For information on how to prepare and ship un-frozen samples, please see the instruction manual on Remote Access at Elevated Temperatures and Controlled Humidity.

A crystal dehydration experiment may improve the diffraction resolution of ill-behaved macromolecular crystals. During these experiments, crystals are equilibrated at decreasing values of relative humidity (RH) in a stepwise fashion. Diffraction images are collected at each step to compare the diffraction quality and resolution.

The Arinax HC-lab is a scientific instrument designed to regulate the relative humidity of macromolecules crystals while mounted on the goniometer. Installed in the same way as a standard cryogenic stream head, the HC-lab blows a controlled humid air stream onto the sample. The air stream is generated by using the Dew Point Method to remove water from a hot and saturated air stream so that it will be at the correct RH when it reaches the crystal. The device provides a user-friendly sample environment to collect data at room temperature without the need of capillary mounting. It also enables crystal dehydration experiments with the aim of improving crystal diffraction.

The HC-lab comprises the machine body connected via a 1.5 m tube to a nozzle, which points the humid stream onto the sample (Figure 1).

Any RH value between 99.8% and 50.0% can be set using the Humidity control in the Blu-ice Sample Tab interface (Figure 2). If you cannot see the humidity control options, ask your user-support scientist to enable humidity control for your experiment. Once the RH value is set, wait until the air stream reaches this value and it is stable

The green area displays the current relative humidity. To change the set point, type a value in the entry box (which will be in red text) then click “Move” to change in set point (the “set point” text will then turn black).

Further integration of the HC-lab control software into the Blu-Ice software is in progress.

Regardless the type of experiment you are planning, the first step is to measure or estimate the RH value of your crystallization solution.

1. Center an empty loop perpendicular to the plane of the image and put a drop of the mother liquor (crystallization condition) on it.
2. Observe the drop size behavior: if the drop size increases/decreases the mother liquor RH is below/above the humid flow RH value, respectively.
3. Adjust the RH set point until the drop size remains stable in time. This can take several minutes. Once the drop size is stable, the mother liquor and the humid flow have the same RH value. This RH value is the starting point for RT data collection or dehydration experiments.

1. An estimate of the the RH Starting Point value can be based on the composition of the crystallization condition. The RH values for the most common precipitants and cryo-protectants have been empirically measured in this Article.
2. For a more accurate estimate, ESRF has developed a Series of Equations that can be used to determine RH Starting Point values for your experiment.

Protocol for Crystal Dehydration Experiments Optimizing Diffraction Resolution

1. Set the RH value to the appropriate starting point.
2. Mount and center a crystal.
3. It is best if any excess of mother liquor around the crystal is removed with a paper wick.
4. Collect a diffraction image to characterize the initial state (space group, unit cell, mosaicity, etc).
5. Decrease the RH value by steps of 1%. At each RH step let the crystal reach the equilibrium. It takes about 5 min to stabilize the system (reaching the set point, stabilizing the drop size).
6. Collect a diffraction image in order to compare it with the previous ones.
7. In case of improved diffraction quality further optimization may be necessary, e.g. chose smaller step sizes for the RH set points.

Complete datasets may be collected from crystals at optimized humidity. Multiple crystals may be required.

Alternatively, a new rapid nozzle switcher has been recently developed at SSRL that will be installed on some of the crystallography beamlines. Using this device, crystals at optimized humidity can be flash-frozen to collect complete datasets.