Humidity control device (HC1)
Synopsis
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.
The device is fully described in Sanchez-Weatherby et al. (2009) Acta Cryst. D65, 1237-1246, and recent examples are described in Russi et al. (2011) J. Struct. Biol. 175, 236-243, please use these references to cite the device in any paper that reports or involved experiments performed with the HC1b.
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 using the forms here. 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.
Figure 1. The HC1b Humidity Control Device. a; The HC1b head mounted
on a standard ESRF MX Beamline. All standard elements are in place, including
an open MiniKappa. Red tape shows the vertical movement required in the
cryo-mount to accommodate the new head. b; Schematic of the HC1b design. c;
Zoomed viewof the sample environment showing the spatial constraints that constrained the design of the nozzle head (note the beamstop and collimating aperture are not shown). d, The 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 4 steps. Each of the four quadrants of the image
show the diffraction pattern at each 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.
After a year of access to the device there are now several examples of systems that have benefited from the device.
The device is fully described in Sanchez-Weatherby et al. (2009) Acta Cryst. D65, 1237-1246, please use this reference to cite the device in any paper that reports or involved experiments performed with the HC1b.
Protein Crystal Dehydration links
The Diffraction Instrumentation Group at the EMBL
The original dehydration device
Dehydration of F1-ATPase crystals
A review of crystal dehydration