The HC1 Humidity control device


The controlled (or uncontrolled) dehydration of protein crystals has been shown to improve the diffraction qualities of many proteins.  Increases in diffraction limit, decreases in mosaic spread and changes in space group have been well documented.  The Humidity Control device (HC1) from the Diffraction instrumentation group at the EMBL provides an easy to use dehydration setup that can be used in conjunction with the world class MX beamlines at the ESRF.   Crystals are mounted on meshes (from Mitegen or MDL) and kept in a stream of humidified air from a modified cryostream nozzle.  Once crystals have been conditioned they can be immediately cryocooled by simply unmounting them with the sample changer.


Please use these references in any paper that reports or involves experiments performed with the HC1:

The HC1 device is described in detail in Sanchez-Weatherby et al. (2009) Acta Cryst. D65, 1237-1246, recent examples can be found in Russi et al. (2011) J. Struct. Biol. 175, 236-243 and a definitive description of the methods can be found here Bowler et al. (2015) Cryst. Growth Des, 15, 1043-1045


The HC1 can now be controlled through the beamline GUI MXCuBE using a workflow that links gradient design, data collection and analysis of changes during dehydration, see here for more information.

There are two HC1 devices available at the ESRF, please see here for booking information.


General procedure

The first step in these experiments is to define the relative humidity in equilibrium with the mother liquor of the system under study; this can often be quite time-consuming. In order to reduce the time spent on this stage of the experiment, the equilibrium relative humidity for a range of concentrations of the most commonly used precipitants has been measured. The relationship between the precipitant solution and equilibrium relative humidity is explained by Raoult's law for the equilibrium vapour pressure of water above a solution. The concentration of buffers, additives and detergents used will have a negligable effect on the RH in equilibrium with the mother liquor and is dominated by the primary precipitant.

For the concentrations of high molecular weight PEGs most commonly used, the starting point will be a RH of 99.5%. 

For PEGs, salts and other solutes the theoretical RH equilibria can be calculated based on buffer composition using these forms:

The measurement of the RH in equilibrium with common precipitant solutions and the equations that predict the RH equilibrium points are described in Wheeler, M.J. et al. (2012) Acta Cryst. F68, 111-114 and Bowler et al. (2015) Cryst. Growth Des, 15, 1043-1045




Figure 1. The HC1b Humidity Control Device. (a) The HC1b head mounted on a standard ESRF MX Beamline. (b) Schematic of the HC1b design. (c) GUI view. (d) The HC1b device installed in the experimental hutch of ID14-2.




Figure 2. Changes in X-ray diffraction of F1-ATPase crystals during dehydration.
A crystal was conditioned in four steps. Each of the four quadrants of the image
shows the diffraction pattern at one stable dehydration stage (1, 3, 4 and 5, where
the arc of the circle is at the following resolutions: 3 Å, 3.8 Å, 4 Å and 2.5 Å). The
inserts show a magnified view of the same area on the detector, demonstrating the
improvement in Bragg peak profile after dehydration.



Some useful dehydration links:


The Diffraction Instrumentation Group at the EMBL

The original dehydration device

The FMS from Proteros

Dehydration of F1-ATPase crystals

A review of crystal dehydration