Fragment-based screening is now well-established as a powerful approach to early drug ("lead") discovery.
A 60 sec data collection strategy has been implemented as the standard for all XChem samples regardless of crystal geometry and quality. A series of ongoing dose optimisation experiments are currently underway, with the first results for 'high resolution' crystals i.e. crystals that diffract to a resolution of 2.0Å or better in an XChem experiment detailed here. This initial dose reduction experiment has enabled us to increase the throughput of XChem samples and provide more appropriate data collection strategies to our users e.g. minimising radiation damage.
Further experiments will be performed on 'low resolution' crystals and the results willl be added to these webpages as the data is analysed.
Dose reduction experiments were performed on a P 21 21 21 crystal system with unit cell dimensions a=40, b=45, c=70, alpha/beta/gamma = 90, which routinely diffracts to 2.0Å (kindly provided by an XChem user).
Two rounds of dose reduction experiments were performed. The first was performed on 320 fragment-soaked crystals at 30sec and 60sec doses. The second experiment was performed using a subset of 32 fragments from the first round.
Dose Experiment Data collection strategies:
Ground state maps were generated from DMSO soaked crystals before analysing data from the fragment soaked crystals using the PanDDA algorithm to identify bound fragments.
Left to right: The ground state FWT PHWT at 1 rmsd for the 15sec (magenta mesh), 30s (blue mesh) and 60s (yellow mesh) doses, with DELFWT PHDELFWT at 3 rmsd in green/red mesh.
The same 8 fragments were identified in each of the three doses that were screened. Two of the fragments were identified at multiple sites within the crystal (fragments x627 and x628) and only a single additional fragment (x630) was identified in the 30sec and 60sec dose.
Below is an example of fragments that were identified at each dose. Event maps are depicted as blue mesh contoured at 1 rmsd (analogous to 2mFo-DFc map) and z-maps as green/red mesh contoured at +/- 3 rmsd (analogous to mFo-DFc map). Details of all the event/z-maps can be viewed in the Low Dose Supplementary Information here.
Below are box and whisker plots showing the data collection statistics for the 15sec (magenta), 30sec (blue) and 60sec (yellow) doses.
We recommend that users with the same or higher symmetry crystals, diffracting to 2.0Å or less should use a lower dose strategy, particularly if radiation damage is a concern. The data shows that there are no major differences in the quality of the maps and placement of ligands at lower doses can be done with the same confidence as those from the 60sec dose.
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