Protein Crystallization Hits

What are readers of this 'Protein Crystallization Hits' blog most interested in? Below is the list of the top 11 blog posts that were requested often during this year. If this is close to what you're interested in, you may as well sign up to get immediate alerts whenever a new post appears here on this site. Alternatively, you can subscribe via RSS (see on top of this page).

So, here are the top eleven:

Table: 'Clickable' most viewed blog posts in 2011

So, what are 'Crystallization Hits' blog readers interested in? Out of 11 of these top posts, 4 articles are on membrane proteins and their crystallization (one on the role of detergent for soluble protein crystallization), and the majority of the remaining posts cover protein crystallization 'tips & tricks'.

Seems you want more of this. 

Your request will be my mission.

Peter

What's a good systematic way to vary all components of a crystallization hit in a single 96-well crystallization tray? For a typical 3 component formulation, Paul Reichert a crystallographer at Schering-Plough, has worked out a remarkably useful schema. Here's what he came up with: At first divide the 96 well tray into 4 quadrants, with 24 wells in each quadrant: 

Reichert systematic optimization quadrant schema for optimizing protein crystallization. Starting point is a single, 3 component hit.

The image above tells much of the story: In quadrant II (beige) the pH and salt concentration are varied systematically in a gradient-like fashion while the precipitation concentration is kept constant. In quadrant III (green) the precipitation and salt concentration is varied while the pH is kept at its original pH and, in quadrant IV (orange) the precipitation concentration and pH are varied while the salt concentration is kept constant. What about quadrant I? Here only the precipitation concentration is changed, keeping pH and salt constant. These are not conditions to be tested in triplicates but allow to assay three different protein concentrations.

Applying this scheme to single crystallization hits provides a lot of meaningful data because it isolates 4 different crystallization factors: (i) pH, (ii) protein, (iii) salt and (iv) precipitant concentration. While not perfect, this is an efficient schema that has often resulted in greatly improved crystal quality.

Since this has worked so well, Emerald provides this type of customized optimization screen as "Reichert Optimization Screen". This is one of several optimization screen services we offer. Just tell us about your hit condition and we'll design, prepare and send you the screen.

Enjoy your crystals,
Peter

P.S. We have recently incorporated this optimization schema into Escreen Builder, a free online tool to calculate an optimization screen using this tool.

How much protein do you need? - Classic first question when a protein biochemist starts a collaboration with a crystallographer. How many milligrams do you need to grow protein crystals and determine the crystallographic structure of my target protein? A simple question that does not have a simple answer, unfortunately. Other than protein-specific reasons, the mg estimate depends in large part on the equipment at one's disposal. For example, the volume of individual crystallization experiments, the number of screens to test, minimum required crystal size, etc. There doesn't seem an end to screening possibilities. If you happen to work in a high-throughput protein crystallization environment it is straight forward to give an estimate for protein requirements for a first screening pass since the protocols and procedures are usually standardized. The high-throughput screening lab at the Hauptman-Woodward Institute for instance, asks for 600 microliters of protein sample at a concentration of ca. 10 mg/ml for an exploratory crystallization screen. That's 6 mg. Depending on your tools you may need 2-5 x that quantity to run a similar screen. But you may not even run that large of a trial and require less than 6 mg. Even in the best case where you obtain crystals from the first crystallization plate it may still be a long way - and many protein preps - to an experimental model on the computer screen.
It is the accumulation of the uncertainties in protein expression, purification and crystallization success that make risk assessment very hard for a single protein structure determination project. This is not at all like converting sheep wool into yarn and the yarn into a pullover. When you think about it, what we're doing in crystallization is akin to throwing darts. Fortunately there's one good rule to go by: the more darts you throw, the higher our chances to hit the bull's eye.