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When trying to decide which additive might be useful during crystallization, try the following. If one has crystals and wants to try using additives to improve the crystal size or quality go back to the initial crystallization screening plates. Review the plates, looking for conditions where no precipitate nor crystals were/are observed. Now, review the results, looking for a common reagent ingredient present where no precipitant or crystals are found. For example, one might find that all drops with isopropanol remained clear. One could then try adding isopropanol to the current crystallization condition to see if the isopropanol could improve the crystal size and/or quality. If one observes a difference in the crystal in the presence of isopropanol, then one might consider evaluating other additives in the class of alcohols such as ethanol, methanol, tert-butanol and others. If one has no crystals, but plenty of precipitate, phase separation and clear drops, follow the above analysis and try adding the common reagent ingredient found in clear drop to those drops which contained precipitate or phase separation. It is possible this agent could improve or at least manipulate sample solubility. The above tip was submitted from Jarmila Jancarik from the laboratory of Sung Ho Kim at the University of California Berkeley. Thank you Jaru! When using this approach it might be reasonable to discern between concentration independent and dependent precipitation when trying to decide which agent to pursue as an additive. Try evaluating the concentration independent agents first and then look at the other agents if sample quantity permits. For example, if one observe precipitate in 15 to 40% PEG but not in 5 to 10% PEG, it might simply be a concentration. However, if one observes no precipitate in 45 to 60% v/v MPD one could guess that MPD is a reasonable agent to evaluate as an additive.
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Although Hampton Research does offer a specifically formulated Additive Screen (HR2-428), here is a tip when one already has a sparse matrix screen or two laying about the lab. When screening additives try adding 50 microliters of each Crystal Screen reagent to 950 microliters the "best" crystallization conditions thus far in order to see if any of the reagents in Crystal Screen might serve as good additives. Crystal Screen 2 is an especially good kit for this technique since Crystal Screen 2 contains numerous divalent cations, Jeffamine Reagent, and a few other "novel" agents. Jeffamine is a registered trademark of the Huntsman Petrochemical Corporation Reference Crystallization of foot-and-mouth disease virus 3C protease: surface mutagenesis and a novel crystal-optimization strategy. Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):646-50. Epub 2005 Apr 20. Birtley JR1, Curry S.
Try benzylkonium chloride (Fluka 12060). This cationic surface active agent has been reported to be useful as a crystallization additive with membrane proteins and may be useful for soluble proteins. We've been using it in the drop between 1 and 3% w/v in water. Try a 10% w/v stock solution in water and dilute into the drop to 1-3%.
Try dissolving the small molecule additive into paraffin or silicon oil, and use this mixture to cover the sample drop. This can be used with sitting drop vapor diffusion or with microbatch under oil. The oil acts as a reservoir that may contain excess small molecule that (you hope) will be fed into the crystals.
Aggregation can be a deterrent to the crystallization of biological macromolecules including proteins, peptides, and nucleic acids. The presence of sample aggregation can be detected by either dynamic light scattering or native gel electrophoresis. Aggregation might be caused by hydrophobic patches on the surface of the sample, differently charged isoforms, differently phosphorylated isoforms, mixtures of methylated and non-methylated samples, glycosylation, as well as electrostatic interactions. Aggregation can be due to autologous aggregation where the protein is aggregating with itself or heterologous contamination where the sample is aggregating with other proteins. In the case of heterologous contamination, further purification of the sample should be seriously considered. In the case of autologous aggregation that precludes crystallization one might consider: Using molecular biology to manipulate intra and inter molecule interactions by modifying the sample sequence (alter, add, or delete residues). Use chemical additives to manipulate sample-sample and sample solvent interactions. Detergents Chaotropes (urea, guanidine hydrochloride, hydrochloric acid, etc) Electrostatic agents Alcohols (isopropanol, methanol, ethanol, etc) Salts (sodium chloride, potassium chloride, sodium fluoride, etc) Polyols (glycerol, PEG 400, etc) Ligands, inhibitors, co-factors, and metals Use temperature to prevent aggregation (0C and 60C) Consider a fusion protein Remove C-terminus or N-terminus Truncate domains Remove His-tag In some cases aggregates can be removed by centrifugation or filtration. In some cases the aggregates can be removed by mixing the sample with the crystallization reagent, allowing the sample to incubate for 15 minutes, centrifuging the sample/reagent mixture, removing the precipitate and setting the drop with the supernatant.
We've had good success using the well solution directly as the foundation of a cryobuffer in several situations where crystals cannot be grown directly in the presence of cryoprotectant, and where crystals don't tolerate transfer to artificial mother liquors. The basic protocol is as follows:
4. Transfer the crystal to aliquot 1, equilibrate for 3 minutes, then to aliquot number 2, then freeze. We've had a few crystals that routinely crack or blow up when transfered to artificial mother liquor that behave well when transfered to well solution plus glucose. We assume that there is some aspect of the crystal drop (pH, ionic tension, precipitant concentration) that is more effectively reproduced within the well than by separately prepared mother liquors. The nice thing about the protocol above is that you don't get much of a volume increase when dry dextrose is dissolved in the well solution, so the components in the solution are not diluted.
High molecular PEGS are also good cryoprotectants. If crystals are obtained from relatively high concentration of PEGS (e.g., 30% of PEG 3350), you can cryoprotect them simply by raising the concentration of the PEG a little bit (e.g., 40% of PEG 3350). J. Appl. Cryst. (2006). 39, 244-251 Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov, N. S. Husseini, O. A. Alsaied and R. E. Thorne Synopsis: Critical concentrations required for vitrification of aqueous solutions are determined for fourteen common cryoprotectants, for sample volumes ranging over four orders of magnitude and covering the range of interest in protein crystallography.
To mount very thin crystals onto cryoloops, first dip the nylon loop into 0.5% Formvar solution (Fluka # 09819) to form a thin film. The film provides extra support for fragile crystals, and can result in much sharper reflections with just slightly higher background. To clean the loop, dip it in alcohol to dissolve the support. Two notes: (1) the technique works only for crystals grown without organic solvents, and (2) take precautions not to breathe vapor from the formvar solution--the solvent is 1,2 dichloroethane. Formvar is a standard support for electron microscopy grids.
Reduced radiation damage. Decreased thermal motion and disorder. Potential for improved resolution. Increased crystal lifetime. Crystals can be stored and shipped.
(Be aware of the potential for) detergent concentration mismatch between your mother liquor and the cryosolution. This particularly happens with vapor diffusion setups: there is a delicate balance of "free" detergent in the mother liquor versus the proportion of the detergent which is bound to the protein. Dropping a xtal into the cryosolution shocks the crystal with a bolus of extra free detergent. Hence, and counterintuitively, you may need to reduce the detergent concentration in the cryosolution to keep everything in balance. Try titrating down from 1% to even as low as 0.4% in the cryosolution. Under the conditions you are using the CMC of bOG is suppressed below the usual 0.67% (w/v). Also, the behavior of many of the alkyl glycoside detergents is very temperature sensitive. So be careful about the temperature of all the solutions you use. R. Michael Garavito, Ph.D. Submitted to CCP4 bulletin board February 2007 Edited by Hampton Research Corp.
Got ice rings and want to get rid of them? While the crystal is in the beam, carefully try the following. Try annealing the crystal by blocking the stream for a few seconds, allowing the crystal to thaw, then unblock the stream to cool the crystal again. (Reference: New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium, B. Leif Hansona, Constance A. Schallb and Gerard J. Bunick, Journal of Structural Biology Volume 142, Issue 1, April 2003, Pages 77-87) Try washing the crystal with liquid nitrogen. Using a pipette, carefully aspirate liquid nitrogen into the pipette, then dispense several drops or even a gentle stream over the crystal and mount. A liquid nitrogen wash can also be used to remove creeping ice from caps, pins and loops.
Many salts are cryosalts, including malonate, formate, citrate, tartrate, acetate, Tacsimate and other organic acids, ammonium sulfate (>3.5 M), lithium sulfate, lithium chloride and other alkylammonium salts. For cryo try increasing your salt concentration by 20%. (Cryosalts: suppression of ice formation in macromolecular crystallography, K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 doi:10.1107/S0907444900007587.