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 is very high. >> Nanodrops will yield "overnucleation". >> >> To conclude dear Zq, listen to all the advice, but unless you really >> understand your protein you will >> find 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 Office >> Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab >> 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 >
