 percent in my crystal growth solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green
Re: [ccp4bb] Question about glove boxes for protein crystallization
Hi Mathews One of our customers, Bret Dillard at the University of Georgia, has had great success with one of our robots in a Bactron X anaerobic chamber. In this case, Bret mainly used the microbatch-under-oil method, which works very well for anaerobic work: (1) the oil protects the sample and reduces exposure to oxygen, (2) the amount of work is far less because you can keep several degassed screens in the chamber to use many times with different protein samples. With vapor diffusion you will have to degass the solutions every for every few samples (if not every sample). Bret won our competition for this work last year. You can find his report at http://www.douglas.co.uk/news.htm, plus more info below Patrick _ Douglas Instruments has announced the winner of the second round of its competition for the best new crystallization technique. Congratulations to Bret Dillard from the University of Georgia, for his winning entry, Automatic Protein Crystallization in an Anaerobic Environment. Bret placed an Oryx1-6 robot in a Bactron X anaerobic chamber, and used mainly microbatch-under-oil crystallization to crystallize four proteins that are not stable in an environment with oxygen. He found that microbatch avoided the need for frequent degassing of solutions which reduced the work-load enormously. The oil also provided extra protection from oxidation. One of the proteins crystallized is rubrerythrin from the hyperthermophilic archaeon, Pyrococcus furiosus. The native protein contains iron, but is unstable in oxygen. A previously-reported structure was determined in the presence of oxygen, but the iron had been replaced by zinc. Using the anaerobic system, Bret has now obtained the native form containing iron. _ On 2/23/07, Mathews, Irimpan [EMAIL PROTECTED] wrote: Dear Friends, Sorry for the non CCP4 question. We are planning to purchase a small glove box to setup crystallization trays under anaerobic conditions. If you have used glove boxes for crystallization, would you please give me some idea? We are thinking of getting the 815 series from Plas-labs (link below). http://www.plas-labs.com/ Thank you very much, Mathews Ps: If others are interested, I will post a summary.
Re: [ccp4bb] Main topic of the day: Protein crystallization
Ronaldo I have a database of crystallization conditions extracted from the PDB. I was able to parse the data to extract the concentration of protein in around 900 cases. The lowest 9 protein concentrations were as follows: PDB ID TYPEDATEProtein conc, mg/ml 1EYMISOMERASE. 07/05/2000 0.75 1ADQCOMPLEX (IMMUNOGLOBULIN/AUTOANTIGEN)18/02/1997 1 1W5GFOUR HELIX BUNDLE. 06/08/2004 1 1UU4HYDROLASE. 15/12/2003 1 1W2UHYDROLASE. 08/07/2004 1 1DXPSERINE PROTEASE.13/01/2000 1 1DY8SERINE PROTEASE.18/01/2000 1 1DY9SERINE PROTEASE.31/01/2000 1 1DG6APOPTOSIS. 23/11/1999 1.2 The maximum protein conc was 200 mg/ml (1BLF) My database goes up to October 2004. Janet Newman and Tom Peat have done this job far more conscientiously and could probably give you better and more up-to-date data. Patrick On 2/23/07, Ronaldo Alves Pinto Nagem [EMAIL PROTECTED] wrote: Dear all, As protein crystallization is the main topic of the day may I include another question? What was the minimun protein concentration reported with success in crystallization trials? I ask that because the protein I am trying to crystallize is much less soluble than the one mentioned in the lasts emails. Thanks Ronaldo.
[ccp4bb] Demonstration and support specialist
Douglas Instruments is seeking a motivated individual with practical aptitude to demonstrate and support its automatic crystallization systems in Europe and/or N America. Knowledge of macromolecular crystallization will be an advantage, scientific (or engineering) experience essential. No salesmen please. Please reply to the address below, or e-mail. Patrick Shaw Stewart -- [EMAIL PROTECTED] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Help with reducing crystal mosaicity
Just a thought, Mary - going back to your original question about MPD. I extracted the crystallization conditions from REMARK 280 of 3939 PDB entries a couple of years ago. The average concentration of the MPD used was high - 38.6%, while PEGs tended to be used at lower concs, e.g. PEG400 25.7%. You can see the data at www.douglas.co.uk/top14.htm I thought this information could be useful if you want to replace some of the MPD with another precipitant (or cryoprotectant). Best wishes Patrick Shaw Stewart -- [EMAIL PROTECTED] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://douglas.co.uk or http://www.douglasinstruments.com Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Mary Fitzgerald Sent: 09 July 2007 23:05 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help with reducing crystal mosaicity Help please! I'm looking for some new ideas. I have crystals that come out of a sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium acetate and MPD for the well solution. The MPD concentration is sufficient to act as a cryoprotectant. Currently, I directly freeze these crystals in liquid nitrogen. When I collect data, I typically have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is further complicated with a weakly diffracting crystal (4-5 A) that has a long unit cell axis of ~500 and often twinning. It has been suggested to me that the cryoprotectent is a problem. I haven't checked the diffraction at room temperature, yet. Please no suggestions of finding a different crystal form as that's not a consideration at the moment. I have my reasons. I did find one crystal that has lower mosaicity (0.5 to 0.8) but had weaker diffraction then the typical crystal. Attempts at flash cryoannealing have not helped. So, what's a good way to change the cryoprotectant if the cryoprotectant is the precipitant? I've considered trying dehydration but wasn't certain if that would help with the mosaicity. Thanks for any ideas, Mary X. Fitzgerald Postdoctoral Associate
[ccp4bb] Fwd: [ccp4bb] crystallisation robot
One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up.. On 1/17/08, Oganesyan, Vaheh [EMAIL PROTECTED] wrote: Mark, What was the state of the larger drops when tiny counterparts had crystals? My guess - they all precipitated. I'm trying to understand why some proteins or some conditions require change in protein concentration while others do not when migrating from smaller drops to larger ones. If it is protein dependent then I'm afraid there might be no one answer; if it is not then there should be a trend and explanation of phenomena. Vaheh From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, January 16, 2008 8:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. :-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL Demetres Which, indirectly, brings up an interesting (but not relevant to the Oryx) question. Nucleation is a process that does have a stochastic aspect. Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample). any comments ? (let the flames begin). A. PS1 another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ... PS2 I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask. On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote: David Briggs wrote: I'll defend the honour of the phoenix... (again) Bernhard Rupp 100+100 nl Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl Others.. Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage. Dave -- David C.
Re: [ccp4bb] isopropanol as a precipitant
Shivesh It's often very difficult to harvest crystals from conditions that contain isopropanol because the alcohol is volatile - especially with 50% or over! A solution that has worked several times it so use the Vapor Batch plates. http://www.douglas.co.uk/vb.htm You can cover the drops with oil, and then put isopropanol solution into the moat of the plate. The alcohol diffuses through the oil into the drops, while the oil acts as a buffer, reducing loss of alcohol during harvesting and freezing. For a case study see http://www.douglas.co.uk/winner1.htm Please let me know if anyone wants samples. Patrick -- [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart, James Smith http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of shivesh kumar Sent: 05 March 2008 07:23 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] isopropanol as a precipitant Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.The interesting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh
Re: [ccp4bb] about microbatch
Jiamu Although we no longer focus on microbatch-under-oil, we have a small utility called VDTOMB that converts from vapor diffusion conditions to microbatch. I have tested it with known published and in-house crystallization conditions and it seems to work remarkably well. Go to http://www.douglas.co.uk/tipsntech.htm (you must use Internet Explorer, not Firefox or something) and click on the link near the middle of the page. In most cases where the main precipitant is PEG there will be very little equilibration in the VD experiment, so the MB conditions are almost the same as the VD conditions. The typical size for manual dispensing is 1 + 1 ul. For robotic dispensing, 0.3 + 0.3 is typical. As long as you have a couple of mm of paraffin (or silicone mixed with paraffin for screening) over the drops you will be OK. If you or anyone else would like plates for microbatch, please ask me for samples - see http://www.douglas.co.uk/vb.htm. They're about $4, which is cheaper than most VD plates. MB often works well for proteins that are sensitive to oxidation, and for membrane proteins. Generally, less protein is lost on the surface of the drop, and the crystallization conditions are better known. It is also great for conditions with isopropanol or other volatile liquids - see http://www.douglas.co.uk/winner1.htm Good luck with your project Patrick -- [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jiamu Du Sent: 26 July 2008 12:39 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] about microbatch Dear all, I want to screen crystal using microbatch method. I do not have standard microbatch plate, so I have to using a 96 well cell culture plate instead. My question is below: What is a typical drop size for microbatch? 1ul protein + 1ul mother solution or larger? How much paraffin oil is sufficient for cover the drop? Thanks. -- Jiamu Du, Ph.D. State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 P. R. China Tel: +86-21-5492-1117 E-mail: [EMAIL PROTECTED]
Re: [ccp4bb] Query on program for creating random crystallization screen
Dear Rajakumara I forgot to say, there's a very simple way to do this by aliasing. You write (let's say by hand) one or more random screens with solutions called e.g. Precipitate1, Precipitate2, PrecipitateN; Buffer1 to BufferN; Additive1 to AdditiveN etc. Each time you want to make a screen you simply replace Precipitate1, Precipitate2 etc. with the precipitates you want to use, say 30% PEG, 2M AS etc. It's almost trivial to do the whole thing in Excel using a look-up table - or just links If you have a robot you can run the same script each time, just put the appropriate solutions into the stock wells. Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups.google.com/group/oryx_group/members_invite?hl=en [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: 12 September 2008 15:17 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Query on program for creating random crystallization screen Dear All I want to create a random crystallization screen using given set of crystallization parameters (pH, Precipitant, salt, additive.) for protein-DNA complex crystallization. I was using Brent Segelke's CRYSTOOL program, not accessible online now. Please can you suggest any other such programs and/ how do I can access the CRYSTOOL (sort of registration or purchase?) Thanking you in advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your preferred Email name! Now you can @ymail.com and @rocketmail.com. http://mail.promotions.yahoo.com/newdomains/aa/
Re: [ccp4bb] Crystallography plates, hanging drop but templated sealing film.
You lose about 1 microlitre of water per well per day with polystyrene Linbro plates - it goes through the plastic Polypropylene and COC are better (or maybe worse if the evaporation encourages crystallization) -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups.google.com/group/oryx_group/members_invite?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Diana Tomchick Sent: 15 January 2009 21:56 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystallography plates, hanging drop but templated sealing film. Are you saying that you'd like to re-use the plate with the original screening solutions, or that you plan to clean the plates, then dispense fresh screening solutions? If you would like to re-use the original screening solutions, beware...after many weeks, they will not be the same concentrations (and in some cases, not even the same pH) as they were when first dispensed into the plates. Slow evaporation of water occurs through the plastic of the plates as well as through the tape. Diana On Jan 15, 2009, at 3:34 PM, Francis E Reyes wrote: Does such a thing exist? A 24-well microplate configuration where in substitution of glass cover slips, you have a roll of tape templated such that there are circular areas where you can add your protein where there is no adhesive, but there is adhesive everywhere else? This may be a nightmare for plate manufacturers, but to reuse the plate, you just throw away the film and tear a new one. Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D * * * * * * * * * * * * * * * * * * * * * * * * * * * * Diana R. Tomchick Associate Professor University of Texas Southwestern Medical Center Department of Biochemistry 5323 Harry Hines Blvd. Rm. ND10.214B Dallas, TX 75390-8816, U.S.A. Email: diana.tomch...@utsouthwestern.edu 214-645-6383 (phone) 214-645-6353 (fax)
Re: [ccp4bb] microbatch vs hanging drop
Rui Microbatch - which I take to mean crystallization under oil with no reservoir - has many advantages, but with some robots it's a little slower to set up (20 mins on ours). So most people I know start off with vapor diffusion and only move to microbatch if they have problems with VD. It seems to find as many hits as vapor diffusion, but in different conditions - see http://www.douglas.co.uk/mbnvdall.htm http://www.douglas.co.uk/mbnvdall.htm Main advantages: 1. Gives thinner skins on drops and less protein is lost on the surface 2. The oil often protects sensitive proteins such as membrane proteins or oxygen sensitive proteins 3. You can change temperature freely (no condensation etc.) 4. Very good for use with volatile organics, see e.g. http://www.douglas.co.uk/winner1.htm http://www.douglas.co.uk/winner1.htm And you can use it with MMS microseeding just like VD - very important I believe. I guess the main disadvantage is that it can be hard to harvest crystals through the oil (although some say the oil makes it easier because you can take your time) People are using smaller and smaller reservoirs so I guess one day they'll realize that you don't need a reservoir ;-) Good luck Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of rui Sent: 22 April 2009 17:06 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
Hi Mo What you need to remember is that a relatively large amount of protein is lost from smaller drops. The ratio of surface area to volume is greater. With 100 + 100 nl drops about half of the protein is lost, either as skin on the drops or on the plastic of the plate. So when you scale up you need to reduce the protein by about half. (Another approach, suggested by Heather Ringrose, is to put extra protein into the drops at the screening stage, e.g. 200 nl protein + 100 nl reservoir solution. The hits found can usually be scaled up by dispensing 1 + 1 microlitre drops.) This is counterintuitive because people expect the small drops to dry out more quickly - so they expect, if anything, to get more precipitation in the small drops. Instead they get precipitation when they scale up, assuming they keep the ratio of protein to reservoir constant. It can also help, when you scale up, to increase the salt by 50 to 100% - this is indicated by data mining but I'm not sure what the mechanism is Hope that's helpful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mo Wong Sent: 18 August 2010 16:18 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate Hi all, I know scaling up from a hit found from a high throughput screen is an empirical process, but does anyone have a good rule of thumb as a starting point when it comes to scaling up from a hit observed in an Intelliplate to a Linbro plate (i.e., change in volume ratios, amount to add to reservoir, etc)? I've Googled around but haven't seen anything which either suggests I shouldn't be asking this question, I've not looked hard enough, or it really is a case of try and see. Thanks
Re: [ccp4bb] turn granular to crystal
Rui Assuming that they are protein, this is a perfect candidate for MMS microseeding into random screens You can do it with a robot or by hand See D'Arcy et al. Acta Cryst. (2007). D63, 550 - 554. 'An automated microseed matrix-screening method for protein crystallization' A very good recent article is Obmolova et al. Acta Cryst. (2010). D66, 927-933 'Promoting crystallization of antibody-antigen complexes via microseed matrix screening' (See also our website at http://www.douglas.co.uk/mms.htm) Yibin, what proteases do you use, what conc, how much do you add? Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of rui Sent: 25 August 2010 13:37 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] turn granular to crystal Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot.
[ccp4bb] Fab purification and crystallization
Again you should read the spectacular paper by Obmolova and co. where they solved the structures of three Fab-antigen complexes using MMS microseeding (seeding into random screens), starting with one hit containing clusters of needles - which could not themselves be optimized The paper is available on open access (free) http://journals.iucr.org/d/issues/2010/08/00/issconts.html http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf Acta Cryst. (2010). D66, 927-933 [ doi:10.1107/S0907444910026041 http://dx.doi.org/10.1107/S0907444910026041 ] Promoting crystallization of antibody-antigen complexes via microseed matrix screening G. Obmolova http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Obmo lova%2C%20G%2E , T. J. Malia http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Mali a%2C%20T%2EJ%2E , A. Teplyakov http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Tepl yakov%2C%20A%2E , R. Sweet http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Swee t%2C%20R%2E and G. L. Gilliland http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Gill iland%2C%20G%2EL%2E Synopsis: The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. Online 14 July 2010 The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: 10 September 2010 04:00 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
Re: [ccp4bb] crystallizing a complex that's sensitive to ionic strength
Hua Peter Sun and S. Radaev wrote a paper a few years ago where they showed by data mining that the crystallization conditions of complexes heavily favor PEG rather than salt conditions, so you are not alone. Radaev and Sun, Crystallization of protein-protein complexes J. Appl. Cryst. (2002). 35, 674-676 You could try microseeding *into random screens* using the crystals that you have to make a seed stock. See Acta Cryst. (2007). D63, 550–554 and http://www.douglas.co.uk/mms.htm. This approach has sometimes worked before for complexes, eg seeding crystals of an antigen-Fab complex with crystals of just the Fab. You can use this approach by hand or with a robot (spin the seed stock if you us a non-contact robot). One problem is that you would normally put quite a lot of Amm Sulf into your wells because you would suspend the seed crystals in the reservoir solution where the crystals used to make the seed stock were found. The conventional volumes to use are 0.3 protein + 0.2 reservoir solution + 0.1 seed stock, so typically 1/3 of the precipitant would be Amm Sulf. My colleagues and I recently submitted a manuscript to Crystal Growth and Design where we look in great detail at suspending seed crystals in other precipitants (instead of Amm Sul in this case). We found that this normally works, eg you can usually make a seed stock with PEG instead of say salt conditions. You can predict whether the PEG (or any other solution) will work for suspension by incubating the crystals in PEG for 24 hours. If the crystals look unchanged after that, you can almost certainly make a seed stock from the solution in question. (If the crystals dissolve, shatter or crack, you MAY be able to make a seed stock with the solution.) We used 100% PEG600, although in restrospect I think 30% - 50% PEG 3000 might be better. I can send you a preprint, but the gist of the relevant part is above. For a review of more radical approaches, see Bing Xiao et al., Optimizing Protein Complexes for Crystal Growth. Crystal Growth Design, Vol. 7, No. 11, 2007 2215 I hope that helps Patrick On Sun, Feb 27, 2011 at 2:24 AM, Hua Yuan foxso...@gmail.com wrote: Dear CCP4 community members, I've been trying to crystallize a protein complex that's very sensitive to ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the complex but higher salt (~0.5 M) breaks the complex apart. The interaction that holds the complex is probably mainly ionic type. The crystals I got so far has only one component of the complex from which all the crystallization conditions have high salt such as 2M Ammonium Sulfate in them. Besides repeatly screening many crystallization conditions, I was wondering whether is any way to work around this problem. Your suggestions would be greatly appreciated! Thanks, Hua -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Reproducing crystals.
Jun Yong and Jobichen (I've mentioned this before, but ) - both of your projects jump out as very good targets for microseeding with random screens. This method often gives extra hits and better crystals because it is more likely that crystals will grow in the metastable zone. It often reduces the *need *for optimization. Read Allan D'Arcy's excellent paper, plus Obmolova et al for a spectacular example. Our web site has some recent tips that may help you too. Good luck Patrick D'Arcy, A., Villard, F., Marsh, M. An automated microseed matrix screening method for protein crystallization, 2007, Acta Crystallographica, D63, 550-554. G. Obmolova, T. J. Malia, A. Teplyakov, R. Sweet and G. L. Gilliland. Promoting crystallization of antibody-antigen complexes via microseed matrix screening Acta Cryst. (2010). D66, 927-933 http://www.douglas.co.uk/mms.htm * * On Tue, Apr 12, 2011 at 1:07 PM, Jun Yong Ha j...@princeton.edu wrote: Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] small lysozyme crystals?
James One thing you should be aware of is that bugs growing in the lysozyme stock solution can have a dramatic effect on the number and size of crystals. (Way, way) back in 1993 when I was in David Blow's lab we noticed an ageing effect with the lysozyme stock solution; if we dissolved freeze-dried lysozyme and set up experiments immediately we got a few large crystals. If, however, we did exactly the same thing except that we kept the lysozyme protein stock overnight in an Eppendorf tube (using a certain batch of tubes), then set up experiments the next morning, we grew dozens of small crystals. The effect seemed to be caused by fungi that were growing in the tubes because fungicides eliminated the effect whereas antibiotics didn't, see below. I have often wondered whether people designing e.g. microgravity experiments that are stored for several days before launching are aware of this! Best wishes Patrick See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2142306/ Abstract This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample. Summary of results 1. Under the particular conditionosf these experiments, growth of lysozyme crystals depends on thep resence of a nucleating agent. 2. Aging of lysozyme in solution gives rise to enhanced nucleation. 3. The aging effect is independent of the source of the lysozyme powder. 4. Filtration or centrifugation prior to aging does not eliminate the aging but lessens the number of crystals grown. 5. The agent responsible for the enhanced nucleation can be removed by centrifugation or filtration even after aging has occurred. 6. Desalting increases aging. 7. Aging is prevented by antifungal agents but is not affected by either class of antibiotics. 8. Aging is eliminated when a filtered solution is stored in a sterile vial. 9. Nucleation (and consequently the number of crystals grown) can be controlled by altering the ratio of freshly filtered and aged protein solution just prior to crystallization. On Tue, Jul 26, 2011 at 6:56 PM, James Holton jmhol...@lbl.gov wrote: Does anyone out there have a protocol of growing HEWL crystals that are all 50-100 microns wide? I gave this project to a summer student recently, thinking it would be easy, but it is turning out to be more difficult than I thought. Keep getting sphereulites instead of small crystals. Yes, I know you can smash a large lysozyme crystal with a hammer, but that is not exactly what I was going for. What I was hoping for was a well-defined protocol for growing reference crystals that stay evenly illuminated in our x-ray beams as they rotate. The beam is 100 um wide. I'm sure someone has done this before? -James Holton MAD Scientist -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Hi Ivan Did you use microseeding with *random *solutions? If not see the following paper by Obmolova and Co about exactly this, microseeding with Fab complexes, http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf For more subtle variations in using microseeding with complexes see http://pubs.acs.org/doi/abs/10.1021/cg2001442 Good luck Patrick On Thu, Jul 28, 2011 at 4:41 AM, xaravich ivan xaravich.i...@gmail.com wrote: Hi everyone, I have been trying to crystallize Fab:antigen complex( 50kda:90kDa) complex and initially got needle clusters which after microseeding gave me single crystals but they are very small and I could not repeat the results. I have been using HEPES buffer at pH 6.8 to do the final SEC purification step of the complex before setting trays. I was wondering whether there are some other buffers (that one could suggest eg tris-hcl etc) which have given decent positive results when crystallizing Fab complexes.Though I have gone through individual papers (case by case) to get some idea, It would be great if anyone could direct me to a comprehensive literature towards studying the crystatllization conditions of Fab complexes. Equally, people who have considerable experience could suggest a list of must do steps for such problems which have routinely been practiced in their lab Also what is a good storage condition for the excess complex that you want to use later? I would really appreciate any suggestion,help, direction. Thanks ivan -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Fab:antigen complex crystallization!!!
Ed (and Ivan) Peter Sun and colleagues published two papers where they show that crystallization conditions for protein-protein complexes are strongly biased towards PEG-based rather than high-salt or organic-solvent-based conditions. This includes antibody-antigen complexes. http://www.ncbi.nlm.nih.gov/pubmed/16699187 http://scripts.iucr.org/cgi-bin/paper?do0016 I have heard anecdotally that the same is true of protein-peptide and protein-small molecule complexes, although I don't know of any systematic study. Can anyone shed light on this? I guess we can look in the Marseilles database Best wishes to all Patrick On Thu, Jul 28, 2011 at 2:32 PM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2011-07-28 at 05:07 +0100, Sean Seaver wrote: Spoiler - Fabs like ammonium sulfate. Not really - in my hands the ammonium sulfate was one hit out of 7. While Ivan's question is about Fab complexes with protein antigen, I think it brings up a more general question of protein class-dependent crystallization bias. While some general trends exist for classes of biopolymers (e.g. MPD is number one precipitant for DNA; protein:DNA complexes tend to crystallize in PEG-based conditions), a general idea of assigning a preferred precipitant to a protein class is, imho, pointless. Fabs are a good example - one would think that with half of the protein more or less the same in all instances some general trends should exist. And perhaps they do, as this http://scripts.iucr.org/cgi-bin/paper?S0907444999016224 seems to suggest. But alas, Fab crystallization conditions, once you look into it, appear to be just as diverse as the same for proteins in general. Crystallization conditions may change radically upon point mutation, so why would one expect that a class of proteins sharing some 50% identity will show unusual love for PEG, ammonium sulfate, sodium malonate or any other miracle precipitant? Consider this. Thanks to great engineering at the Douglas Instruments, we can routinely set up ~1000 drops for a given protein. If one of them shows a crystalline shower, we celebrate. To me, the fact that we try wrong crystallization conditions 99.9% of the time, proves that any attempt to predict crystallization conditions beyond vague things like keep pH close to protein pI, sodium malonate is cool, PEG and ammonium sulfate are two most successful precipitants in history of protein crystallography, etc., is futile. Time wasted on looking into what is the most common precipitant for a particular class of proteins is better spent on setting up more trays. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Crystals with Organic solvents
Hi Eswar Firstly, I would certainly try crystal seeding into random screens if you haven't already tried it. Refs below. Secondly, it's very convenient to grow the crystals under oil, and to soak the organic solvents into the drops, through the oil. This makes it much easier to harvest the crystals because the oil becomes saturated with the solvent and stops it from evaporating when you pick up the crystals. This approach can be used at the screening stage too. See Mortuza, et al. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. Also see http://www.douglas.co.uk/winner1.htm Good luck Patrick Allan D’Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007) 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D66 (2010) 568-576. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444910005512 Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 See also http://www.douglas.co.uk/mms.htm On Fri, Aug 26, 2011 at 11:55 AM, eswar reddy eswar.uo...@gmail.com wrote: Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] UV imaging of crystals
microscopes use glass. And you can't see 280 nm (and its not good for your eyes) so you need some kind of phosphor screen to view the image? Bosch, Juergen wrote: I'm replying here to myself :-) So in an off-board discussion it turns out that the microscope in question was a special emitted light and not a UV microscope. So real UV microscopes might be better for the purpose of detecting real crystals. Sorry for the confusion - had too much sun today :-) Jürgen On Sep 15, 2011, at 4:19 PM, Jürgen Bosch wrote: I once tested such a commercial system in Seattle about 4 years ago. It did not impress me. In particular the discrimination between salt and protein did not work for about 10 different proteins from which we already had collected data. sure those were small between 10 and 100 micrometer. Excuse was to few tryptophans So in theory it is nice but a cheaper variant might be to add Gfp to your protein and screen for something green. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 15, 2011, at 16:03, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: A while ago I was trying to be cheap, so we played around with it quite a bit in the lab. After rediscovering some of the basics of signal-to-noise and microscope transmission efficiency and that sort of rot, I realised that the commercial systems may not be all that ridiculously overpriced after all. Not if one wants to be able to say something useful about really really small crystals -- the only ones that really matter in the grand scheme of things (big ones are quick to test; little ones must first be optimized = money+time). But maybe I was just being incompetent. Happens. phx. On 15/09/2011 20:50, Andrew Purkiss-Trew wrote: Quoting Harman, Christinechristine.har...@fda.hhs.gov: Hi All, I was curious if any of you have tried or even know if it is possible to adapt a stereoscope (in my case an Olympus SZX10 model) so as to view protein crystals with UV illumination. Basically, I want a cheap manual version of what a Rock UV Imager does. I know this is probably a crazy dream. However, I would greatly appreciate any comments, advice or experience any of you may have. Molecular Dimension do such an adaptor which fits to existing microscopes. See http://www.moleculardimensions.com/shopdisplayproducts.asp?id=121cat=X%2DtaLight%3Csup%3E%99%3C%2Fsup%3E+100+%2D+UV+for+Microscope+ This message was sent using IMP, the Internet Messaging Program. .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Complex seeding
Hi Peter This idea was discussed at the recent RAMC meeting, and there is at least one example where it has worked. Generally, cross-seeding can work as long as you have homology. See e.g. Obmolova et al. Acta Crystallogr. 2010, D66, 927–933. The same group has reported seeding a complex with crystals of one of the monomers. One thing to bear in mind is that there is no point in adding a seed stock (with e.g. crystals of one of the monomers) if the seed stock destabilizes your complex. This is all discussed in great detail and suggestions are made for finding alternatives in a paper that I mentioned here earlier (which we published this year) ref below. Good luck Patrick _ “Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization”. Shaw Stewart et al, Crystal Growth and Design, 2011, 11 (8), p3432. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442 On Wed, Sep 21, 2011 at 6:04 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I've been trying to crystallize a 3 protein complex recently with little success. However, crystals of each subunit have previously been crystallized. I was wondering if any one knows of any literature/experiences where people have used seeds from an individual subunit to seed for a complex and succeeded? Or is this just a crazy/bad idea? Thanks in advance for any input. Peter -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] neutron data measurement
Third, the size of crystal needed for successful neutron diffraction is right at the limit of the size of crystal that can be successfully flash-cooled without inducing excess mosaicity. Can't the crystal be flash-cooled at high pressure? The inventors of the Crystal Harp in Zurich use a machine that does this automatically for cryo e.m., which many universities already have. On Fri, Sep 23, 2011 at 4:40 PM, Leif Hanson leif.han...@gmail.com wrote: This thread caught my attention several days ago and I now have enough time to add my two cents worth. These are my own biases and probably do not reflect the views of my friends and colleagues at various neutron facilities. With respect to the size of crystals for neutron diffraction, a good rule of thumb is that there should be at least 10exp24 uniformly ordered unit cells in a D2O exchanged crystal to have successful diffraction on par with rotating anode data measured on a crystal with a tenth the volume. Several data sets have been measured from smaller crystals, and perdeuteration lowers the volume needed to extract useful information. Most of the neutron data has a resolution cutoff of 1.8 to 2.0Å, which permits unambiguous placement of deuterons and solvent molecules, especially when completing dual refinement of X-ray and neutron data from the same crystal. There have been a limited number of low temperature neutron diffraction experiments for several reasons. First, of the available neutron beamlines for macromolecular data measurement there are only one or two with open flow cryostats available, limiting the locations for standard macromolecular cryocrystallography. Second, there are a tremendous number of important structures that can be done at room temperature. It is difficult to justify the time needed for low temperature work to experimental review panels when crystals are available to resolve a knotty enzyme mechanism problem. Third, the size of crystal needed for successful neutron diffraction is right at the limit of the size of crystal that can be successfully flash-cooled without inducing excess mosaicity. Most neutron beamlines use some form of quasi-Laue data collection strategy. Mosaicities in excess of 0.5º render most crystals unusable for neutron data measurement. Remember that a lot of uniform unit cells are needed to get a usable diffraction signal from neutrons. Often a large flash-cooled cooled crystal appears to have low mosaicity when exposed to 0.5mm x-ray beam. However, when placed in the 3mm neutron beam, limited streaky low-resolution diffraction appears. It is difficult to judge the quality of flash-cooled neutron diffraction sized crystal without placing it in the neutron beam. Returning to point 2 it is difficult justify the time needed on fishing expedition. So far the only large crystals I have been able to flash-cool that met the demands of size and crystal perfection had very low solvent content or were grown in high levels of cryoprotectant. That said, several critical problems cry out for low temp neutron studies so there is every reason to persevere. I would be pleased to answer any questions off-line for those of you with more interest in neutron cryocrystallography. Finally with respect to radiation damage, Benno Schoenborn has had a myoglobin crystal in sealed capillary that he has used as a “standard candle” for testing neutron beamlines. There has been no discernable degradation of the crystal in all the years he has used it. The neutrons used for neutron diffraction are ‘cold’ neutrons, usually with energies of 1 – 10 meV. Damage could come from activated nuclei, but these are usually very limited on a molar basis within the crystal. As can be seen with Benno’s myoglobin crystal, 30 years of iron activation has yet to produce a measurable defect. Leif Hanson University of Toledo -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein melting temperatures
I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein melting temperatures
Susan and everyone, I should apologise for any confusion that I may have caused. Rajiv actually asked his question a year ago, and I accidentally replied to it a year too late! It's an interesting question though Patrick On Wed, Sep 28, 2011 at 5:03 PM, Linda Schuldt lschu...@mb.au.dk wrote: ** Dear Raji, what exactly do you mean when you say the melting temperature is 45deg. Did you only test one buffer, or did you test many buffers and 45deg is the most stable one? If you have only tested one buffer you should run a screen testing different buffer systems (pH) and e.g. NaCl concentration and glycerol concentrations (or ligands, if your proteins binds any). Then you identify the buffer which is stabilizing your protein the most. I have seen big impacts on protein stability and crystallization when optimizing my buffers like this. I think you should not only consider the melting temperature alone, but also how the curve looks like. Do you get a high initial flourescence (which often indicates partially unfolded protein or hydrophobic patches) or do you have very low initial flourescence (which is a good sign for compact protein). Another thing to look at is if your transition is sharp (the steeper the better). Taking all this together you can judge if your protein is happy or not. Hope this helps you! Linda Patrick Shaw Stewart wrote: I actually think you *can *make comparisons between different proteins. We heard a very nice talk by Jose Marquez about exactly this at the RAMC meeting recently. Basically, 45C seemed to be the dividing line. If your protein melts below this it's a bad sign for crystallization and may point to setting up your crystallization experiments at lower temperatures. Patrick On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis a.perra...@nki.nlwrote: ** Hello - The excellent paper of McCrary, uses differential scanning calorimetry, which will give an absolute measure of thermostability. Using Thermofluor I would be afraid you can only assess the relative thermostability of one protein in different conditions. As your fluorescence reporter would interact differently with exposed hydro[hobic patches in different proteins, I would be a bit more careful in comparing the Thermofluor results between different proteins ... I am not aware of anyone correlating differential scanning calorimetrywith Thermofluor data, but I must admit I have not looked up that literature recently. A. On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : Raji I suggest having a look to this paper: McCrary et al. J. Mol. Biol. 264(1996) 784 where you will find an interesting study on protein stability and an interesting comparison with other proteins. Philippe Dumas Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University McCrary-JMB264(1996)784.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *** Dr. Linda Schuldt Department of Molecular Biology University of Aarhus Science Park Gustav Wieds Vej 10c DK-8000 Århus C Denmark -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] IUCr committees, depositing images
Could you perhaps use the principle of capture storage that is used by wild-life photographers with high-speed cameras? The principle is that the movie is written to the same area of memory, jumping back to the beginning when it is full (this part is not essential, but it makes the principle clear). Then, when the photographer takes his finger off the trigger, the last x seconds is permanently stored. So you keep your wits about you, and press the metaphorical store button just *after *you have got the movie in the can so to speak Just a thought Patrick On Wed, Oct 26, 2011 at 2:18 PM, John R Helliwell jrhelliw...@gmail.comwrote: Dear Frank, re 'who will write the grant?'. This is not as easy as it sounds, would that it were! There are two possible business plans:- Option 1. Specifically for MX is the PDB as the first and foremost candidate to seek such additional funds for full diffraction data deposition for each future PDB deposiition entry. This business plan possibility is best answered by PDB/EBI (eg Gerard Kleywegt has answered this in the negative thus far at the CCP4 January 2010). Option 2 The Journals that host the publications could add the cost to the subscriber and/or the author according to their funding model. As an example and as a start a draft business plan has been written by one of us [JRH] for IUCr Acta Cryst E; this seemed attractive because of its simpler 'author pays' financing. This proposed business plan is now with IUCr Journals to digest and hopefully refine. Initial indications are that Acta Cryst C would be perceived by IUCr Journals as a better place to start considering this in detail, as it involves fewer crystal structures than Acta E and would thus be more manageable. The overall advantage of the responsibility being with Journals as we see it is that it encourages such 'archiving of data with literature' across all crystallography related techniques (single crystal, SAXS, SANS, Electron crystallography etc) and fields (Biology, Chemistry, Materials, Condensed Matter Physics etc) ie not just one technique and field, although obviously biology is dear to our hearts here in the CCP4bb. Yours sincerely, John and Tom John Helliwell and Tom Terwilliger On Wed, Oct 26, 2011 at 9:21 AM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Since when has the cost of any project been limited by the cost of hardware? Someone has to implement this -- and make a career out of it; thunderingly absent from this thread has been the chorus of volunteers who will write the grant. phx On 25/10/2011 21:10, Herbert J. Bernstein wrote: To be fair to those concerned about cost, a more conservative estimate from the NSF RDLM workshop last summer in Princeton is $1,000 to $3,000 per terabyte per year for long term storage allowing for overhead in moderate-sized institutions such as the PDB. Larger entities, such as Google are able to do it for much lower annual costs in the range of $100 to $300 per terabyte per year. Indeed, if this becomes a serious effort, one might wish to consider involving the large storage farm businesses such as Google and Amazon. They might be willing to help support science partially in exchange for eyeballs going to their sites. Regards, H. J. Bernstein At 1:56 PM -0600 10/25/11, James Stroud wrote: On Oct 24, 2011, at 3:56 PM, James Holton wrote: The PDB only gets about 8000 depositions per year Just to put this into dollars. If each dataset is about 17 GB in size, then that's about 14 TB of storage that needs to come online every year to store the raw data for every structure. A two second search reveals that Newegg has a 3GB hitachi for $200. So that's about $1000 / year of storage for the raw data behind PDB deposits. James -- Professor John R Helliwell DSc -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Disappearing crystals
-- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] No diffraction
Theresa You should also try microseeding into *random screens *by making a seed stock with the crystals that you have, to use with both microbatch and vapor diffusion experiments. You will often pick up new and better conditions and you're more likely to get well-formed crystals right out of the screens. I hope it works Patrick _ Refs: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica section D 63 (2007), 550–554. Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and Gary L. Gilliland. 'Promoting crystallization of antibody–antigen complexes via microseed matrix screening.' Acta Crystallographica Section D 66 (2010) 927–933. Open-access at http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F. Harris. 'Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias.' Acta Crystallographica Section D 66 (2010) 568-576. Gregory Ireton and Barry Stoddard. 'Microseed matrix screening to improve crystals of yeast cytosine deaminase'. Acta Crystallographica section D60 (2004) 601–605. Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442. * If you don't have a subscription to Crystal Growth and Design, click **here*http://www.douglas.co.uk/mms.htm#CGDFreeCopy * to obtain a free copy (we're limited to 50 downloads in the first year).* On 26 January 2012 15:33, Theresa H. Hsu theresah...@live.com wrote: Dear crystallographers I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and vapor diffusion method in 24 hours but no diffraction at home source. Dissolved crystals was confirmed to be the protein with mass spec. Any suggestions to improve diffraction would be welcome. Thanking you in advance. Theresa -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Dye for protein affinity measurement
Jiahong Thermo sells a series of kits called DyLight Fluor for fluorescent labelling of antibodies or other proteins. They have everything you need and they're very convenient and easy to use. You can pick the excitation and emission wavelength. If you label both A and B (or C) with different colors you will be able to see if both are in your crystals (assuming crystallization is part of your approach). You need only label a small percentage of your protein or peptide to see whether the protein is present in a crystal. Patrick http://en.wikipedia.org/wiki/DyLight_Fluor Forsythe, E.L., Achari, A., and Pusey, Marc L. (2006), Trace Fluorescent Labeling for High Throughput Crystallography, Acta Cryst. D62, 339-346. We used DyLight 350 NHS Ester to check we had protein crystals - see methods section of *Cryst. Growth Des.*, 2011, *11* (8), pp 3432–3441 2012/2/8 Jiang Jiahong jiang_jiah...@126.com Dear all, I am looking for some kind of dye for protein affinity comparison, but do not know which to choose. I know protein A can contact B to form a complex,now I hope to find something simiar with A to act as an inhibitor to block the process of A-B complex formation. Maybe a short peptide, a segment of protein A or even some organic molecule. Because here is a poor access to ITC nor Biacore, I can only rely on some dye to check the competence between A and inhibitor candidates. If any one can offer any suggestions. That would be so grateful! Any way,thank kind-hearted people in advance! Regards Jiahong -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Membrane Protein Crystallization Plates
Yuri I know this isn't quite what you're asking, but it's helpful to use the COC (UV permeable) version of plates if you're crystallizing samples that contain detergent. It's just that the drops tend to spread very thinly if you use the normal polystyrene PS plates. The COC is more hydrophobic. (On the other hand we prefer PS plates for normal proteins with no detergent.) Patrick On 25 February 2012 20:35, Yuri Pompeu yuri.pom...@ufl.edu wrote: Hello Everyone, I am considering the purchase of crystallization plates for membrane proteins. I would love to hear what some of the community thinks or has experienced with these. Particulalrly the monoolein and monoolein/cholesterol coated plates ( I am not sure I can mention the vendor here but it should not matter) So fire away. Is it worth it? Any succes stories? Bad experiences? I appreciate the input Best, Yuri -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Desalting columns
We did some data mining from remark 280 of the PDB in 2004. Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml. See table 3 at http://www.douglas.co.uk/PDB_data.htm . Patrick On 27 February 2012 16:25, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? ** ** For crystallization, the concentration needs to be **a) **high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) **b) **high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. ** ** There is ample evidence for proteins crystallizing below 3 mg/ml. ** ** The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. ** ** Sometimes the shape of a distribution matters ;-) ** ** BR ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Sangeetha Vedula *Sent:* Monday, February 27, 2012 8:02 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Desalting columns ** ** Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha. -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] microseeding
Rajesh If you set up the volumes you suggest you will probably get precipitation. This is counterintuitive until you realize that (as Ed says) you will be losing a lot of protein with those small drops. When you scale up the surface area to volume ratio is lower, so a smaller proportion of the protein is lost. Therefore you go *up* on the phase diagram and get precipitant or very small crystals. Normally halving the amount of protein for the hits from 200 nl drops works (suggesting that half the protein is lost from such small drops). Try say 500+1000+500 (don't reduce the volume of seed stock because the solution that you suspended the crystals in may be important). Or dilute the protein and use 1000+1000+500. For the hits from the 450 nl drops you could reduce or dilute the protein by say 25.%. Or make plenty of seed-stock and try seeding into a random screen again with larger drops, say 1.5+1+0.5 ul Those tiny crystals should be good for seeding, don't worry about that (provided they are protein of course). Streak seeding may work but bear in mind that roughly a third of the precipitant comes from the seed stock in your 250 nl drops. You can add the seed stock with a syringe and needle if you don't have suitable robot ;) Experience and data-mining suggests that reducing the salt precipitant (in high-salt drops) or salt additive (in PEG drops) by around 50% may be helpful too when scaling up - I'm not sure why this works. Good luck Patrick For the hits in the 250 nl drops you are probably losing On 19 March 2012 20:31, Rajesh kumar ccp4...@hotmail.com wrote: Dear All, I have few papers in hand which explain me about microseeding, matrix microseeding, and cross seeding. I have also read few earlier threads and some more literature in google. Using Phoenix robot, I did a matrix micro-seeding and matrix cross seeding. I have few hits with this. In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed) in separate expts. I have hard time to plan to translate this 96 sitting drop well plate to 24 well plate to refine the conditions to get better crystals. only 1-2 hits are small crystals and they are tiny. I wonder in 24 well plate, if I should do- 1) for Example 500+500+50nl (I am sure I cant add less that 500 nL precisely) 2) to a drop of 500+500 nL do microseeding/streaking with a hair I appreciate if you could advise and share some practical ways to further my experiment. Thanks in advance Regards, Rajesh -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays
Hi Matt Rajesh asked a similar question last week, below Essentially, you have to *reduce *the protein concentration when you scale up because you lose proportionally more protein from smaller drops. This usually works very well and we see no reason to use more than 0.3 + 0.3 for initial screening. There's a page on our web site which explains this in much more detail, see http://www.douglas.co.uk/Scaling_Up.htm Best wishes Patrick -- Forwarded message -- From: Patrick Shaw Stewart patr...@douglas.co.uk Date: 19 March 2012 22:29 Subject: Re: [ccp4bb] microseeding To: Rajesh kumar ccp4...@hotmail.com, ccp4bb@jiscmail.ac.uk Rajesh If you set up the volumes you suggest you will probably get precipitation. This is counterintuitive until you realize that (as Ed says) you will be losing a lot of protein with those small drops. When you scale up the surface area to volume ratio is lower, so a smaller proportion of the protein is lost. Therefore you go *up* on the phase diagram and get precipitation or very small crystals. Normally halving the amount of protein for the hits from 200 nl drops works (suggesting that half the protein is lost from such small drops). Try say 500+1000+500 (don't reduce the volume of seed stock because the solution that you suspended the crystals in may be important). Or dilute the protein and use 1000+1000+500. For the hits from the 450 nl drops you could reduce or dilute the protein by say 25.%. Or make plenty of seed-stock and try seeding into a random screen again with larger drops, say 1.5+1+0.5 ul Those tiny crystals should be good for seeding, don't worry about that (provided they are protein of course). Streak seeding may work but bear in mind that roughly a third of the precipitant comes from the seed stock in your 250 nl drops. You can add the seed stock with a syringe and needle if you don't have suitable robot ;) Experience and data-mining suggests that increasing the salt precipitant (in high-salt drops) or salt additive (in PEG drops) by around 50% may be helpful too when scaling up - I'm not sure why this works. Good luck Patrick For the hits in the 250 nl drops you are probably losing On 19 March 2012 20:31, Rajesh kumar ccp4...@hotmail.com wrote: Dear All, I have few papers in hand which explain me about microseeding, matrix microseeding, and cross seeding. I have also read few earlier threads and some more literature in google. Using Phoenix robot, I did a matrix micro-seeding and matrix cross seeding. I have few hits with this. In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed) in separate expts. I have hard time to plan to translate this 96 sitting drop well plate to 24 well plate to refine the conditions to get better crystals. only 1-2 hits are small crystals and they are tiny. I wonder in 24 well plate, if I should do- 1) for Example 500+500+50nl (I am sure I cant add less that 500 nL precisely) 2) to a drop of 500+500 nL do microseeding/streaking with a hair I appreciate if you could advise and share some practical ways to further my experiment. Thanks in advance Regards, Rajesh -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Experimental design and response surface methods for crystal optimization
Hi Christian We often use a simple approach where people design their experiments using rational multivariate design, but then simply use the best well that is found as the centre of the next round of experiments. This means that you don't have to score all the wells - which is time-consuming! There's more info at http://www.douglas.co.uk/rat_des.htm Patrick -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Benda, Christian Sent: 15 May 2009 15:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Experimental design and response surface methods for crystal optimization Dear all I recently came across the experimental design method for systematic optimization of multiparametric problems like macromolecular crystal growth. (The response surface concept has been introduced to protein crystallization by Carter et al. (e.g. Meth. Mol. Biol, vol. 363) and should theoretically enable optimization of a multiparatmetric problem by simultaneous variation of several variables). I am wondering if anyone is actually making use of this method on a regular basis and what their experiences are. It would also be interesting to now if tools (web-based) are available to help in designing and evaluating experiments. Any comment appreciated! Thanks very much Christian
Re: [ccp4bb] How to improve crystal which is twinning?
If you haven't already used it, I would certainly try the approach of microseeding into screens since you already have crystals http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 -- patr...@douglas.co.uk mailto:patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of HanJie_HCT Tai Sent: 17 May 2009 13:27 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to improve crystal which is twinning? Hi, I have a 22kDa protein that the floopy N C terminus have been deleted. It was crystallized in 35%MPD/0.1m Tris (pH 8.5)/0.2M/(NH4)2SO4 in 2 - 3 days. Previously, a few small twining crystals were grown in this condition. I tried Hampton Research 96-additive screen . Additives such as glycerol, dioxane, etc didn't work well to improve/reduce twining issue. The 0.3% DMSO is the best additive I found that can grow a single crystal in the 1(pro)+0.8(buff)+0.2(add) drop. However, If looking careful under microscope it may be some other crystals growing inside that single crystal(hardly see under the microscope, but I can see it in other drops). I conducted the x-ray diffraction experiment for this 0.1x 0.1 mm crystal for which no cryoprotectant is required. The highest resolution is 2A but there are a lot of smear on the diffraction pattern. Thus, the index procedures failed due to the crystal quality. Do you have any brilliant ideas to improve the crystal growing condition in my case in order to get a truly single crystal? regards, Heng Windows Live(tm): Keep your life in sync. Check it out. http://windowslive.com/explore?ocid=TXT_TAGLM_BR_life_in_synch_052009
Re: [ccp4bb] anaerobic crystallization
Chris Although generally less popular nowadays, microbatch-under-oil has great advantages for anaerobic work. You can keep your stock solutions in the chamber all the time, so you only need to degas your protein. Microbatch finds roughly as many crystals in screening experiments as vapor diffusion (summary at http://www.douglas.co.uk/mbnvdall.htm ). Of course the oil helps to protect the protein too - generally less is denatured on the surface For an automatic setup that fits in an anaerobic chamber conveniently see the winning entry in Douglas Instrument's competition (2006), from the University of Georgia, Athens http://www.douglas.co.uk/presentations/winner2_files/v3_document.htm Hope that's useful Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Christopher Rife Sent: 02 September 2009 18:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] anaerobic crystallization Hi, Does anyone have tips or suggestions for getting started with anaerobic crystallization? Searching through the archives, I was able to find some discussion about various glove box options (http://tinyurl.com/ccp4-glovebox), but not about the process itself (we already have a box). To simplify things, would it be worthwhile to perform the initial screens in something like the pre-filled Qiagen screens (http://tinyurl.com/qiagen-xseal)? Do I need to worry about evaporation while solutions are being degassed (esp volatiles)? Better/easier to try microbatch? Thanks for any input. Chris ___ _ ___ This is an e-mail from Danisco and may contain confidential information. If you are not the intended recipient and you receive this e-mail by mistake, you are not allowed to use the information, to copy it or distribute it further. Please notify us and return it to Danisco by e-mail and delete all attachments. Thank you for your assistance. ___ _ __
Re: [ccp4bb] stabilizing solution
James Generally you can use the reservoir solution to make your seed stocks, as is routinely done with the MMS microseeding method. However, the seeds will not be completely stable because there will not be much protein in the solution, so you must keep your seed stocks on ice, and freeze them as soon as you've finished using them I imagine that MPD would stabilize your seed crystals as well as anything else Reference below Best wishes Patrick [2] Allan D'Arcy, Frederic Villarda, May Marsh. 'An automated microseed matrix-screening method for protein crystallization'. Acta Crystallographica section D63 (2007), 550-554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of james09 pruza Sent: 06 September 2009 09:14 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] stabilizing solution Dear CCP4bbers, Please comment on the stabilizing solution for seed stocks. If the crystal is in 30% MPD and coming after 2-3 days, what should be the stabilizing solution for seeding. Thanks. James
[ccp4bb] Vapor diffusion calculator
Jacob and CCP4bb It's not exactly what you're looking for, but my colleague Peter Baldock wrote a program called VD to MB a few years ago that does part of the job. It was a program to convert vapor diffusion crystallization conditions into microbatch-under-oil conditions. I tried it back then with several published and unpublished examples where crystallization had been optimized in both vapor diffusion and microbatch. I was originally sceptical, but it seemed to work remarkably well, generally getting the numbers right to within 5 or 10%. The program uses a parameter called 1/e equilibration time for 1M salt (days). I originally used a value of 1, but I think we changed it to 0.5 for 96-well plates. Obviously the value will depend on the geometry of the plates. Does anyone have any practical information about equilibration times? Different salts have very different effects on vapor pressure. I found a list in an old Rubber Book (CRC Handbook of Physics and Chemistry) part of which I have pasted below. I tried quite hard to make sense of this list - hoping that it would also shed light on the protein-precipitating effect of salts - but concluded that the numbers are pretty random apart from obvious valence effects. (Someone here may be able to explain them, though!) You can download the program from http://www.douglas.co.uk/tipsntech.htm near the middle of the page. There's also an equivalent Excel spreadsheet for playing around with. See also http://www.douglas.co.uk/convert.htm for some (very old but still valid!) examples and comments. Also bear in mind that - with many proteins - roughly half of the protein is lost on the surface of VD drops for 100 + 100 nl drops. Peter also wrote an algorithm - now in our optimization software - that calculates the pH of a solution with any number of buffers in it, where the buffers can be at different concentrations too. A sort of super Henderson-Hasselbalch. If anyone is interested I'm sure Peter could show you the maths or pass on the code (it took him about a month of lunch-breaks!) Best wishes Patrick _ Reduction of vapor pressure in mmHg due to the presence of 1M salt at 100C (at which temperature the vapor pressure of water is 760 mmHg) From Handbook of Physics and Chemistry, 76th Ed CdSO4 8.9 ZnSO4 10.4 MnSO4 10.5 FeSO4 10.7 MgSO4 12 CdI2 14.8 CdBr2 17.8 ZnCl2 18.7 CdCl2 18.8 KNO3 21.1 NH4NO3 22 NaNO3 22.5 NH4Cl 23.7 NH4Br 23.9 NH42SO4 24 KCl 24.4 Na2SO4 25 NaCl 25.2 KI 25.3 LiCl 25.5 LiNO3 25.9 NaBr 25.9 LiBr 26.2 BaNO32 27 Li2SO4 28.1 LiI 28.6 K2CrO4 29.5 Na3PO4 30 Li2CrO4 32.6 MnCl2 34 CaNO32 34.8 CoCl2 34.8 Al2S043 36.5 BaCl2 36.7 NiCl2 37 NiNO32 37.3 BaBr2 38.8 MgCl2 39 CaCl2 39.8 MgNO32 42 MgBr2 44 CaBr2 44.2 AlCl3 61 StDev 10.7 Av 27.7 CV 0.4 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 02 February 2010 22:34 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Vapor diffusion calculator Dear Crystallographers, Is anybody aware of a calculator for vapor diffusion experiments to plot concentrations of various solvent components as a function of time? For a simple example, what happens when I mix a protein solution containing 50mM NaCl 1:1 with a reservoir containing 50% MPD but no salt? Where is the vapor diffusion equilibrium, and how does the drop composition change as a function of time? More complicated would be experiments involving volatile components other than water, as I think, for example, ethanol would almost instantly equilibrate, then the water diffusion would kick in over a longer time scale. Even more complicated would be pH-dependent volatilities such as acetate. I don't think this would be impossible to figure out, but it would be nice if there were a pre-existing tool/server to do such. Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] 24-well sitting-drop plates
We have had dozens of boxes of XtalQuest 42-2 plates sitting on the shelf for three years and I've just been told we've only sold three boxes so far We'll accept any reasonable offers! -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Patrick Shaw Stewart Sent: 25 February 2010 16:37 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] 24-well sitting-drop plates Jacob There is the MRC Maxi, and also XtalQuest plate, both with 48 wells. Less confusing for filling by hand than 96 wells -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 24 February 2010 00:02 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] 24-well sitting-drop plates Dear Crystallogrpahers, what are the best 24-well sitting drop plates on the market? I have used the standard pilar-type ones from Hampton, but I would like to have the ability to set up a few drops per well, and also those plates have a lot of background polarization. Also, the reason I am looking for 24-well plates is that I would like to set them up by hand, and 96 wells seems like a lot to do before sealing! Best Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] freezing crystals grown in isopropanol condition
Chris Isopropanol is said a very good precipitant but it's unpopular because it's so difficult to harvest crystals However there's a very convenient way to grow and harvest crystals in isopropanol using Vapor Batch plates. Basically you set up microbatch-under-oil drops containing all the ingredients except for the isopropanol, and cover with Al's Oils (silicone mixed with paraffin oil). Then you put e.g. in your case 25% isopropanol in the reservoir around the outside of the Vapor Batch plate. Over a few hours the isopropanol will diffuse through the Al's Oils into the drops. The great advantage is that the oil also becomes saturated with isopropanol (they are only partially miscible in fact) so that the oil acts as a barrier when you're harvesting crystals. You can usually convert vapor diffusion conditions to microbatch in a single 24-well experiment where you vary protein against precipitant. You don't need to remove the oil to collect data. This is all described in Lesley Haire's winning entry to a competition we organized a few years ago: http://www.douglas.co.uk/winner1.htm See also Mortuza et al. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. The Vapor Batch plate can be seen at http://www.douglas.co.uk/vb.htm If you or anyone on the b.b. would like to try some samples of Vapor Batch plates just let me know Best wishes Patrick On Fri, Apr 9, 2010 at 9:53 AM, Chris Meier crystallogra...@christophmeier.com wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] control of nucleation
Hi Zq Deng No-one has mentioned microseeding into random screens, the so-called Microseeding Matrix Screening (MMS) method , or just matrix seeding. This is exactly the kind of situation where the method works really well. You will very often get many new leads, as well as bigger crystals. See the excellent paper by D'Arcy et al., Acta Cryst. (2007). D63, 550-554, and also the original paper of Ireton and Stoddard, Acta Cryst. (2004). D60, 601-605. See also the practical and theoretical comments at http://www.douglas.co.uk/mms.htm Best wishes Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of zq deng Sent: 06 May 2010 09:04 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] control of nucleation hello,everybody . due to excess nucleation,I often get many tiny crystals instead of few,large crystals.i wana optimize the condition, does anyone have adivce about this? Best regards.
Re: [ccp4bb] frozen pellet insoluble protein
Andreas, you probably know all this, but I only understood quite recently. What happens is that as ice crystals form you get brine rejection, the same thing that happens in the arctic when sea water freezes. Therefore you can have protein concentrated in pockets of high salt. Fine for some proteins, but others don't like it. And it can happen during (slow) thawing as well as during freezing. - Patrick On 29 September 2014 16:02, Andreas Förster docandr...@gmail.com wrote: Dear all, I've encountered people who refuse to freeze cells and always lyse fresh pellets. Better protein, they say. I've never had reason to do so myself, or even to believe in their voodoo. Up until now, maybe. My protein expresses well and is almost all in the soluble fraction in an expression test from a fresh pellet. The large-scale expression from the same pellet, now frozen and thawed, yielded 90% insoluble protein. If it's the freezing that dooms the protein, I'm happy to redo the fermentor run. Are there other examples out there of this? Thanks. Andreas -- Andreas Förster Crystallization and X-ray Facility Manager Centre for Structural Biology Imperial College London -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] fastening crystal formation
Michael (and some others) You haven't mentioned - and I guess don't use regularly - the random MMS approach, where crushed seed-crystals are added to random screens. This really is a terrific method, and it will on average roughly double productivity. It's the first thing I would think of in a case like Vijaykumar's (as I told him this morning!). Galina Obmolova and her colleagues published a great paper earlier this year about MMS. They were working with a set of 16 Fab fragments that comprised all combinations of 4 different heavy chains and 4 different light chains. Three structures were generated without MMS, but by various very creative uses of microseeding they were able to get all 16/16 structures. Ref below. Best wishes, Patrick __ Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., Gilliland, G. L. (2014). Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. *Structural Biology and Crystallization Communications*, *70*(8). On 31 October 2014 16:07, R. M. Garavito rmgarav...@gmail.com wrote: Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry 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) 353-9334Email: rmgarav...@gmail.com garav...@gmail.com* ** On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri vijaypkuma...@gmail.com wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation
Hi Thomas We use a very simple solution. We simply shorten a normal needle to 6.5 mm and push it into another syringe that has *two *of the white PTFE ferrules made by Hamilton. (We salvage the extra ferrule from another needle.) We use a 22 gauge needle (not 22S, where the S stands for *small *inner diameter). Now you can push the two syringes together and make your LCP. It helps if you tighten the knurled nose-piece to compress the PTFE ferrules before you join the syringes. You have to keep a little pressure on the two syringes to hold them together while you mix up the LCP, or use eg tape. (Which is why we've made a little mixer on a rail, but that's another story.) One nice feature is that you hardly waste any LCP and the needle for dispensing it is already attached to the syringe. Let me know if you need more info or you'd like me to send you a short needle. Best wishes, Patrick On 16 December 2014 at 02:14, Thomas Cleveland thomas.clevel...@gmail.com wrote: Hi everyone, Thanks for the advice. I had, in fact, already ordered some commercial couplers, but they haven't come in yet, and there was an experiment I wanted to do today. Here's what I found: 1. The steel ferrules have an orientation, with a tight side and a loose side. They can be removed by sliding in one direction, but not the other. Even then, it's pretty difficult. I had to use pliers, and pieces of needle broke off during the process. The tight side of the ferrule then needed to be reamed open slightly with a steel tool before I was able to slide the ferrule onto the other needle. 2. Soldering stainless steel is really a pain. In the end I got a coupler that seems to work well, but it was a pain, and it's a bit charred looking. Thanks again, Tom On Mon, Dec 15, 2014 at 6:01 PM, Aaron Thompson aaron.a.thomp...@gmail.com wrote: I agree with Jim – purchasing the couplers will get you up and running much quicker. TTP also sells nice couplers: https://www.ttplabtechstore.com/ttp_ecom/cc/ItemDetails.jsp?@where.ItemID@EQ=3072-01050sessionkey=# Aaron On Mon, Dec 15, 2014 at 12:38 PM, Bernhard Rupp hofkristall...@gmail.com wrote: Tried the RN kludge at least I did not get it to work. You cannot tighten the plastic swage lock type sleeves tight enough. On operation the pressure drives the PEEK tubing out of the compression fit. Maybe if you have a jig that holds the 2 syringes in a fixed position so they cannot move apart it can work. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Daniel Anderson Sent: Monday, December 15, 2014 8:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Fabricating hamilton syringe coupler for LCP preparation Here's my addition to Jim Fairman's reply: You could use a pair of RN compression fittings (www.hamilton dot com part number 55751-01) and a segment of HPLC tubing. HPLC tubing within my field of view can have an inside diameter as small as 0.005 inch. hope that helps, Happy Merry, etc., Dan On 12/15/2014 11:09 AM, Thomas Cleveland wrote: Hi all, I'm trying to put together some homemade syringe couplers following the published instructions from the Caffrey group. I'm having a bit of trouble with this part: The stainless steel ferrule of the second needle is removed and placed on the free end of the coupling needle such that the double thumb nut is held symmetrically between the two steel ferrules Has anyone done this, and if so, how did you remove the stainless steel ferrule from the second needle in order to place it over the first? The stainless steel ferrule appears to be firmly attached and I'm having trouble removing it. Thanks, Thomas Cleveland -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box
Hi Steve I have one more comment for this thread. The microbatch-under-oil method is very handy for anaerobic work: 1. You can keep the microbatch stock solutions in normal microtitre plates (polypropylene is best to reduce evaporation) for months, which hugely reduces the amount of degassing that you need to do. You will only use say 0.5 ul of stock per drop. 2. The oil offers a surprising amount of protection from oxidation, which may be helpful eg in harvesting. 3. Microbatch can be automated - in parallel to vapor diffusion if desired It's amazing how often (aerobic) microbatch produces far superior crystals to V.D. for no obvious reason - it's well worth trying for both screening and optimization. Best wishes Patrick On 11 March 2015 at 10:17, Stephen Carr stephen.c...@rc-harwell.ac.uk wrote: Dear CCP4BBer's Apologies for the off-topic post, but the CCP4BB seems to be the best place to ask about crystallisation. I am looking to set up crystallisation in an anaerobic glove box and wondered how other people did this, specifically the crystallisation stage. My initial thoughts were to place a small crystallisation incubator inside the box, however the smallest I have come across so far (~27L) is still rather large. Has anyone come across smaller incubators? Alternatively are incubators even neccessary if the glove box is placed in a room with good air conditioning and stable temperature control? Any recommendations would be very helpful. Thanks in advance, Steve Carr Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 This email 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 authorized 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 this email. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Research Complex at Harwell. There is no guarantee that this email 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. We use an electronic filing system. Please send electronic versions of documents, unless paper is specifically requested. This email may have a protective marking, for an explanation, please see: http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm . -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box
Thank you for your comments everyone. The CCP4BB is a wonderful resource and it has answered several questions that have been bothering me for years! Tristran has given us the correct conclusion as well as the important facts: the capacity of oil for holding O2 is high, but the diffusion rate is low. This makes complete sense of the observations reported. The O2 is (slowly) diffusing OUT of the permanganate drop, and the oil is already saturated with O2, therefore it takes a long time for the purple colour to be lost. The O2 is diffusing INTO the drop in the dithionite experiment, and presumably the oil that Julia used was already loaded with O2, so the reducing environment was quickly lost. I hadn't figured out how to take advantage of the protection of oil - I just had a vague feeling that it might be helpful. Now however I can see that it's useful, because the oil will provide quite good protection against a pulse of O2 e.g. if someone accidentally lets air into the chamber. (Or moves plates from one glovebox to another?) O2 will start to diffuse into the oil, but most of it will diffuse out again if the O2 pulse is short. And the lids that are standard on microbatch plates will help a lot. (The oil is almost the ideal barrier, although you *might* prefer something with a very low solubility since it *might *give a lower O2 flux in the steady state - as Tristran says it's complicated. And I'm guessing that the O2 flux through the thin plastic tape used in vapor diffusion setups would be quite high. Does anyone have a friend who works in food science?) There's an even more exciting conclusion: we should degass our oil even for use with *aerobic *microbatch setups. I have heard of a case where diffracting crystals were only obtained for aerobic targets in a glovebox, and I think the skins on drops are, or can be, related to oxidation. There may even be mileage in microbatch with the zip lock bag approach for targets that are not overly sensitive - *if* you degas your oil before you start a vacuum should do it. I agree that Al's Oil (silicone) should be avoided from this point of view - although I would certainly use it anyway for screening experiments (whether aerobic or anaerobic). Riveting stuff. Thx to all, Patrick On 18 March 2015 at 18:58, Tristan Croll tristan.cr...@qut.edu.au wrote: It's a little complicated. It's true that oxygen is more soluble in most oils than in water - but in a high viscosity mineral oil the diffusion rate is orders of magnitude lower. So the combination of an oil overlay and a reducing agent in your buffer should protect your sample much longer than the reducing agent alone - as long as your oil was degassed to start with. Note that silicon oils are a bad choice for this - silicones have an enormous affinity for oxygen (so much so that they've been explored as artificial blood substitutes), and it diffuses through them very readily. Tristan Croll Lecturer Faculty of Health School of Biomedical Sciences Institute of Health and Biomedical Engineering Queensland University of Technology 60 Musk Ave Kelvin Grove QLD 4059 Australia +61 7 3138 6443 This email and its attachments (if any) contain confidential information intended for use by the addressee and may be privileged. We do not waive any confidentiality, privilege or copyright associated with the email or the attachments. If you are not the intended addressee, you must not use, transmit, disclose or copy the email or any attachments. If you receive this email by mistake, please notify the sender immediately and delete the original email. On 18 Mar 2015, at 11:49 pm, Edward A. Berry ber...@upstate.edu wrote: Do you have evidence that the oil blocks diffusion of O2? O2 is a nonpolar molecule, generally much more soluble in oils than in water. I'm not sure about silicone oils, but I would think they also dissolve O2 readily. eab On 03/18/2015 08:02 AM, Patrick Shaw Stewart wrote: Hi Steve I have one more comment for this thread. The microbatch-under-oil method is very handy for anaerobic work: 1. You can keep the microbatch stock solutions in normal microtitre plates (polypropylene is best to reduce evaporation) for months, which hugely reduces the amount of degassing that you need to do. You will only use say 0.5 ul of stock per drop. 2. The oil offers a surprising amount of protection from oxidation, which may be helpful eg in harvesting. 3. Microbatch can be automated - in parallel to vapor diffusion if desired It's amazing how often (aerobic) microbatch produces far superior crystals to V.D. for no obvious reason - it's well worth trying for both screening and optimization. Best wishes Patrick On 11 March 2015 at 10:17, Stephen Carr stephen.c...@rc-harwell.ac.uk mailto:stephen.c...@rc-harwell.ac.uk wrote: Dear CCP4BBer's Apologies for the off-topic post
Re: [ccp4bb] Choice of stereomicroscope
Ronnie I see a lot of cheap and expensive microscopes, and I notice that expensive is not always better for protein crystallization. Almost the most important thing is that illumination comes from *one particular direction*. Often this means that the light source is small and far from the sample stage. What does not work well is to have a large light source (eg multiple LEDs, large white screen, mirror or sintered transparent sheet) that is close to the sample - even with the best optics in the world, you won't see crystals well. Dark ground (see only scattered light) and ordinary transmission mode can both work well - good to have both if possible. Good luck, Patrick On 27 March 2015 at 13:08, Ronnie Berntsson ronnie.bernts...@medchem.umu.se wrote: Dear all, I’m currently looking in to buying a new microscope for viewing crystal plates, mounting crystals etc, and would love some input into what I should get. What I would like is a microscope that has a high quality image, that is easy to work with and which is ergonomical. It does not have to have a fixed digital camera, but it should be possible to attach a digital camera to take pictures. Price is obviously also important... I’ve been looking at the standard microscopes that Molecular Dimensions sell, and also on a Nikon SMZ18. I remember that we used to have a Leica microscope in a previous lab that I liked, but can’t seem to find the model at the moment. I am also interested in getting a UV source, to inspect crystals under UV to see if you fluoresce (and hence are protein crystals). Molecular Dimension used to sell XtalLight 100, but doesn’t seem to do so anymore. Do any of you have other suggestions regarding the possibility of adding UV to a stereomicroscope? Suggestions and thoughts are more than welcome! Thanks, Ronnie -- Ronnie Berntsson, PhD Assistant Professor Department of Medical Biochemistry and Biophysics Umeå University 90187 Umeå Sweden e-mail: ronnie.bernts...@medchem.umu.se -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] how to reduce protein solubility
I use a syringe with a needle and poke through the tape into the reservoir and top it off Another approach is to poke holes in the tape with a needle, and just let the drops slowly evaporate and dry out. I'm told it works! On 17 February 2015 at 09:19, Bernhard Rupp hofkristall...@gmail.com wrote: Just a possibility for salvage of your already set-up drops: You can spike the reservoirs with some highly concentrated precipitant (no matter what as long as it sucks more water out of your drop). It does not solve your problem but maybe you can revive a few drops and get more information from your experiment. I use a syringe with a needle and poke through the tape into the reservoir and top it off with the high conc. precip. The tiny hole is easy to re-tape and does not hinder observation. Best, BR *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of * Mattiroli,Francesca *Sent:* Tuesday, February 17, 2015 5:23 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] how to reduce protein solubility Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] handling crystals in volatile solvents
Hi Len In every case that I know of this problem has been solved by working under oil. The oil becomes saturated with the volatiles, and it prevents crystals from doing backstroke, breaststroke or front crawl during harvesting. The most convenient way to get going is to dispense the wells without the volatile solvents, then allow solvents to difuse through the oil and into the drops. We suggest our old Vapor Batch plates, and you should place the same concentration of the solvent that you want in the drops into the large moat around the plate. If you put in higher concentrations you may dehydrate your drops :-( A very nice feature is that you can set up e.g. a random screen, then try soaking in one or more volatile solvents if nothing grows in the first round. The approach was invented by Lesley Haire, and you can find her presentation at http://www.douglas.co.uk/winner1.htm See also Mortuza, Gulnahar B., et al. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. *Nature* 431.7007 (2004): 481-485. The plates look like this: http://www.douglas.co.uk/products.htm#Vapor Batch Plates We'll send you some sample plates to try. Best wishes, Patrick On 12 June 2015 at 22:11, Thomas, Leonard M. lmtho...@ou.edu wrote: Hi All, We have gotten some very nicely formed crystals out of a couple of different volatile solvents recently. Besides looking for something easier to work in does anybody have any tips on handling crystals from these types of solvents. It is very hard to loop a crystal while it is doing the backstroke in the well with all of its buddies. Thanks in advance. Len Leonard M. Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center 101 Stephenson Parkway Norman, OK 73019 405-325-1126 lmtho...@ou.edu http://barlywine.chem.ou.edu http://structuralbiology.ou.edu -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Crystals grow at the bottom of the tube.
Hi Lingyuan I would certainly make a seed stock and use it with RANDOM screens - this will (usually) allow you to pick up new conditions and control the number of crystals per drop. However you say your crystals melt easily when you harvest them. I don't know how David stabilized his seedstock (if he did) but you may be able to stabilize yours with a precipitant. In all the cases that we looked at we found that if you soaked uncrushed crystals in a precipitant (or cocktail) and the crystals were unchanged after 24 hours, then the precipitant/cocktail could be used to make a seedstock. In practice 100% PEG 600 worked for 5 of the 6 model proteins that we looked at. The remaining seedstock worked with seed crystals suspended in 4M amm. sulfate. I hope it works for you. Best wishes, Patrick ___ Ref for stabilizing seed stocks with various precipitants: Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock, P.F., 2011. Random microseeding: a theoretical and practical exploration of seed stability and seeding techniques for successful protein crystallization.*Crystal Growth & Design*, *11*(8), pp.3432-3441. Ref for random microseeding: D'Arcy A, Villard F, Marsh M. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica Section D: Biological Crystallography. 2007 Apr 1;63(4):550-4. On 19 October 2016 at 15:20, Hargreaves, David < david.hargrea...@astrazeneca.com> wrote: > I have been presented with crystals grown in nmr tubes on two separate > occasions. Both diffracted very well and thankfully, neither required a > magnetic field for optimisation. On the first occasion I did use the > initial sample as a seed stock. > > > > Best wishes, > > > > David > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of * > kiki > *Sent:* 18 October 2016 11:42 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Crystals grow at the bottom of the tube. > > > > Dear all users, > > > > I got my crystals at the bottom of the tube on ice just before I started > to screen crystals. Those crystals' shapes were good but easy to melt when > I harvested them. I shot these crystals. They only diffracted to 7A to 8A. > Has anyone ever met this situation before? How to improve? > > > > Thanks, > > Lingyuan > -- > > AstraZeneca UK Limited is a company incorporated in England and Wales with > registered number:03674842 and its registered office at 1 Francis Crick > Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0AA. > > This e-mail and its attachments are intended for the above named recipient > only and may contain confidential and privileged information. If they have > come to you in error, you must not copy or show them to anyone; instead, > please reply to this e-mail, highlighting the error to the sender and then > immediately delete the message. For information about how AstraZeneca UK > Limited and its affiliates may process information, personal data and > monitor communications, please see our privacy notice at > www.astrazeneca.com > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Slightly OT: crystallization teaching resources for kids
Yes, but the kids will want to know WHY there is an energy barrier. I prefer my explanation below. Happy New Year to all Patrick _ > I am especially needing help with the concept of nucleation, and why nucleation is slower and then crystal growth faster once nuclei have formed. I always explain this by pointing out that, when the crystal is very small, each new molecule that lands on the surface of the nascent crystal can only be attached on one or two sides. When the crystal is larger, molecules that land are more likely to become attached on two, three or more sides. Atomic force microscopy images of "islands" that appear on the faces of crystals are also helpful. Islands are more likely to appear on larger faces. Once they appear they can rapidly spread to the edges of the crystal, which is (one mechanism explaining) why crystals have flat faces. On 4 January 2017 at 16:45, Nicolas FOOS <nicolas.f...@esrf.fr> wrote: > Dear Evette, > > If I was is your situation (explaining nucleation and other concept). I > will discuss in terms of energy. > > I mean obtaining the initial nuclei is the "costly" step in terms of > energy. To represent that, out the classical curve of energy, I will use a > metaphoric representation such as jump over a barrier and run after. > > With this analogy, it's possible to explain that the first step is > difficult and the second more accessible. If the barrier is to high, it's > impossible to continue and run. If you don't have any barrier it's easy to > run and if you only have a small barrier is not to difficult to jump over > and run. But It also allow you to explain that if you facilitate the > apparition of the first "surface" thanks to appropriate method (seeding, > dust...) you can help the first step (to continue with the barrier story, > it like you have ladder to help, or the ability to decrease the size of the > barrier. > > For why the crystal and how, I will maybe use the example of orange > pyramid in the food store. Orange are stable together because they have > enough contact, because they have relatively homogeneous shape. If you > mixed orange with water melon it's difficult to obtain nice pyramid. > > For crystallization experiment which work, I have no Idea out of the one > you already mentioned. > > > Hope this help. > > Nicolas > > Nicolas Foos > PhD > Structural Biology Group > European Synchrotron Radiation Facility (E.S.R.F) > 71, avenue des Martyrs > CS 40220 > 38043 GRENOBLE Cedex 9+33 (0)6 76 88 14 87 <+33%206%2076%2088%2014%2087>+33 > (0)4 76 88 45 19 <+33%204%2076%2088%2045%2019> > > On 30/12/2016 11:06, Radisky, Evette S., Ph.D. wrote: > > Can anyone point to some especially useful resources to help explain to > kids (pre-chemistry, ~age 10-12) how and why molecules crystallize? Maybe a > good online movie or animation? I am especially needing help with the > concept of nucleation, and why nucleation is slower and then crystal growth > faster once nuclei have formed. I have been supervising some experiments > growing sucrose crystals from supersaturated solutions, which have worked > really well, but I am having more difficulty in explaining the underlying > fundamental concepts in a way that is understandable to the kids. > > Thanks! > Evette > > Evette Radisky, PhD > > Associate Professor of Cancer Biology > > Mayo Clinic Cancer Center > > Griffin Cancer Research Building > > 4500 San Pablo Road > > Jacksonville, FL 32224 > > tel: 904-953-6372 > > fax: 904-953-0277 > > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystallization optimization
Vicky, streak seeding is a very good method, but it can be quite a lot of work. Before he tries that, why wouldn't we suggest to Liuqing that he should try MMS - that is, adding a liquid seed stock to random screens? That way he is likely to end up with seeds in wells with similar conditions that happen to be in the metastable zone of the phase diagram, and also with seeds in *completely unrelated conditions *that are also in the metastable zone. That way you can cast your net very wide. One simple experiment does so much - it is also an additive experiment : ) Part of the art of crystallization is to try lots of things without thinking too much. Patrick On 12 July 2017 at 07:50, Vicky Tsirkone <vtsirk...@gmail.com> wrote: > Dear Frank, > > I may see in the attached pic several nucleation points and a considerable > amount of microcrystals. Based to my knowledge decreasing the concentration > of the precipitant and/or the protein concentration would be a reasonable > approach to refine the initial hits. > By checking the diagram as you correctly mentioned you may see that the > fine tuning of protein and precipitant concetration may lead to the > desirable result without reaching the precipitation zone. > > Patrick just check your screens. Just a rule of thumb, if you see > precipitation in the ~60% of your drops then you should definitely reduce > the protein concentration. > > ps dont forget to try the *streak seeding*, as well. > > Have a nice day and again good luck. > > Vicky > > On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft < > frank.vonde...@sgc.ox.ac.uk> wrote: > >> Actually, you should try *increasing* the protein concentration - a >> lot. But be prepared to drop the precipitant concentration to almost >> nothing (1 or 2% isn't "low"). >> >> To understand why, look at the phase diagram and what we assume about >> vapour diffusion. (Which I'm assuming is what you're doing.) >> >> >> On 12/07/2017 06:28, Vicky Tsirkone wrote: >> >> Dear Patrick, >> >> You may reduce the protein concentation, as well. >> Another option could be the *streak seeding* by exploiting the drop of >> your initial condition. >> >> Good luck, >> >> V.T. >> >> On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart < >> patr...@douglas.co.uk> wrote: >> >>> >>> Microseed them into two or three random screens. >>> >>> Search for MMS and rMMS online. >>> >>> Good luck >>> >>> Patrick >>> >>> >>> >>> >>> On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com> wrote: >>> >>>> hello everyone! >>>> I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my >>>> protein grow small needle like crystals, how can i optimize it to get >>>> bigger crystals? the attach is the crystals figure. >>>> thanks in advance >>>> sincerely >>>> Liuqing Chen >>>> >>> >>> >>> >>> -- >>> patr...@douglas.co.ukDouglas Instruments Ltd. >>> Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK >>> Directors: Peter Baldock, Patrick Shaw Stewart >>> >>> http://www.douglas.co.uk >>> Tel: 44 (0) 148-864-9090 <01488%20649090>US toll-free >>> 1-877-225-2034 <%28877%29%20225-2034> >>> Regd. England 2177994, VAT Reg. GB 480 7371 36 >>> >> >> >> > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystallization optimization
Oii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgCC5qcLQd_xWchIlNmFOVWl0_LVlkIZxPEbwqt58Rj4K7aTJtr9yEI6PJ5njvoBPKMTo4XRqpLcm6BTWwlUxXiR1lz4f5CR8PWDaB6APo_1xcZzogykGtw4prvYZyqr3_Xe6jlmQ%3D%3D=http%3A%2F%2Flcrbw.pharmacie.univ-paris5.fr%2Fspip.php%3Farticle18> > -- > > > > > -- > > *De :* Patrick Shaw Stewart <patr...@douglas.co.uk> > *À :* CCP4BB@JISCMAIL.AC.UK > *Envoyé le :* Mercredi 12 juillet 2017 17h28 > *Objet :* Re: [ccp4bb] crystallization optimization > > > > > > Alun > > > > I agree Frank's point is very interesting - and he intriguingly refers us > to the phase diagram. > > > > Is the point that Line A is longer than Line B ? > > > > Best wishes > > > > Patrick > > > > > > > > > > [image: Inline images 2] > > > > > > > > > > > > > > On 12 July 2017 at 14:40, Alun R Coker <alun.co...@ucl.ac.uk> wrote: > > Hi Everyone, > > Franks point is really interesting. We routinely reduce the protein > concentration when we see too many precipitated wells, but we never dilute > the screen. Has anyone tried this? > > All the best, > > Alun > > > > On 12/07/17 08:48, Frank von Delft wrote: > > The point I was failing to make: reducing either protein or precipitant > concentration will indeed reduce nucleation, but often won't get you bigger > or more single crystals: it will just make the appearance of crystals less > reliable. > > The way to get big single reliable crystals is to *increase* protein and > *greatly* reduce precipitant. > > (Even better: do seeding. Like Vicky said. Incredible how often people > don't bother to do seeding, yet it solves so many problems.) > > phx > > > > On 12/07/2017 07:50, Vicky Tsirkone wrote: > > Dear Frank, > > > > I may see in the attached pic several nucleation points and a considerable > amount of microcrystals. Based to my knowledge decreasing the concentration > of the precipitant and/or the protein concentration would be a reasonable > approach to refine the initial hits. > > By checking the diagram as you correctly mentioned you may see that the > fine tuning of protein and precipitant concetration may lead to the > desirable result without reaching the precipitation zone. > > > > Patrick just check your screens. Just a rule of thumb, if you see > precipitation in the ~60% of your drops then you should definitely reduce > the protein concentration. > > > > ps dont forget to try the *streak seeding*, as well. > > > > Have a nice day and again good luck. > > > > Vicky > > > > On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft < > frank.vonde...@sgc.ox.ac.uk> wrote: > > Actually, you should try *increasing* the protein concentration - a lot. > But be prepared to drop the precipitant concentration to almost nothing (1 > or 2% isn't "low"). > > To understand why, look at the phase diagram and what we assume about > vapour diffusion. (Which I'm assuming is what you're doing.) > > > > On 12/07/2017 06:28, Vicky Tsirkone wrote: > > Dear Patrick, > > > > You may reduce the protein concentation, as well. > > Another option could be the *streak seeding* by exploiting the drop of > your initial condition. > > > > Good luck, > > > > V.T. > > > > On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart < > patr...@douglas.co.uk> wrote: > > > > Microseed them into two or three random screens. > > > > Search for MMS and rMMS online. > > > > Good luck > > > > Patrick > > > > > > > > > > On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com> wrote: > > hello everyone! > I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my > protein grow small needle like crystals, how can i optimize it to get > bigger crystals? the attach is the crystals figure. > thanks in advance > sincerely > Liuqing Chen > > > > > > -- > > patr...@douglas.co.ukDouglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > http://www.douglas.co.uk > <https://clicktime.symantec.com/a/1/25dI3WrmU_REz0R2AqEuuVcDP3s_fHPB3ls19C25QnM=?d=ydHuK2RzVboI0S1f0-VMFO47v0jLw-bGOuPIOXlvgxVovpKjpVbqlqHHe92eD9EXP1yEiYalPcDJ38NT0DPj0JHU1ay-7q9vqP6cG6Jh_Y2NFwbuGut6uA1GqbEf11-ROhtHIifuMGSuiUO4Yxf9ZKoKqiIJZmSrtwlOii1OALADi7UtKkI_mrwwLYA4QugHgmtOUOK5Mw1HMEYTM0eLrh1UB5KlzbR1gdvNDPZRm1oDPNOSmHAOma5Mdveve6ZlC5WH6mgC
Re: [ccp4bb] crystallization optimization
Microseed them into two or three random screens. Search for MMS and rMMS online. Good luck Patrick On 10 July 2017 at 15:47, Liuqing Chen <519198...@163.com> wrote: > hello everyone! > I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my > protein grow small needle like crystals, how can i optimize it to get > bigger crystals? the attach is the crystals figure. > thanks in advance > sincerely > Liuqing Chen > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Granada Crystallization Boxes?
Gloria, would you be interested in used ones? I don't actually have any - we threw them out a few months ago, I've just checked - but someone might have some. Best wishes, Patrick On 11 July 2017 at 19:04, Gloria Borgstahl <gborgst...@gmail.com> wrote: > I have recently found out that these are no longer being manufactured or > sold commercially. But, as fortune has it, we have just been funded to fly > some large quartz capillaries crystallization experimente up to the > International Space Station for neutron crystallography. Our experimental > design is to fly the experiments in the Granada Crystallation Boxes! NASA > has already approved the 3x10x0.5 cm plastic Granada boxes for flights. > Does anyone have any in their lab supplies that they do not plan to use? > We would be willing to buy them from you! Thanks, Gloria > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein or DNA crystals
We have sometimes used Michael Garavito's excellent suggestion of diffusing glutaraldehyde into the drops - mentioned on this board a few days ago. Our protein crystals either turned brown or turned into brown goo. Michael and others, what effect would you expect glutaraldehyde to have on DNA crystals? Patrick On 19 June 2017 at 15:20, Joseph Ho <sbddintai...@gmail.com> wrote: > Dear all: > > I would like to seek your opinion on our crystal hits. We are working > on protein/dsDNA complex. By changing different protein and DNA > (14-22bp) constructs, we recently got some hits from commercial > screens using sitting drop vapor diffusion (very small xtals). The > precipitant is PEG and the picture of crystals are attached. In this > particular condition, it is 30%PEG3350, sodium succinate pH5.5 and > 100mM NaCl. The crystal seems floating and sit in the bottom. We do > some test shot from other conditions and it is not salt crystals. The > crystals can suck in izit dye. I do some google and it seems izit dye > also turns dsDNA crystal into blue. We also do UV/Vis microscope but > no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp. > > This is our first time to work on protein/DNA complex crystals and we > are not certain if this is just DNA or protein/DNA crystals. Can you > provide your comments on our hits? > > Thank you for your help > > Joseph > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystallization screen for protein-protein complex
Hena There was a very interesting paper by Peter Sun and coworker from 2002. They pointed out that there is a very strong bias towards crystallizing protein-protein complexes with PEG rather than salt as the main precipitant. Patrick ___ Radaev and Sun. Crystallization of protein-protein complexes. J. Appl. Cryst. (2002). 35, 674-676 On 20 May 2017 at 22:14, Hena Dutta <hdutt...@gmail.com> wrote: > > Dear Members, > I am trying to crystallize a protein-protein complex. Both are soluble proteins and form complex as observed in FPLC either co-purifying or separately purifying and then mixing with equi-molar ratio. > Looking for suggested screens to try for crystallization. > > So far tried with Qiagen - ProComplex Suite, Classics Suite and Classics II Suite > No success yet. > > Is there any guidance which screen to try? > > Looking for your help... > Regards, > Hena. -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Regarding Patents
There are some interesting anti-patent initiatives https://en.wikipedia.org/wiki/Patent#Anti-patent_initiatives including prizes as an alternative to patents https://en.wikipedia.org/wiki/Prizes_as_an_alternative_to_patents#Other_areas_for_prize_models_over_patents On 4 November 2017 at 15:08, Bernhard Rupp <hofkristall...@gmail.com> wrote: > > to publish it so the world can benefit from it. > > Isn’t that exactly the idea of a patent? Instead of keeping the invention > > a trade secret (occasionally a viable alternative) you publish the > invention, > > and the inventor (and in general, the supporting institutions) can get > > rewarded if someone plans to use the idea commercially. Someone > > (in academia often the tax payer) did pay for the work after all, and > having > > an option to recover the money (or god forbid, make a profit…) seems > > a reasonable proposition…. > > > > Best, BR > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Abhishek > Anan > *Sent:* Saturday, November 4, 2017 05:31 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Regarding Patents > > > > I second Gert's thoughts > > Best, > > Abhishek > > > > On Sat, Nov 4, 2017 at 10:21 AM, Gert Vriend <gerrit.vri...@radboudumc.nl> > wrote: > > A related question. If you have a crystal structure and found a novel > ligand binding site that can be used to regulate protein activity, could > you patent such "binding site"? If not, how to make the best use of such > findings? > > > I would say that the best one can do with important novel > data/information/knowledge/insights is to publish it so the world can > benefit from it. > > Gert > > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Na-Binding Protein?
That's very interesting. I guess it's an unusual manifestation of the Hofmeister series. It might give guidance to developers of screens for both crystallization and cryoEM. Thx, Patrick On 9 January 2018 at 15:44, Andrew Mesecar <amese...@purdue.edu> wrote: > Dear Jacob, > > > > One of my favorite examples of monovalent cation discrimination is by the > enzyme Pyruvate Kinase. It prefers K(+), NH4(+), Rb(+) and Tl+ for maximum > catalysis and then activity falls off as the monovalent cation sizes get > larger, Cs(+) or smaller Na(+) >> Li (+). The conformations of pyruvate > kinase are known to be altered by binding of monovalent cations and it has > been studied for over 50 years by a variety of approaches. A number of the > X-ray structures are with K(+). I spent a few years of my life studying > this enzyme a couple of decades ago. It will hopefully provide some > information for your project. > > > > Best of luck, > > > > Andy Mesecar > > On Tue, Jan 9, 2018 at 9:42 AM, Keller, Jacob <kell...@janelia.hhmi.org> > wrote: > >> Dear Crystallographers, >> >> >> >> Is anyone aware of a soluble protein which changes large-scale >> conformation +/- Na+, and is specific for Na+ per se, or at least ignores >> K+ and Ca++? E.g., Rb+ or Li+ might be okay. Structural info would be a >> plus, but not a sine qua non. >> >> >> >> Similarly, what about with K+ or Cl- specificities, but oblivious to >> similar common ions? >> >> >> >> All the best, >> >> >> >> Jacob Keller >> >> >> >> + >> >> Jacob Pearson Keller >> >> Research Scientist / Looger Lab >> >> HHMI Janelia Research Campus >> >> 19700 Helix Dr, Ashburn, VA 20147 >> <https://maps.google.com/?q=19700+Helix+Dr,+Ashburn,+VA+20147=gmail=g> >> >> (571)209-4000 x3159 <(571)%20209-4000> >> >> + >> >> >> >> The content of this email is confidential and intended for the recipient >> specified in message only. It is strictly forbidden to share any part of >> this message with any third party, without a written consent of the sender. >> If you received this message by mistake, please reply to this message and >> follow with its deletion, so that we can ensure such a mistake does not >> occur in the future. >> >> >> > > > > -- > *Andrew D. Mesecar* > Head, Department of Biochemistry > Walther Professor of Cancer Structural Biology > Deputy Director, Purdue Center for Cancer Research > E-Mail: amese...@purdue.edu > _ > *Department of Biochemistry Contact Information:* > 175 S. University Street > <https://maps.google.com/?q=175+S.+University+StreetW.+Lafayette,+IN+47907=gmail=g> > W. Lafayette, IN 47907 > <https://maps.google.com/?q=175+S.+University+StreetW.+Lafayette,+IN+47907=gmail=g> > -2063 > 765-494-1607 > -- > *Research Lab Contact Information:* > Hockmeyer Hall of Structural Biology > Room 311 > <https://maps.google.com/?q=311+240+S.+Martin+Jischke+Drive+West+Lafayette,+IN+47907=gmail=g> > 240 S. Martin Jischke Drive > <https://maps.google.com/?q=311+240+S.+Martin+Jischke+Drive+West+Lafayette,+IN+47907=gmail=g> > West Lafayette, IN 47907 > <https://maps.google.com/?q=311+240+S.+Martin+Jischke+Drive+West+Lafayette,+IN+47907=gmail=g> > -1971 > 765-494-1924 > _ > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] crystals that dont diffract :( :(
Hi Careina Expanding on what Tim says, try crushing your crystals to make a seed stock, and adding it to a few *random screens - *preferably screens that you have already tried with this target. Search for instructions for MMS or rMMS online. Good luck, Patrick On 14 August 2018 at 11:34, Tim Gruene wrote: > Dear Careina, > > you could use the old crystals, that did not diffract, for microseeding > to regrew nicer crystals. Once you have them, try to use them as quickly > as possible. Three weeks can be a long time for crystals. > > Storage in liquid nitrogen should not be the problem. > > Best, > Tim > > > On 08/14/2018 11:58 AM, Careina Edgooms wrote: > > I got the most beautiful crystals I have ever seen and they don't > > diffract at all. Not poor diffraction, NO diffraction. Anyone know why > > this could be and how I can go about fixing it? I had three beautiful > > crystals and not one diffracted. I did leave them in the drop for about > > 3 weeks before harvesting and in liquid nitrogen for about a month > > before diffracting. Could that be a factor? If I regrew more beautiful > > crystals and diffracted straight away could that help? > > Careina > > > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > -- > -- > Paul Scherrer Institut > Tim Gruene > - persoenlich - > OSUA/204 > CH-5232 Villigen PSI > phone: +41 (0)56 310 5297 > > GPG Key ID = A46BEE1A > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
Hi Thomas
One problem with conventional approaches is that you tend to get high
mosaicity when harvesting from drops with isopropanol.
A very good method, invented by my old friend Lesley Haire, that has always
worked (so far!) is to use microbatch-under-oil with a plate called the
Vapor Batch plate.
- Set up the drops with everything in them except for the isopropanol.
- Cover the drops with 50:50 paraffin and silicone oil ("Al's Oil").
- Put a few mL of 27% isopropanol in the "moat" around the outside of
the plate.
The isopropanol will saturate the oil and then diffuse into the drops. You
can take your time and harvest the crystals through the oil. The oil
protects the crystals, preventing evaporation of isopropanol.
Let me know if you (or anyone) would like some sample plates to try this
out.
See example and ref. below.
Best wishes, Patrick
___
Example: https://www.douglas.co.uk/winner1.htm
*G. B. Mortuza et al.. High-resolution structure of a retroviral capsid
hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. (This
paper describes the use of the vapor batch plate with isopropanol.)*
Sent from mobile
Patrick Shaw Stewart
+44 7901 548 201
On Wed, 15 Aug 2018, 08:41 Yvonne Thielmann, wrote:
> Dear Thomas,
>
> maybe you can try to overlay your drop with LV CryoOil from Jena
> Bioscience. Then the evaporation of the solvent is slowed down and the
> crystals are directly cryoprotected when you move the crystals through
> the oil. We had quite good results when we cryoprotected crystals in
> this oil.
>
> Best wishes,
> Yvonne
>
>
> --
> Dr. Yvonne Thielmann
> Max Planck Institute of Biophysics
> Molecular Membrane Biology
> Max-von-Laue-Strasse 3
> 60438 Frankfurt / Main
> Germany
>
> Office +49 69 6303 1056
> Lab +49 69 6303 1074
> Fax +49 69 6303 1002
>
> Am 15.08.2018 um 08:05 schrieb Kajander, Tommi A:
> > Yes sorry, i meant paratone-N also.
> >
> > Tommi
> >
> > Kohteesta: ferrer
> > Lähetetty: keskiviikko 15. elokuuta klo 0.41
> > Aihe: Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
> > Vastaanottaja: ccp4bb@jiscmail.ac.uk
> >
> >
> > Dear Thomas,
> >
> > Alternatively you can try shooting on crystals in the drop, in situ. So
> > fishing, no cryo. But potentially high radiation damage. Can be
> > considered if you have enough crystals, and if your crystallization
> > plate makes it possible.
> >
> > Regards
> >
> > JL
> >
> > On 14/08/2018 20:58, Thomas Krey wrote:
> > Dear crystallization experts,
> >
> > We have 3D protein crystals grown from a microseed matrix screening
> > vapor diffusion experiment in either
> >
> > 15% (v/v) Reagent alcohol
> > HEPES Na pH 7.5
> > 0.2 M MgCl2
> >
> > or in
> >
> > 27% Isopropanol
> > 0.18 M MgCl2
> > 90 mM HEPES Na pH 7.5
> > 10% Glycerol
> >
> > Upon opening the corresponding wells these crystals move quite a bit –
> > presumably due to the volatility of the alcohols. Does anyone have a
> > good suggestion to stabilize the swirling movements? Does anyone have
> > experience, whether these conditions alone can serve as cryo-protectant
> > (i.e., do we really have to fish, move into cryo solution and fish
> again)?
> > Any suggestion or input would be highly welcome.
> >
> > Thank you very much in advance.
> >
> > Thomas
> >
> >
> > Prof. Dr. Thomas Krey
> > Hannover Medical School
> > Institute of Virology
> > Structural Virology Group
> > Carl-Neuberg-Str. 1
> > D-30625 Hannover
> > phone: +49 (0) 511 - 532 4308
> > email: krey.tho...@mh-hannover.de <mailto:krey.tho...@mh-hannover.de>
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
> > -- Jean-Luc Ferrer Institut de
> Biologie
> > Structurale 71 Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 -
> > FRANCE Ph.: +33 (0)4 57 42 85 22 Cell: +33 (0)6 89 45 13 57 email:
> > jean-luc.fer...@ibs.fr <mailto:jean-luc.fer...@ibs.fr>
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
> >
> >
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
>
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Oxford University Press
evier is the place to start as their profit > margins are like those of Apple, and of competition there is none. > > > Elsevier: Like Apple, but without the design sense. > > > But seriously, Adrian makes an excellent point. And the large profit > margins wouldn’t be quite so galling, if only the publishers were able to > provide competent and helpful administrative support; but in my recent > experience, not-for-profit scientific society journals are actually > providing better experiences for reviewers and authors than the big > commercial ones. > > Pat > > > --- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St > <https://maps.google.com/?q=245+N.+15th+St=gmail=g>., > Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > > (215) 762-7706 > > pjl...@gmail.com > > pj...@drexel.edu > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > -- > Prof. George M. Sheldrick FRS > Dept. Structural Chemistry, > University of Goettingen, > Tammannstr. 4, > D37077 Goettingen, Germany > Tel. +49-551-39-33021 or +49-5594-227312 > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Please share your experience about "ugly" crystals showing good diffraction
Hi All I have a comment, based on my old supervisor's explanation, which seemed to make sense. Crystals usually grow layer by layer. Once a new layer is formed it quickly expands to cover the whole surface. That's why crystals normally have flat surfaces and sharp edges - the layers/steps expand rapidly until they get to the edges. However it doesn't have to be like that. Sometimes new layers can form roughly as quickly as the previous layers can spread. The result is crystals with curved surfaces - or even just blobs. Just because the new layers form at a rate that is comparable to the spreading doesn't mean that the crystals won't be ordered, and won't diffract well. Once I understood that I understood what I was seeing better when I checked my drops. Best wishes Patrick On 5 July 2018 at 22:06, Sanishvili, Ruslan wrote: > Hi Anirban, > > It would be great if you could share the compilation of relevant responses > to your request. I think many others in the community could use these > examples for educational purposes. > > Best, > > Nukri > > > Ruslan Sanishvili (Nukri), Ph.D. > Macromolecular Crystallographer > GM/CA@APS > X-ray Science Division, ANL > 9700 S. Cass Ave. > Lemont, IL 60439 > > Tel: (630)252-0665 > Fax: (630)252-0667 > rsanishv...@anl.gov > > > > -- > *From:* CCP4 bulletin board on behalf of Anirban > Banerjee > *Sent:* Thursday, June 28, 2018 7:07 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Please share your experience about "ugly" crystals > showing good diffraction > > > Dear all, > > Apologies for the non-CCP4 related question. > > If you have concrete experience about visually unappealing, i.e. ugly > crystals ( your own take on ugly is fine) diffracting better than > comparably similar sized nicer looking crystals of the same protein, will > you please share here ? Even better if that led to a published structure. > Might you also have pictures ? > > We have all heard anecdotes about not using visual appearance to judge the > quality of crystals as far as their ability to give good diffraction data > is concerned but I am trying to gather some concrete pointers here to > motivate trainees. > > Thanks very much for any help. > > Best, > > Anirban > > P.S. I know that there is probably a lot of thought and wisdom on this > this specific topic but I am really looking for actual experience. > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] suggestions on a microscope for Crystallography
Hi Chandra My only comment is be careful of modern microscopes that have a frosted glass screen with LEDs behind it, just below the plate. For looking at crystals you need *directional *light. I've seen some very expensive modern microscopes with illumination that just doesn't work for crystallization. If I come across that situation I normally make a platform and raise the plate up by a few inches - it can dramatically improve the quality of images. You can also cut a round hole in e.g. a piece of aluminium foil and use it to make the area of the light source smaller. On the other hand illumination mustn't be *too *directional because the drop itself acts as a lens. If you have a light source that is small and too far from the sample you'll get black regions around the outside of the drop where you can't see crystals. It's all about the solid angle of the light hitting the sample - I'm sure others can explain better than I can. Good luck, Patrick On 7 March 2018 at 02:06, Chandramohan Kattamuri < 1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk> wrote: > Hi > > I'm looking for suggestions on a good microscope for looking at crystals, > which includes polarization, light source (fiber optics), crosshairs and > camera mount. What Models and make? > > Thanks in advance > > Chandra > > > > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Anderson?Evans polyoxotungstate (TEW)
I notice that Molecular Dimensions offers a similar compound https://www.moleculardimensions.com/shopcontent.asp?type=crystallophoreno1 Are there any others? On 30 March 2018 at 19:24, Nikolay Dobrev < nikolay.dob...@bzh.uni-heidelberg.de> wrote: > Dear all, > I apologize for my off-topic question. > I want to ask if anyone has so far used the Anderson-Evans > polyoxotungstate (https://www.jenabioscience.co > m/crystallography-cryo-em/screening/xp-screen/x-tew-anderson > -evans-polyoxotungstate). > Did someone see an improvement in crystal quality/diffraction? > Any comments will be highly appreciated. > > Nikolay Dobrev > > > > Nikolay Dobrev > PhD Student in AG Sinning & AG Fischer > Biochemie Zentrum > Heidelberg University > Im Neuenheimer Feld 328 > 69120 Heidelberg > Germany > Phone: +49 6221 54 4796 > Email: <nikolay.dob...@bzh.uni-heidelberg.de > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?
t; > > > #### > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Optimization from needle shaped crystals
Hi Chitra Needles usually work very well for making seed stocks for random Microseed Matrix Screening (MMS). Your protein probably crystallizes well, but it is growing too fast in one direction. MMS has lots of advantages. If it's going to work it will almost certainly work within 12 hours. Also, it allows you to control the number of crystals per drop by diluting the seed stock. Another excellent (open-access) paper that gives lots of examples of seeding, cross-seeding, dilution, optimization etc within a defined set of fabs is here: https://scripts.iucr.org/cgi-bin/paper?nj5193 Good luck, Patrick On Sun, Sep 8, 2019 at 1:46 PM David Briggs wrote: > 4. Matrix microseeding. Make a seed stock from these crystals and then > re-run your primary screens. > > https://www.ncbi.nlm.nih.gov/m/pubmed/25195878/ > > -- > Dr David C. Briggs > Senior Laboratory Research Scientist > Signalling and Structural Biology Lab > The Francis Crick Institute > London, UK > == > about.me/david_briggs > > -- > *From:* CCP4 bulletin board on behalf of chitra > latka > *Sent:* Sunday, September 8, 2019 12:12:28 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Optimization from needle shaped crystals > > I can share what has worked for my crystals : > > 1. You can put a grid across your condition with same or altered drop > ratios. > > 2. You can try micro seeding. (This has given me the best results so far). > > 3. You can try Hampton's additive screen. > > On Sun, Sep 8, 2019 at 10:09 AM Prem Prakash wrote: > >> Dear all, >> Sorry for a trivial query. I am trying to Co-crystallize my protein with >> its substrate (peptide) using commercial screenings. In one condition of >> JCSG plus (Molecular Dimension) that contains 0.2 M Magnesium chloride >> hexahydrate, 0.1 M Tris 8.5 50 % v/v Ethylene glycol, I got needle like >> crystals (picture attached). Does anyone have idea to optimize such needles >> into better crystals. I would appreciate all your suggestions. >> >> Thank you >> With kind regards, >> Prem Prakash (Ph.D.) >> >> >> -- >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 >> <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7C9e5bd9b3c9e7485ed8af08d7344ee01c%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637035385686224007=635CdZ1OnWv%2BoTW6YrzI%2BJaPg64OrVyWzz7p9HiAt2c%3D=0> >> > > > -- > Regards > Chitra > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2Fwebadmin%3FSUBED1%3DCCP4BB%26A%3D1=02%7C01%7C%7C9e5bd9b3c9e7485ed8af08d7344ee01c%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637035385686234001=iT7qhIjkuHu1eO%2BqZMthtr4gWL%2B3pI0eYOfJOR0Jm9I%3D=0> > > The Francis Crick Institute Limited is a registered charity in England and > Wales no. 1140062 and a company registered in England and Wales no. > 06885462, with its registered office at 1 Midland Road London NW1 1AT > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Intergrown crystals and excess of nucleation
Hi Nikolaus I completely agree with Claude's comments. Microseeding is the first thing to try. You would like to find conditions where you *don't *get crystals without seeding, but you *do *get crystals with seeding. Then, just by diluting the seed stock, you can control nucleation. Look at D'Arcy and Obmolova's papers, links below (second time!). I also agree that crystallization in agarose can work well - if seeding doesn't solve your problem. The only person that I know who tried containerless crystallization ended up with more crystals rather than fewer. Actually your case is not very unusual - when you scale up you tend to get *more *crystals. The reason is that the surface-area-to-volume ratio is greater for smaller drops. This means that you lose a greater proportion of the protein on the plastic or glass surface of your plate, and also on the air-drop interface. Therefore you should dilute the protein and/or the precipitant when you scale up. Good luck, Patrick http://scripts.iucr.org/cgi-bin/paper?S2053230X14015507 https://scripts.iucr.org/cgi-bin/paper?nj5193 On Mon, Sep 9, 2019 at 4:45 PM Claude Sauter wrote: > Le 09/09/2019 à 17:22, Nikolas a écrit : > > Dear crystalgrowers, > > I am currently working with a protein that appeared to be friendly but > turned out it was not the case. > I found myself to face -in the scale up- the opposite of the usual problem > of nucleation (I really love how this topic finds new ways to make fun of > me). In 24-well plates, hanging-drop, for the same condition but in > different drops I found few big but intergrown crystals and/or a full with > microcrystals. Sometimes also in the same well, when having more drops. > I already decreased the concentration to less than 4mg/mL, made small > adjustments in the optimizations - both with apo and ligand samples, used > Al's oil. > > I have read about the "containerless crystallization" but since I cannot > obtain the sample myself I would like to know if there are any experiences > and/or if there are suggestions for solving this problem. > > Many thanks! > > Best regards, > Nikolas > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > Dear Nikolas, > > since you have some crystal stock, I would definitely try seeding to > better control nucleation events in your drops. Then, instead of using the > containerless approach which requires two types of oils to prepare floating > drops, I suggest the crystallization in agarose gel. Easy to perform, it > favors the 3D growth of well separated crystals in ideal convection-less > conditions. In addition, the gel provides a physical protection of your > crystals during handling, mounting and cryocooling. For more details, see > "Crystal > growth of proteins, nucleic acids, and viruses in gels. Lorber et al. Prog. > Biophys. Mol. Biol. (2009), 101: 13-25." > > Happy crystallization! > > Claude > > -- > Dr Claude Sauter > Institut de Biologie Moléculaire et Cellulaire (IBMC-ARN-CNRS) > Biologie des ARNt et pathogénicité tel +33 (0)388 417 102 > 2 allée Conrad Roentgen fax +33 (0)388 602 218 > F-67084 Strasbourg - France http://cj.sauter.free.fr/xtal > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Importance of temperature during initial crystallization screening
Hi Sergei We did some data-mining on this way, way back, in 2004. See the second section in this link https://www.douglas.co.uk/PDB_data.htm When you consider the *non-standard *temps - ie NOT 4C or 20C - it *looks* as though the higher-end temps *may *work better. But of course it's hard to make sense of the results of a martingale. Thx Patrick PS Janet (Newman) do you have anything more up-to-date on this? On Thu, Aug 1, 2019 at 10:24 AM Sergei Strelkov wrote: > Dear all, > > > I wondered if someone could point me to a recent study on the importance > of temperature during initial search for crystallization conditions. It > would be interesting to see any real statistics on this subject. > > > We typically try to perform screening at at two temperatures, such as > duplicating a given kit screen at 20C and 4C if there is enough sample. My > 'gut feeling' is that this is not as important as sampling the chemical > space though. > > > Thank you! > > Sergei > > > Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of > Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 > bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 > 32 Lab pages: http://pharm.kuleuven.be/Biocrystallography > <http://pharm.kuleuven.be/anafar> > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] challenges in structural biology
I take the view that I'm trying to communicate with as many people as possible, without distracting them with my spelling . . . So go for US spellings. Sent from mobile On Tue, 23 Jul 2019, 22:39 Goldman, Adrian, wrote: > ..and responding in the same vein: > > my OED says that its etymology also comes from the Latin sulfur, sulphura > in the plural. So there is an etymological basis for the ph, even if it > doesn’t come from Greek. > > Plus, since when has etymological logic has _anything_ to do with English > spelling? > > Finally, it may be how the RSC is spelling it, but I would take a fair bet > that writers of English prose today (pace America), contemplating an stinky > inferno, will write “sulphurous flames”, not the unattractive and less > stinky “sulfurous ones”. > > Adrian > > > On 23 Jul 2019, at 22:21, CCP4BB < > 193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote: > > Hi > > Going off at a tangent... > > The accepted spelling by the Royal Society of Chemistry (i.e. the > professional body representing chemists in the U.K.) since at least the > early 1990s has been "sulfate" too. "Sulphur", etc, has been deprecated for > quite some time. Why? Well, there's no good etymological reason for the > "ph" in "sulphate". My 1984 copy of Greenwood and Earnshaw's "Chemistry of > the Elements", written in Yorkshire, uses "sulfur" etc throughout. > > "Phosphorus" comes from the Greek, so retains the "ph"s on both sides of > the pond. > > Element 13 appears to have started life as "alumium", mutated to > "aluminum", and finally (in the English speaking world outside North > America) settled down as "aluminium". > > Harry > -- > Dr Harry Powell > > On 23 Jul 2019, at 17:12, Engin Özkan wrote: > > On 7/23/19 3:35 AM, melanie.voll...@diamond.ac.uk wrote: > > No longer those 20 odd names for ammonium sulphate > > > You mean ammonium *sulfate*. As it is called across the pond. :) > > On a related note on common nomenclature for recording crystallization > experiments that Janet brought up: > > I find it odd that we still do not report cryo-protection methods and > conditions in PDB depositions. Given that a large fraction of the small > molecules observed in crystal structures are derived from the > cryo-protectants, one would think that reporting the contents of that > solution (and pH) would be paramount to a PDB deposition. Surely, the > crystallographic experiment has changed since 1990/use of synchrotron > sources, which PDB has adjusted well to in most other aspects (e.g., > including reporting of synchrotron x-ray optics and all the new > detectors during submission). > > Engin > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] challenges in structural biology
On a completely different tack, isn’t the most pressing requirement in current structural biology a really good method of characterizing macromolecular samples *before *they are put onto cryoEM grids – ie analysing *and screening them *in solution. For one thing I’m told those huge microscopes are quite prone to breaking down, which makes the queues (lines) to get onto them even longer. That method might be (micro-scale) DLS – or something completely different. Thx, Patrick On Mon, Jul 15, 2019 at 8:44 PM Holton, James M < 270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote: > Hello folks, > > I have the distinct honor of chairing the next Gordon Research > Conference on Diffraction Methods in Structural Biology (July 26-31 > 2020). This meeting will focus on the biggest challenges currently > faced by structural biologists, and I mean actual real-world > challenges. As much as possible, these challenges will take the form of > friendly competitions with defined parameters, data, a scoring system, > and "winners", to be established along with other unpublished results > only at the meeting, as is tradition at GRCs. > > But what are the principle challenges in biological structure > determination today? I of course have my own ideas, but I feel like I'm > forgetting something. Obvious choices are: > 1) getting crystals to diffract better > 2) building models into low-resolution maps (after failing at #1) > 3) telling if a ligand is really there or not > 4) the phase problem (dealing with weak signal, twinning and > pseudotranslation) > 5) what does "resolution" really mean? > 6) why are macromolecular R factors so much higher than small-molecule > ones? > 7) what is the best way to process serial crystallography data? > 8) how should one deal with non-isomorphism in multi-crystal methods? > 9) what is the "structure" of something that won't sit still? > > What am I missing? Is industry facing different problems than > academics? Are there specific challenges facing electron-based > techniques? If so, could the combined strength of all the world's > methods developers solve them? I'm interested in hearing the voice of > this community. On or off-list is fine. > > -James Holton > MAD Scientist > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
