Bernhard, I'm often amazed at how forgiving protein crystallization is, or to put it another way, how efficient screens are at picking up crystallization hits even when you do everything wrong.
However if you do go down to 20 + 20 nl drops you have to remember that probably 3/4 of your protein will be lost by sticking to the plastic of the plate or forming a skin at the air interface. You generally get roughly the same number of hits from 1 + 1 ul drops (where most of the protein is still in solution) as from 100 + 100 nl drops (roughly half the protein is lost), although the hits will be in different places. This implies that, within broad limits, things like protein concentration don't matter too much. My view of crystallization is that there's a group of proteins (lysozyme and many others) where all you have to do is gently push the protein out of solution, while taking care of nucleation (think microseeding). For these proteins it's a matter of *not *adding something that interferes with crystallization. Then there are others where you have to add something to the mix that stabilizes the crystal lattice. A smaller group requires 2 additives, a few may need 3 .... At the end there's a bunch that will never crystallize no matter what you do. It seems that protein concentration is of secondary importance in screening - although I accept that it may be crucial in optimization. Patrick On 11 June 2013 08:48, Bernhard Rupp <hofkristall...@gmail.com> wrote: > Hmmmm….the real message from figure 3. The protein concentration…**** > > seems to be that >70% of proteins do NOT crystallize in the 10-12.5 mg bin, > **** > > i.e. the ‘right’ concentration is an individual protein-in-that-buffer > property - and**** > > all one can say is that the concentration needs to be high enough so that > the crystallization **** > > conditions (either right away in case of batch, or vapor-diffusion > assisted) can drive**** > > the system across the solubility limit into supersaturation. **** > > Most people actually working with their protein have already a reasonably > developed idea what**** > > their protein can take in various buffer systems (valuable parameter!)**** > > … once they have scraped if off a centricon membrane, for example…**** > > The pre-screening originated from structural genomics when one had no idea > what**** > > the ‘customer’ actually sent to the facility. If one knows the material it > might be more**** > > efficient (i.e. more information from the same precious material) to set > up a plate of **** > > 96 20+20 nl nanodrops than spending 2 ul on prescreening.**** > > ** ** > > Just an opinion sans statistics, BR**** > > ** ** > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Debasish > Chattopadhyay > *Sent:* Tuesday, June 11, 2013 1:02 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Protein concentration for crystallization**** > > ** ** > > Perhaps my question was not expressed well. I wanted to know if proteins > crystallize more frequently when the protein concentration is in the range > 5-30mg/ml.**** > > The answer pointed out by my colleague Todd Green is on the page**** > > http://www.douglas.co.uk/PDB_data.htm**** > > ** ** > > Thanks for your inputs.**** > > ** ** > > Debasish**** > > ** ** > > *From:* Orru, Roberto [mailto:roberto.o...@emory.edu<roberto.o...@emory.edu>] > > *Sent:* Monday, June 10, 2013 5:04 PM > *To:* Debasish Chattopadhyay > *Subject:* RE: Protein concentration for crystallization**** > > ** ** > > Dear Debasish, > > On my memory there are 2 way (but I cannot say that are the only 2!) > First: if you have the structure and you know the water content, you can > guess the amount of protein crystallized in your drop by calculating the > volume of the crystals. > Second (if you can waste your drops): Fish all the crytsals in any drop > for a given concentration, load a sds page w/ silver staining developing > and compare it with a calibration curve done with your same protein in the > same gel. > > Best > R.**** > ------------------------------ > > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Debasish > Chattopadhyay [debas...@uab.edu] > *Sent:* Monday, June 10, 2013 10:49 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Protein concentration for crystallization**** > > What would be a convenient way to estimate what percentages of proteins > have been crystallized in a concentration range, for example 5-30 mg?**** > > **** > > Debasish Chattopadhyay**** > > **** > > University of Alabama at Birmingham**** > > CBSE-250**** > > 1025 18th Street South, Birmingham, Al-35294**** > > USA**** > > Ph: (205)934-0124; Fax: (205)934-0480**** > > **** > > ** ** > ------------------------------ > > > This e-mail message (including any attachments) is for the sole use of > the intended recipient(s) and may contain confidential and privileged > information. If the reader of this message is not the intended > recipient, you are hereby notified that any dissemination, distribution > or copying of this message (including any attachments) is strictly > prohibited. > > If you have received this message in error, please contact > the sender by reply e-mail message and destroy all copies of the > original message (including attachments).**** > -- 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
