This workflow tool allows the design of dehydration protocols that are then coupled to data collection and on-line data analysis through MXCuBE. The dehydration of crystals of macromolecules has long been known to have the potential to increase their diffraction quality.  A number of methods exist to change the relative humidity that surrounds crystals, but for reproducible results, with complete characterisation of the changes induced, a precise humidity control device coupled with an X-ray source is required. The EMBL Grenoble outstation has developed a humidity control device, the HC1, that is available on the ESRF MX beamlines (Sanchez-Weatherby et al. (2009) Acta Cryst. D65, 1237-1246, Russi et al. (2011) J. Struct. Biol. 175, 236-243), and many systems have shown major improvements in diffraction quality using the device. For details on how to perform these experiments please see here.

 

Contact Matthew Bowler for help.

 

The workflow allows dehydration protocols to be designed and launched in MXCuBE coupled to data collection and data analysis. The integration allows experiments to be standardised and performed in a highly reproducible manner. Charts of the results are provided automatically.

 

The workflow is started by selecting the "Dehydration" workflow in the Advanced tab and adding it to the queue. The user then selects the starting relative humidity (this can be determined from these equations) and a dehydration protocol is also defined - the key factors are the step decrease in RH and the equilibration time between each step.  We generally recommend for an initial experiment a step size of 0.5-1% RH and an equilibration time of 5 minutes.  Once changes have been characterised these parameters must be adapted to the system under study.

 

mxcube_WF_111.png

 

Once the gradient has been defined the process is launched. First an image is collected - this is important as the initial diffraction quality of each crystal must be assessed at the beginning of each experiment. At this point the gradient is launched and the user is given the options to continue automatically (recommended to prevent clicks for each dehydration step) and whether to collect data or not - this option is useful when a protocol is known but radiation damage needs to be avoided, see below.  At the end of this gradient these options will again be presented allowing users to move to a new RH and to collect data or not.  This allows multiple rounds of dehydration with or without data collection and can include gradients that increase the RH as well. If the experiment is finished the process can be ended. Graphs can be viewed by double clicking a data collection in the queue:

 

HC1_112.png

 

Images are stored in a sub-directory call /dehydration_### and the results of processing are stored here as well.  This is in the form of graphs that are produced at each step and updated as new results are obtained, some examples are shown below as well as a video of the process:

 

 

 

 

Dehydration chart3

Dehydration chart4