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

Christian G Feiler,Dirk Wallacher,Manfred S Weiss

doi:10.3791/59722

Abstract

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

Highlights

The knowledge of the three-dimensional structure of biological macromolecules constitutes an important cornerstone in all basic biological, biochemical and biomedical research
There are ongoing efforts to use capillaries, in particular from counter-diffusion crystallization experiments, for low-temperature diffraction work[7,8], but, irrespective of that, it became the standard approach in macromolecular crystallography to mount macromolecular crystals held by a thin film of mother liquor inside a thin wired loop[9,10]
For sample holders of types 2 and 3, it is recommended to set a maximum of two drops of maximum volume 2 μL each. 24 sample holders can be fitted onto one 24well Linbro plate (Figure 3D)

Summary

Introduction

The knowledge of the three-dimensional structure of biological macromolecules constitutes an important cornerstone in all basic biological, biochemical and biomedical research. The major prerequisite of X-ray crystallography, which is at the same time its major limitation, is that diffraction-quality crystals have to be produced and prepared for the diffraction experiment This step still constitutes one of the major bottlenecks of the method. There are ongoing efforts to use capillaries, in particular from counter-diffusion crystallization experiments, for low-temperature diffraction work[7,8], but, irrespective of that, it became the standard approach in macromolecular crystallography to mount macromolecular crystals held by a thin film of mother liquor inside a thin wired loop[9,10]. It may be safely stated that most diffraction data collections on macromolecular crystals nowadays still rely on this approach[5]

Methods

Results

Conclusion