The development of acoustic levitation for time resolved protein crystallography experiments at XFELS

P. Docker, R. Morris, M. Newton, J. Kay, J. Beale, D. Axford, A. Orville, D. Stuart
Diamond light Source,
United Kingdom

Keywords: acoustic levitation, XFE, L X-ray diffraction, time resolved


This body of work is building on the systems described in a recent publication by Tsujino and Tomizaki 1. They describe the application of acoustic levitation to support protein crystals mounting for X-ray diffraction experiments carried out at synchrotron light sources. For clarity, a typical acoustic levitation system generates intense acoustic waves which are reflected back from a mirroring surface set at a distance matched to the frequency of sound being used. The reflected acoustic waves generate locations of minimum pressure or ‘nodes’ which can position micron sized particles or droplets containing protein crystals. Multiple nodes can be generated and the nodes can also be made to process. These levitating drops provide a number of significant benefits to current sample mounting methodologies. Beam attenuation and shadowing present in physical mounts is eliminated. The method is also enormously applicable to X-ray free electron laser (XFEL) light sources. XFEL sources, such as the LCLS in Stanford, California produce light approximately ten orders of magnitude greater than 3rd generation synchrotrons. XFEL light destroys physical sample mounts which come into contact with the beam. The levitating drops therefore provide a method to reliably deliver sample to a specific location for beam illumination. We are using this technology to develop a sample delivery mechanism for conducting time-resolved X-ray crystallographic experiments at XFEL light sources. A significant barrier in conducting these experiments is currently co-ordinating the localisation of a crystal, the mixing of the substrate with the crystal and then illuminating the crystal with X-rays. In our system we propose to suspend a crystal in a levitating drop and then ‘fire’ a drop of substrate solution into the crystal prior to X-ray radiation. We are currently using this system to aid our understanding of the Stenotrophomonas maltophilia beta-lactamase reaction mechanism which catalyse the hydrolysis of beta-lactam antibiotics. These proteins are ideal for this task as several substrates undergo a colorimetric change during catalysis so the X-ray diffraction data can be obtained whilst monitoring the activity of the enzyme. The principle challenge this work is aiming to overcome is the reduction in crystal size the system can support as smaller protein crystals allow for quicker substrate diffusion and therefore more coherent diffraction data. The size of the trapping potential has a direct relationship to the frequency of the acoustics and increasing this is not trivial. We are also developing the system to introduce the substrate to the crystal droplet using induction based microfluidics. Overcoming these challenges will create a method to facilitate time-resolved crystallography experiments and will enable the unlocking of many of natures secrets. References 1 Ultrasonic acoustic levitation for fast frame rate X ray protein crystallography at room temperature, S Tsujino, T Tomizaki, Scientific reports (nature), may 2016