“Hot” Macromolecular Crystals

Abstract

Transcriptional regulator protein TM1030 from the hyperthermophile Thermotoga maritima, as well as its complex with DNA, was crystallized at a wide range of temperatures. Crystallization plates were incubated at 4, 20, 37 and 50° C over 3 weeks. The best crystals of TM1030 in complex with DNA were obtained at 4, 20 and 37° C, while TM1030 alone crystallized almost equally well in all temperatures. The crystals grown at different temperatures were used for X-ray diffraction experiments and their structures were compared. Surprisingly, the models of TM1030 obtained from crystals grown at different temperatures are similar in quality. While there are some examples of structures of proteins grown at elevated temperatures in the PDB, these temperatures appear to be underrepresented. Our studies show that crystals of some proteins may be grown and are stable at broad range of temperatures. We suggest that crystallization experiments at elevated temperatures could be used as a standard part of the crystallization protocol.

Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions

“…However, only one such report could be found, where the crystals of the transcriptional regulator protein TM1030 from the hyperthermophile Thermotoga maritima as well as its complex with DNA were grown at 4, 20, 37, and 50 °C, but the resulting structures did not show noticeable differences. 4 It is unclear whether the crystal packing force restricted structural variation during crystal growth at different temperatures.…”

Unmet challenges of structural genomics

“…A search of the literature and analysis of PDB deposits [22] shows that the advantage of the unusual stability of many target proteins originating from extremophilic bacteria has not been well exploited to date. Although temperature was shown to be an important factor affecting crystallization, there has been no reported systematic effort to use temperatures above ambient for preparation of proteins from thermophilic organisms such as Thermotoga maritima [22,23].…”

“…Although temperature was shown to be an important factor affecting crystallization, there has been no reported systematic effort to use temperatures above ambient for preparation of proteins from thermophilic organisms such as Thermotoga maritima [22,23]. This is rather surprising, since, based on analysis by the Joint Center for Structural Genomics (JCSG) of the crystallization conditions for several hundred proteins from T. maritima, it has been shown that for the two temperatures of crystallization tested (4 and 20 °C), the higher one gave 25% more hits [24].…”

To automate or not to automate: this is the question

“…There is a rising gap between the growing number of protein models and the ability to process and analyze the resulting data in a complex way. The attempt to analyze even simple fields in the PDB header, such as the temperature of crystallization, shows that existing data are not fully reliable [ 58 ]. The recent retraction of 11 fraudulent PDB deposits [ 59 ] shows the necessity for uniform validation of protein models and uniform validation of experiments that lead to structure solution.…”