Femtosecond Nanocrystallography and Characterization of Photosystem II

Membrane proteins are extremely difficult to crystallize, however they are highly important proteins for cellular function. Photosystem I, one of the most complex membrane proteins solved to date took more than a decade to have a structure solved to molecular resolution. Large, well-ordered crystal growth is one of the major bottlenecks in structural determination by x-ray crystallography, due to the difficulty of making the "perfect" crystal. The development of femtosecond nanocrystallography, which uses a stream of fully hydrated nanocrystals to collect diffraction snapshots, effectively reduces this bottleneck[1] Photosystem II changed our biosphere via splitting water and evolving oxygen 2.5 billion years ago. Using femtosecond nanocrystallography we are developing a time-resolved femtosecond crystallography method [2] to unravel the mechanism of water splitting by determining the conformational changes that take place during the oxygen evolution process. Multiple crystallization techniques were originally developed in order to make the nanocrystals necessary for femtosecond nanocrystallography. For Photosystem II nano/microcrystals a free interface diffusion method, is used to increase yield over traditional methods. These crystals are then characterized by three different methods before being used for collecting diff raction data. The three methods currently used are optical microscopy, dynamic light scattering (DLS), and Second Order Nonlinear Imaging of Chiral Crystals (SONICC).

Detection of protein nanocrystals based on the reversibility of crystallization

A prerequisite for conventional X-ray protein structure analysis is the growth of crystals with a sufficient size in the range of several µm. This is a time consuming and not always successful process, challenging especially when working with membrane proteins. The recently developed technique of femtosecond X-ray crystallography enables structure analysis of crystals with a size in the nm range, thus the process of growing large single crystals can be avoided. Moreover femtosecond X-ray nanocrystallography is a potential method to overcome the radiation damage problem and to perform time-resolved structure analysis (1, 2). Nanocrystals are too small to be detected with an optical microscope, hence crystal growth cannot be monitored with common methods used in crystallography. A powerful technique to screen for nanocrystals is Second Order Nonlinear Imaging of Chiral Crystals (SONICC). This method is based on the principle of second harmonic generation and detects noncentrosymmetric ordered crystals (3). However, the instrumentation is not generally accessible yet. In addition, proteins ordered in higher symmetry crystal classes do not necessarily lead to a positive SONICC signal. In this work, a new method is developed to screen for nanocrystals based on the reversibility of crystallization. We show that dilution experiments performed with a crystallization robot and monitored by a crystallization imaging system enable the distinction between precipitation comprised of nanocrystals and precipitation caused by aggregated protein.