An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

This sample holder can be used for growing crystals and collecting X-ray diffraction data. This sample holder was made for the use on 24-well plates which are widely used in the MX community. The very important aspect is that there is no manipulation needed.

A sample holder featuring these aspects described here did not exist before. Seeing something is always much better than just reading about it. By watching the video, a prospective user can grasp the features and intricacies of the new sample holder immediately.

To begin, create a clean and dust-free surface using a damp, lint-free cloth. Take one sample holder from its box and gently place it, yellow foil facing up, on the clean surface to avoid damage or unwanted puncture of the backside COC foil. Set up the crystallization drops on the yellow foil as it would be done on commonly used cover slides.

Place the drops gently to avoid any rupture or piercing of the foil using a pipette. On type 2 and type 3 sample holders, two drops are the recommended maximum. On a Type 1 sample holder, up to three drops can be placed.

Flip the sample holder over and place it onto a pre-greased cavity of a 24-well Linbro-style plate. Use the positioning aids of the sample holder to guide it to its optimal position. Ensure the correct position of the sample holder in order to avoid unwanted evaporation.

By placing the crystallization plate under a transmission-light microscope, without or without polarizers, monitor crystal growth without any disturbance of the experiment. Perform cryoprotection under a transmission light microscope. Gently pierce the outer COC foil using a fine canula.

Make sure the inner yellow foil remains untouched. The puncture should be right next to the drop that is to be manipulated. Insert a fine paper wick in the poked hole.

Carefully push the wick forward until it touches the yellow polyimide foil. Keep the wick in contact with the perforated foil. The wick will suck away all excess solution.

The time required for complete liquid removal depends on the viscosity of the solutions and the mother liquor composition. After all the liquid is sucked away, gently retract the paper wick. Remember the position of the drop, since it may not be visible after removal of the mother liquor.

Take a standard pipette to apply a small volume of cryo-protectant solution, using an extruded tip through the same hole. To reseal the self-healing COC foil, gently place a protected finger on the hole for about one second and slide it across the puncture. The slight pressure in combination with the elevated temperature will promote the resealing of punctures which are not too large.

Dissolve the ligand in mother liquor in the desired concentration in a reaction tube. Spin the solution for 10 minutes at 12, 000 times g in order to remove insoluble particles. Use a temperature-controlled centrifuge if needed.

Gently place a maximum of three microliters of ligand-containing solution in the gap between the COC foil and the polyimide film using a long, extruded pipette tip;retract the tip. To reseal the self-healing COC foil, gently place a protected finger on the hole for about one second and slide it across the puncture. Incubate the experiment for some time to allow for diffusion across the membrane.

The soaking time highly depends on the viscosity of the diffusing solution and its components. Repeat the ligand soaking steps multiple times to subsequently soak different ligands. To minimize solvent scattering, remove excess solution before data collection.

Ensure a stable, humidity-controlled beamline environment with pre-established conditions. To perform in situ diffraction data collection at ambient temperature, gently lift the transparent COC foil at the designated point using forceps and peel it off. Now gently lift the sample holder from its cavity and insert it immediately into a pre-prepared magnetic sample holder base.

No glue is necessary for this step. Apply gentle pressure to ensure the correct positioning of the sample holder within the base. For sample holders with a removable outer ring, apply gentle pressure by holding on to the outer ring and break it off at the designated break points.

The sample is now ready for centering and diffraction data collection. Mount the sample holder on a beamline goniometer and ensure correct positioning of the holder. Depending on goniometer geometry, the sample holder can be rotated by up to 160 degrees without causing any shadowing during the diffraction experiment.

Use a paper wick and gently touch the yellow polyimide foil from the backside to remove excess mother liquor. The sample is now ready for centering and diffraction data collection. Prepare for in situ diffraction data collection at cryogenic temperature as done for ambient temperature, then plunge the sample into liquid nitrogen and transfer it into a SPINE-compatible vial.

The geometry of the sample holder types 2 and 3 allows their transfer into standard SPINE sample vials, which can be used for robot-assisted sample mounting. The sample holder type 1 and type 2 have been designed to fit onto a well of a 24-well Linbro-style plate. Each individual sample holder contains positioning aids on either side of the outer rim in order to ensure optimal positioning on the rim of the well.

A 24-well Linbro-style plate was used with the type 1 sample holder to equilibrate a drop at 293 Kelvin against 500 microliters of mother liquor. Crystals of 40 to 50 microns were observed after five hours using a transmission light microscope with or without a polarizer. A sample holder with hen egg-white lysozyme crystals was placed in a humidity-controlled airstream on HZB-MX beamline 14.3.

A typical diffraction image is shown here. No elevated background scattering on the diffraction image can be detected. It is critical that contaminations of the polyimide foil must be avoided.

Further, it is important that the sample holder is positioned accurately and crystal manipulation carried out carefully. The sample holder allows the handling of very delicate crystals, one of the most important steps in micromolecular crystallography. Its use greatly decreases the chance of destroying the sample.