> no systematic study has been reported We had a ACA poster many years ago, comparing same drops of various proteins in linbro plates, 98 conditions hanging and sitting drop, on sitting posts, and with added glass shards (attached gif). In summary, comparable overall success rate, but variation in which drops showed results. A few were unique to either setup. This is understandable given the generally poor reproducibility even of identical setups shown later by Janet Newman.
Unfortunately neither our nor Newman's study were designed for rigorous statistical analysis. It is also unclear given the limited number of proteins examined, how much bias exists in this regard. Similar considerations hold for temperature, except that there is more likely a dependence on the specific material (which again one does not know beforehand). So, you have to try, and local practical use considerations may be more important than uncertain statistical arguments. There is one hard statistically verified rule though, and that is that a protein that inherently likes to crystallize does so a) under multiple conditions b) gives better crystals on average This is however also trivial and does not provide predictive guidance, but points towards use of multiple constructs. It is probably impossible to predict crystallization conditions that depend on local protein properties, given only global parameters. I am also skeptical and argue that rational design methods frequently achieve their objective for reasons not related to the ratio behind the design. Consider how many protein properties are changed simultaneously during say surface entropy reduction or lysine methylation. Doing something to the protein obviously *IS* important. In summary, 1) do nothing that is a priori doomed to fail 2) sample anything else comprehensively with a valid design 4) accept that the protein (variant) properties determine success 5) accept that many relevant parameters exist (and vary) which you do not even know (purification, cofactors, detritus) 6) use whatever technique works for your situation. That unfortunately is about as good as it gets for rules. br -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joe Sent: Friday, May 01, 2009 7:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Hanging vs. Sitting Sure, there are differences between these two methods, but no systematic study has been reported showing one is better than the other in terms of getting initial hits. Since we have a crystallization robot, I routinely set up sitting drops for initial screens and hanging drops (manually) for optimization thereafter. As long as I have no problem getting reproducible conditions, I will stick to the one I found most efficient and convenient for myself. Joe Frank von Delft wrote: > Sorry, disagree again: with the right plate type (e.g. SwissCi > plates), it's far far easier from sitting drop, because: > 1. you don't have to muck around with flipping over the cover slip, > instead just cut the seal > 2. you have more time, because your drop does not evaporate as quickly > (see earlier mail) > 3. if the crystal sticks, just poke an acupuncture needle into the > plastic below it: off it pops. > phx > > > > Simon Kolstoe wrote: >> It's also easier to fish the crystals out of the solution with a >> hanging drop. >> >> Simon >> >> >> >> On 1 May 2009, at 06:35, Debajyoti Dutta wrote: >> >>> >>> Hi, >>> >>> >From the experiance of mine I can tell you that the crystal size >>> sometimes matters between these two methods. Hanging drop may yield >>> bigger crystals than sitting drop, that may be due to the >>> evaporation rate(surface area). Hanging drop allow us to set >>> different protocols also like free interface diffusion, area covered >>> by the drp etc. >>> >>> These informations are gained purely by experiance. >>> >>> cheers >>> Deb >>> >>> >>> On Thu, 30 Apr 2009 20:40:35 +0530 wrote >>> >I have noticed that a significant majority of crystallizations are >>> done in >>> >hanging- rather than sitting-drop configuration, and considering the >>> >significant extra labor involved in hanging drops, can only >>> understand this >>> >preference as a historical bias. I understand that sometimes one >>> technique >>> >works and not the other, but all things being equal, why is hanging >>> drop >>> >still "hanging around?" Any insights appreciated... >>> > >>> >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 <mailto:j-kell...@northwestern.edu> >>> >******************************************* >>> > >>> <http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/sign atureline....@middle?> >>> >>> >>
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