Femtosecond nanocrystallography using X-ray lasers for membrane protein structure determination

Abstract

The invention of free electron X-ray lasers has opened a new era for membrane protein structure determination with the recent first proof-of-principle of the new concept of femtosecond nanocrystallography. Structure determination is based on thousands of diffraction snapshots that are collected on a fully hydrated stream of nanocrystals. This review provides a summary of the method and describes how femtosecond X-ray crystallography overcomes the radiation-damage problem in X-ray crystallography, avoids the need for growth and freezing of large single crystals while offering a new method for direct digital phase determination by making use of the fully coherent nature of the X-ray beam. We briefly review the possibilities for time-resolved crystallography, and the potential for making 'molecular movies' of membrane proteins at work.

Figure 1

Principle of the experimental setup used for femtosecond crystallography.. The stream of crystals is provided by the injector with the gas focusing nozzle. The femtosecond X-ray beam intersect with the continous crystal stream; diffraction patterns are recorded with the frequency of the Free Electron Laser at LCLS (60 or 120 Hz) using either a one or two CCD detector setup. In the two detector setup, which was used in the CAMP camber at the AMO beamline at LCLS for the first fs crystallography experiments reported in [•• 24], the high order reflections are detected on the front detector. The back detector, which records the low order reflections, allows the resolution of the shape transforms of the reflections. The figure has been modified after [•• 24].

Figure 2

Example of a diffraction pattern from photosystem I which shows the shape transforms. Note the fringes between the Bragg peaks which indicate the number of unit cells in that direction. Thus the evaluation of the shape transforms can be used to determine the size of the crystals [•• 24], and for phase determination [•• 38]. This Figure has been modified after [•• 24].

Figure 3

Electron density map of Photosystem I at 8 Åresolution, derived by fs crystallography. The phases were obtained using molecular replacement as described in [•• 24]. The section of the electron density in this picture shows transmembrane helices of the large PSI subunits PsaA, PsaB as well as helices of PsaK and PsaF. This Figure has been modified after [•• 25].