Temperature can have a major impact on the conformational state and ligand binding properties of a protein, new research shows. As a result, computer modeling based solely on cryogenic structural data can produce misleading results.

Almost all crystallographic data sets collected for the determination of protein structures are obtained at cryogenic temperatures. The protein crystals are cryocooled with liquid nitrogen, and when a crystal is bombarded with X-rays, the low temperature conditions help limit X-ray damage, allowing users to collect high-resolution data sets with high completeness. Although it can cause more crystal damage, it is also possible to conduct experiments at room temperature. Many proteins are flexible, i.e. have dynamic structures, and certain conformational states may only be accessible at room temperature. Therefore, users may not be able to observe or correctly detect the dominant conformational state of a protein at natural biological temperatures when examining a protein under cryogenic conditions. In addition, if several protein conformations are possible, these can occur in different proportions at different temperatures.

Marcus Fischer from St. Jude Children’s Research Hospital in Tennessee, USA, and colleagues have now carried out the first systematic analysis of temperature artifacts in protein structures. “Maybe people are overinterpreting data that is actually cryogenic artifacts. Just looking at cryo and optimization … you can be misled. You just don’t know, ”he says.

Two protein structures

The team began its study by examining the T4 lysozyme L99A model system, which contains more than 700 structures in the Protein Data Bank (PDB). However, when collecting the largest temperature series to date of nine pairs of data sets at both cryo and room temperature, Fischer’s team observed a new apo-helix confirmation that is relevant for ligand binding. “That is what we are committed to here,” says Fischer. “Room temperature can provide additional insights into the conformational landscape that can be explored for ligand design and discovery.”

Computer modeling is critical to modern drug discovery and design, and in order for ligand docking studies to translate into successful drug discovery, it is critical that the starting protein model matches the in vivo target as closely as possible. By collecting 14 new high-resolution datasets and combining them with datasets from the PDB, the team was able to analyze nine pairs (at cryo and room temperature) of the T4 lysozyme L99A structure. They found that more than a third of the residues, most of which are located near the ligand binding site, responded to changes in temperature. They also examined ligand binding and occupancy in hundreds of data sets, which revealed new ligand binding poses at room temperature. The investigation was extended to four further protein classes and clearly shows that room temperature structures can provide information that cannot be recorded with cryogenic structures. Using the collected data as a starting point for modeling studies shows that alternative features can and perhaps should be carefully modeled rather than discarded as minor states.

‘A structure is a snapshot’

Those who base future experiments on crystallographic results should be aware of the limitations, ”comments Eleanor Dodson, professor emeritus at the University of York, UK, and expert in computational modeling of protein crystallography. “The results are influenced by temperature, crystallization conditions, radiation damage, and so on. A structure is a snapshot. Good modeling tools should take these results into account, but computational modeling must also deal with approximations. ‘

Cryoprotection pioneer Elspeth Garman of the University of Oxford, UK, says computer modeling tool developers should take note of this study: “PDB cryostructures will not be as productive a training set as room temperature structures. With the development of serial synchrotron crystallography and reliable data analysis software to merge data from many different crystals, room temperature crystallography is experiencing a renaissance that will expand the number of room temperature structures in the PDB and increase the room temperature structure Training set for AI-based computational modeling Tools. ‘

While this research highlights the disadvantages of using cryogenic structures, Keith Wilson, an expert in method development for protein crystallography at the University of York, UK, says that room temperature data acquisition is not always possible when studying biologically active macromolecules: “For the For most proteins, data collection at room temperature results in very rapid crystal death and a large number of crystals are required to record a complete data set. Room temperature data collection, including in plate situ techniques, has been applied to crystals for which cryogenic freezing has been shown to be persistent, such as: B. a number of membrane proteins. However, this in turn means that a large number of crystals are required for complete data. I would continue to view cryogenic data collection as the standard for the vast majority of projects. It is just too difficult to record room temperature data for most studies.

However, Fisher would like to encourage more researchers to try experiments at room temperature. “There’s not much to lose other than time and maybe a little money, but the knowledge you gain – it’s almost a new experiment. You just get so many new, different insights that it’s worth trying. “

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