Biology Reference
In-Depth Information
The same can be said about crystallization data performed by trial-and-
error, or so-called “high-throughput crystallization.” However, here
making more or less reasonable decisions on how to continue with
crystallization — in situations where crystals are not available — is
impossible.
It is widely believed among crystal growers that crystallization by
chance and trial-and-error methods is most successful when applied to
biological macromolecules; consequently, thousands of randomly per-
formed trials, generally known as “high-throughput crystallization” or
“deliberate approaches to screening for initial crystallization conditions,”
are thought to guarantee further success in obtaining crystals (Luft et al .,
2003). The high-throughput crystallization offered, for example, by lead-
ing crystals provider Hauptman-Woodward Institute (Buffalo, USA) com-
prises the screening of experiments in 1536-well micro-assay plates and
requires a minimum of 400
l of protein solution at a concentration
10 mg/ml. Moreover, it costs US$1000 per plate, if the protein name is
not released by the customer. (http://www.hwi.buffalo.edu/High-Through/
High_Through.html).
The application of more rational approaches has gained little recogni-
tion among protein crystallographers. Indeed, it has been suggested
to “get rid of all the crystals altogether” from structural biology (Patel,
2002). Instead of protein crystals, it would be sufficient to have only
several molecules of protein in order to perform a full structural determi-
nation. Indeed, turning protein crystallization from an art into a science is
still in the early stages of development (Chayen, 2004).
Optimizing protein concentration is very important when using com-
mercially available screens (see www.hamptonresearch.com for refer-
ences). When using these screens, the protein concentration is constant,
usually
µ
10 mg/ml. However, if the protein concentration is too high,
there is nothing other than precipitate at the outset of all the experiments
(sometimes phase separation may develop as well); however, when the
concentration is too low the drops are usually clear in all trials. The rem-
edy is pre-crystallization testing, requiring the adjustment of the stock
protein concentration used in further trials. If after mixing the protein
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