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.
[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] control of nucleation
Dear zq deng, The standard method, I dare say, in such a case is micro seeding: reduce the precipitant to a concentration so that you just do not get crystals any more, wait until the drop equilibrates (about one day in the case your precipitant is a salt, up to 5-7 days for high MW PEGs), then use a cat's whisker for micro seeding into that drop. Well, that's micro seeding in a nut shell. Tim On Thu, May 06, 2010 at 04:03:46PM +0800, zq deng wrote: 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. -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] control of nucleation
Dear Zq, A few ideas: 1) Vary protein concentration, temperature, or protein : mother liquor ratio. 2) Try dioxane - it is supposed to reduce nucleation. 3) give your protein a good hard spin before you set up drops to remove aggregates. 4) seeded factorial screen. 5) re-purify on gel filtration? Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- Nature composes some of her loveliest poems for the microscope and the telescope. ~Theodore Roszak From: zq deng dengzq1...@gmail.com Reply-To: zq deng dengzq1...@gmail.com Date: Thu, 6 May 2010 09:03:46 +0100 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] control of nucleation
Dear Zq CCP4BB readers,The precipitant is the main component that affects nucleation.In specific cases other factors can be used to modulate nucleation as mentioned before by others: protein concentration, temperature, drop size, initial protein/precipitant ratio etc. All good components of a very long list that will give a student years of work ahead.Just to take the first item: "Protein concentration"Most will think that "Protein concentration" should be reduced to reduce nucleation. Unfortunately,what will happen when the protein concentration is reduced is not so easily predictable.Let's do the opposite! Just for fun, let's increase the protein concentration instead.We will increase the protein concentration while severely reducing the precipitant concentration. The 15 mins lysozyme crystallization is a good example of this. Enrico's 15mins Lysozyme recipeLysozyme concentrations of 100-150 mg/ml are used. The high protein concentration allows crystals to grow rapidly, so each nucleus has a chance to grow before more nuclei are formed. This is because each growing nucleus ends up depleting its local environment and making the nucleation of others nearby less likely. Nucleation requires a higher degree of supersaturation than crystal growth. Getting it to work and controlling it requires accuracy, but it is great fun to do ... and lysozyme is cheap.HOT STUFF crystallization is ! In the case of lysozyme: Do it in a hot room you for better control. This ambiguity persists for other items:Thomas Edwards suggests that: "dioxane - it is supposed to reduce nucleation.""Supposed to" when it does not do the opposite:Ménétrey, J., Perderiset, M., Cicolari, J., Houdusse, A. Stura, E.A. (2007) Improving Diffraction from 3 to 2 Å for a Complex between a Small GTPase and Its Effector by Analysis of Crystal Contacts and Use of Reverse Screening. Cryst. Growth Des. 7:2140-2146.This is the one additive that really increases nucleation in the above paper.Online access: Improving DiffractionThe procedures in "reverse screening" are used to identify what each proposed effector really does and use it toachieve better crystals.As screening is done with smaller and smaller drops, the conditions that will emerge more often are those where the nucleation rate is very high. Nanodrops will yield "overnucleation".To conclude dear Zq, listen to all the advice, but unless you really understand your protein you willfind that you will achieve the opposite of what you are trying to do.Enrico.On Thu, 06 May 2010 10:10:37 +0200, Thomas Edwards t.a.edwa...@leeds.ac.uk wrote: Dear Zq, A few ideas: 1) Vary protein concentration, temperature, or protein : mother liquor ratio. 2) Try dioxane - it is supposed to reduce nucleation. 3) give your protein a good hard spin before you set up drops to remove aggregates. 4) seeded factorial screen. 5) re-purify on gel filtration? Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- Nature composes some of her loveliest poems for the microscope and the telescope. ~Theodore Roszak From: zq deng dengzq1...@gmail.com Reply-To: zq deng dengzq1...@gmail.com Date: Thu, 6 May 2010 09:03:46 +0100 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.-- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 OfficeRoom 19, Bat.152, Tel: 33 (0)1 69 08 9449LabLTMB, 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.-sturahttp://www.chem.gla.ac.uk/protein/mirror/stura/index2.htmle-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] control of nucleation
Dear Zq Deng, I've had success with the dilution method as described by Dunlop and Hazes: http://scripts.iucr.org/cgi-bin/paper?en5016 For me it worked for a couple of projects, and gives you a bit more to permutate than just varying the protein:mother liquor ratios, as Thomas Edwards helpfully suggested. Good luck, Mark On 6 May 2010 09:03, zq deng dengzq1...@gmail.com wrote: 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. -- Skype: markabrooks
[ccp4bb] Fwd: [ccp4bb] control of nucleation
I mistakenly sent this off to Enrico, rather than to the CCP4BB. Apologies to Enrico. Begin forwarded message: From: Charles W. Carter, Jr car...@med.unc.edu Date: May 6, 2010 6:50:23 AM EDT To: est...@cea.fr Subject: Re: [ccp4bb] control of nucleation In fact, there is quite good experimental evidence that the most important parameter affecting the rate of nucleation is the supersaturation ratio, or the [protein]/solubility. Unfortnately, the physical reasons for the unexpected behaviors described by Enrico, which do occur frequently, are that nearly all crystallization experiments are carried out in almost total absence of any knowledge of the solubility curve and how it depends on the concentrations of other reagents in the screen. To my knowledge, the best data on the relationship between the rate of nucleation and the supersaturation ratio have been compiled by Hofrichter, Eaton, and Ross for hemoglobin and by Ataka and Tanaka for lysozyme. The experiment is conceptually quite simple, but almost never performed, because the solubility behavior is unknown. It involves setting up crystallizations and measuring the time it takes to see crystals and then plotting the data on a log-log plot, which linearizes the power law relation, so that the slope is the exponent. For lysozyme, the nucleation rate is proportional to a variable, but high power of the supersaturation ratio. For lysozyme, this power is 5, so that lysozyme seeds appear at a rate proportional to ([Lyso]/S)^5 (Ataka, M. and Tanaka, S. (1986) The growth of large, single crystals of lysozyme. Biopolymers 25, 337–350.). For hemoglobin, the same experiments suggest a much higher power, 35-40 for sickle-cell hemoglobin gelation. This number has been revised downward significantly by further work by Hofrichter and others, because while the experimental data are reliable, the interpretation in terms of homogeneous nucleation is not: sickling involves heavy-duty secondary nucleation, which gives a very high apparent exponent. There is a small literature on efforts to incorporate the power law relationship into empirical screening: Carter and Ries-Kautt, 2006 Improving Marginal Crystals, in Methods in Molecular Biology, Macromolecular Crystallography Protocols: Volume 1,Preparation and Crystallization of Macromolecules edited by S. Doublié, 363:153-174. The success of reverse screening arises primarily because it circumvents the requirement for homogeneous nucleation by providing seeds. On May 6, 2010, at 6:15 AM, Enrico Stura wrote: Dear Zq CCP4BB readers, The precipitant is the main component that affects nucleation. In specific cases other factors can be used to modulate nucleation as mentioned before by others: protein concentration, temperature, drop size, initial protein/precipitant ratio etc. All good components of a very long list that will give a student years of work ahead. Just to take the first item: Protein concentration Most will think that Protein concentration should be reduced to reduce nucleation. Unfortunately, what will happen when the protein concentration is reduced is not so easily predictable. Let's do the opposite! Just for fun, let's increase the protein concentration instead. We will increase the protein concentration while severely reducing the precipitant concentration. The 15 mins lysozyme crystallization is a good example of this. Enrico's 15mins Lysozyme recipe Lysozyme concentrations of 100-150 mg/ml are used. The high protein concentration allows crystals to grow rapidly, so each nucleus has a chance to grow before more nuclei are formed. This is because each growing nucleus ends up depleting its local environment and making the nucleation of others nearby less likely. Nucleation requires a higher degree of supersaturation than crystal growth. Getting it to work and controlling it requires accuracy, but it is great fun to do ... and lysozyme is cheap. HOT STUFF crystallization is ! In the case of lysozyme: Do it in a hot room you for better control. This ambiguity persists for other items: Thomas Edwards suggests that: dioxane - it is supposed to reduce nucleation. Supposed to when it does not do the opposite: Ménétrey, J., Perderiset, M., Cicolari, J., Houdusse, A. Stura, E.A. (2007) Improving Diffraction from 3 to 2 Å for a Complex between a Small GTPase and Its Effector by Analysis of Crystal Contacts and Use of Reverse Screening. Cryst. Growth Des. 7:2140-2146. This is the one additive that really increases nucleation in the above paper. Online access: Improving Diffraction The procedures in reverse screening are used to identify what each proposed effector really does and use it to achieve better crystals. As screening is done with smaller and smaller drops, the conditions that will emerge more often are those where the nucleation rate
Re: [ccp4bb] control of nucleation
Hi I have succeeded by using oil (such as parafin oil ... etc ) at reservoir on top of the reservoir solution. You have to try several trials to find optimum ratio. With regards Syed --- On Thu, 5/6/10, zq deng dengzq1...@gmail.com wrote: From: zq deng dengzq1...@gmail.com Subject: [ccp4bb] control of nucleation To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, May 6, 2010, 1:33 PM 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] control of nucleation
actually,the protein grow in too different condition.both have excess necleation,and one condition is that just puting protein in the drop and not mixing with the precipitants.there is one another problem how to cryo-protect. 2010/5/6 syed ibrahim b_syed_ibra...@yahoo.com Hi I have succeeded by using oil (such as parafin oil ... etc ) at reservoir on top of the reservoir solution. You have to try several trials to find optimum ratio. With regards Syed --- On *Thu, 5/6/10, zq deng dengzq1...@gmail.com* wrote: From: zq deng dengzq1...@gmail.com Subject: [ccp4bb] control of nucleation To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, May 6, 2010, 1:33 PM 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.
