Re: [ccp4bb] protein crystals or salt crystals
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
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] protein crystals or salt crystals
If one tries to use a dye to determine if crystals are protein or salt, then I recommend that they use both a positive and a negative control. So have some handy salt or sugar crystals ready along with some known protein crystals to use as controls. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ganesh Natrajan [ganesh.natra...@ibs.fr] Sent: Monday, February 11, 2013 3:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals 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
Re: [ccp4bb] protein crystals or salt crystals
They look like other phosphate crystals that I've seen, but have to shoot them to tell. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols [nathaniel.ech...@gmail.com] Sent: 07 February 2013 22:30 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals If SPG buffer is what I think it is, that means you have a significant concentration of inorganic phosphate, which forms salt crystals when mixed with divalent metal ions. -Nat On Thu, Feb 7, 2013 at 2:24 PM, amro selem amro_selem2...@yahoo.com 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
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
I didn't see the picture that you attached but if you have more than one crystal you could always run one on a gel to see if it runs the expected size of your protein From: Patrick Shaw Stewart [mailto:patr...@douglas.co.uk] Sent: Friday, February 08, 2013 11:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: 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.ukmailto:frank.vonde...@sgc.ox.ac.uk wrote: 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.ukmailto: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 htmlpfont face = verdana size = 0.8 color = navyThis communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorized signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary./font/p/html
Re: [ccp4bb] protein crystals or salt crystals
Hi Patrick Did you mean that to go to BB? To put pressure on us? :) We've not done that analysis, no - good idea though. No standard purification buffer, though most commonly it's HEPES pH 7.5ish, varying amounts of NaCl and glycerol. Like most people, I assume. There certainly are wells in the screens that are more prone to produce salt - but all wells have produced protein crystals at some point, so we've not thrown anything out. (I'm proud to be superstitious.) That said: X-rays are cheap these days, just do the experiment! (My view at least.) HTH phx On 08/02/2013 09:46, Patrick Shaw Stewart wrote: 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.uk mailto:frank.vonde...@sgc.ox.ac.uk wrote: 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.uk mailto: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
There a lot of articles about salt-protein crystals in google - check them first. How to check crystals: 1) X-ray check (most obvious way) 2) izid dye 3)dry it (protein crystal will break apart) 4)crush it (if you dont know how to check by this method - try to grow and break lysozyme or another model protein crystals - it will take not more than 3 days) . Protein crystals behave rather as gelatine and not as solid 08.02.2013 13:53, Sylvia Fanucchi пишет: I didn’t see the picture that you attached but if you have more than one crystal you could always run one on a gel to see if it runs the expected size of your protein *From:*Patrick Shaw Stewart [mailto:patr...@douglas.co.uk] *Sent:* Friday, February 08, 2013 11:47 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* 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.uk mailto:frank.vonde...@sgc.ox.ac.uk wrote: 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.uk mailto: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 This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorized signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary. -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] protein crystals or salt crystals
Patrick, Something related: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Conditions_prone_to_salt_crystallization Truth be told, we recently had a major breakthrough with the peg/fluoride condition I came to consider a useless salt crystal generator. So tables like these are undoubtedly useful but do not reduce workload. :) This is also a rather interesting finding http://www.google.com/url?sa=tsource=webcd=12ved=0CDEQFjABOAourl=http%3A%2F%2Fwww.aseanbiotechnology.info%2FAbstract%2F21021153.pdfei=N_kUUYfkBafV0gG09ICoAgusg=AFQjCNE7C-m6IkLPUay9gEEM60yJF46ZQg Basically, presence of protein may induce salt crustallization. To me, this means that diffraction pattern is the best indicator. Frank already said exactly that, of course. Cheers, Ed. Original message From: Patrick Shaw Stewart patr...@douglas.co.uk Date: To: CCP4BB@JISCMAIL.AC.UK Subject: 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.uk wrote: 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.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] protein crystals or salt crystals
Like Nat points out, I suspect they are phosphate crystals. I have seen those before. And I agree 100% with Frank, for once. Why would I risk making any guesses no matter how salt like my crystals look after all the time it took me to clone, express, purify and crystallize my precious protein. It takes MUCH less time than all of that slog to stick the crystal on a beam and here are a few possible scenarios: (1) Clear diffraction spots spaced planets apart in case of salt (2) Protein like diffraction (3) Inconclusive no diffraction situation, which could indicate a million things including the possibility that your cryoprotectant was sub-optimal for data collection done using flash cryocooled/flash frozen crystals in a stream of gaseous nitrogen. Note of caution: I may take more time to plan the diffraction test (room temperature, cryoprotectant etc.) if I only ever got one precious crystal and was never able to reproduce the crystallization. Cheers, Raji On Fri, Feb 8, 2013 at 8:18 AM, Ed. Pozharski epozh...@umaryland.eduwrote: Patrick, Something related: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Conditions_prone_to_salt_crystallization Truth be told, we recently had a major breakthrough with the peg/fluoride condition I came to consider a useless salt crystal generator. So tables like these are undoubtedly useful but do not reduce workload. :) This is also a rather interesting finding http://www.google.com/url?sa=tsource=webcd=12ved=0CDEQFjABOAourl=http%3A%2F%2Fwww.aseanbiotechnology.info%2FAbstract%2F21021153.pdfei=N_kUUYfkBafV0gG09ICoAgusg=AFQjCNE7C-m6IkLPUay9gEEM60yJF46ZQg Basically, presence of protein may induce salt crustallization. To me, this means that diffraction pattern is the best indicator. Frank already said exactly that, of course. Cheers, Ed. Original message From: Patrick Shaw Stewart patr...@douglas.co.uk Date: To: CCP4BB@JISCMAIL.AC.UK Subject: 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 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] protein crystals or salt crystals
Raji Edayathumangalam wrote: (3) Inconclusive no diffraction situation, which could indicate a million things including the possibility that your cryoprotectant was sub-optimal for data collection done using flash cryocooled/flash frozen crystals in a stream of gaseous nitrogen. But before throwing it in this category, be sure to take a wide-angle oscillation, as reciprocal space is sparsely populated with small unit cell crystals and you might miss spots altogether in a 1 degree oscillation. I like to take a 5-sec 180* oscillation which gives plenty of spots in a nice pattern for a salt crystal, and I suppose records enough spacings to positively identify the mineral if anyone cared to tak the time. eab
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 09:13 -0500, Edward A. Berry wrote: I like to take a 5-sec 180* oscillation which gives plenty of spots in a nice pattern for a salt crystal Second that It also confuses bystanders really well - what a strange diffraction pattern - half salt (small unit cell) / half protein (lots of spots). Takes your mind off the gloom of your dashed hopes :) -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
So, if you are bored and have nothing else to do (which is how we all are at times; kidding), can you set up a control experiment with everything in the crystal dip except protein (so buffer and whatever)? I know protein plays a role in the process, but I have done this before when I had suspect conditions, and it did show that my buffer formed crystals in that situation. Sometimes it helps. But I am one that never throws a crystal away, and always just puts it in a beam before saying it's salt. Protein crystals are too precious to rule out by over thinking. Good luck Dave On 2/8/2013 9:13 AM, Edward A. Berry wrote: Raji Edayathumangalam wrote: (3) Inconclusive no diffraction situation, which could indicate a million things including the possibility that your cryoprotectant was sub-optimal for data collection done using flash cryocooled/flash frozen crystals in a stream of gaseous nitrogen. But before throwing it in this category, be sure to take a wide-angle oscillation, as reciprocal space is sparsely populated with small unit cell crystals and you might miss spots altogether in a 1 degree oscillation. I like to take a 5-sec 180* oscillation which gives plenty of spots in a nice pattern for a salt crystal, and I suppose records enough spacings to positively identify the mineral if anyone cared to tak the time. eab
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 14:53 +0400, Evgeny Osipov wrote: Protein crystals behave rather as gelatine and not as solid I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids Interesting--do you have a reference quickly on hand for those measurements? I have always been somewhat curious about that JPK Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein crystals or salt crystals
Journal of Crystal Growth 232 (2001) 498-501 and references therein Available at http://pages.physics.cornell.edu/~rthorne/publications/caylorjcg01.pdf Colin From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: 08 February 2013 14:57 To: ccp4bb Subject: Re: [ccp4bb] protein crystals or salt crystals I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids Interesting--do you have a reference quickly on hand for those measurements? I have always been somewhat curious about that JPK Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 09:57 -0500, Jacob Keller wrote: do you have a reference quickly on hand http://www.ncbi.nlm.nih.gov/pubmed/8129868 and references therein http://www.sciencedirect.com/science/article/pii/S0022024801010922 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1300955/ The last reference is not strictly speaking about protein crystals, but also emphasizes how protein as elastic material is more of a solid (with some peculiar properties likely arising from large entropic contribution to its deformation energy. -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
Re: [ccp4bb] protein crystals or salt crystals
Ed, Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. I hate to disagree with the disagreement, but there are many exceptions to this rule. I have seen many protein crystals that are quite malleable and bendable. One protein produced rod-shaped crystals (150x40x40 um) that I could bend by almost 60 degrees and it would slowly snap back. Mounting it old school was a real pain, and their diffraction was mediocre. However, the majority of the crystals I have worked with adhere to the general rule you describe. Where the crystal physical behavior is anomalous, it is often when PEG is used and/or there are multiple components that contribute to the crystal's integrity (as in the case of membrane protein crystals with detergent). In the former case, crystals that sit in PEG solutions too long tend to be cross-linked (most likely due to the aldehydes that can exist in some batches of PEG). One could argue that the crosslinking adds long-range elasticity and a resistance to fracturing. In the latter case, I have observed large beautiful crystals of membrane proteins that have the consistency and malleability of warm butter. Sometimes optimization improved their integrity, and other times a new crystal form is needed. Regards, 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 On Feb 8, 2013, at 9:23 AM, Ed Pozharski wrote: On Fri, 2013-02-08 at 14:53 +0400, Evgeny Osipov wrote: Protein crystals behave rather as gelatine and not as solid I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
Michael, It seems to me we have no disagreement, as we both say that it is *unusual* for protein crystals to be non-fragile. Furthermore, my objection is to gelatin characterization. I may be, as is my custom, wrong, but in terms of elasticity gels are purely entropic. Protein crystals, even the malleable ones you describe, have both enthalpic and entropic components to the deformation free energy (admittedly the entropic component is stronger than in regular materials). Your comment on the PEG-induced slow cross-linking is very interesting. What you describe fits perfectly the behavior of long rods of a deliberately cross-linked crystal - they bend and don't snap. Protein crystals (at least some) are also characterized by an unusually broad range of linear response to mechanical deformation, up to 10% http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143058/ According to the theory in this paper though, you might be destroying diffraction by bending the crystal, since in crystal denaturation is likely irreversible. From what you are describing (60 degrees, 150x40x40 um), the linear deformation should reach almost 50%, definitely enough for partial crystal denaturation. Cheers, Ed. On Fri, 2013-02-08 at 11:22 -0500, R. M. Garavito wrote: Ed, Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. I hate to disagree with the disagreement, but there are many exceptions to this rule. I have seen many protein crystals that are quite malleable and bendable. One protein produced rod-shaped crystals (150x40x40 um) that I could bend by almost 60 degrees and it would slowly snap back. Mounting it old school was a real pain, and their diffraction was mediocre. However, the majority of the crystals I have worked with adhere to the general rule you describe. Where the crystal physical behavior is anomalous, it is often when PEG is used and/or there are multiple components that contribute to the crystal's integrity (as in the case of membrane protein crystals with detergent). In the former case, crystals that sit in PEG solutions too long tend to be cross-linked (most likely due to the aldehydes that can exist in some batches of PEG). One could argue that the crosslinking adds long-range elasticity and a resistance to fracturing. In the latter case, I have observed large beautiful crystals of membrane proteins that have the consistency and malleability of warm butter. Sometimes optimization improved their integrity, and other times a new crystal form is needed. Regards, 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 On Feb 8, 2013, at 9:23 AM, Ed Pozharski wrote: On Fri, 2013-02-08 at 14:53 +0400, Evgeny Osipov wrote: Protein crystals behave rather as gelatine and not as solid I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] protein crystals or salt crystals
If SPG buffer is what I think it is, that means you have a significant concentration of inorganic phosphate, which forms salt crystals when mixed with divalent metal ions. -Nat On Thu, Feb 7, 2013 at 2:24 PM, amro selem amro_selem2...@yahoo.com 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
Re: [ccp4bb] protein crystals or salt crystals
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
