Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser.

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Diffraction by a frustrated system: the triangular Ising antiferromagnet.

Expressions are derived for diffraction by the triangular Ising antiferromagnet, a disordered lattice system consisting of two kinds of scatterer and exhibiting geometric frustration. Analysis of the expressions shows characteristics of the diffraction patterns, including the presence of Bragg and diffuse diffraction, superlattice reflections, and their behavior with temperature. These characteristics are illustrated by numerical simulations. The results have application to diffraction imaging of disordered systems.

Determination of myosin filament orientations in electron micrographs of muscle cross sections.

An automated image analysis system for determining myosin filament azimuthal rotations, or orientations, in electron micrographs of muscle cross sections is described. The micrographs of thin sections intersect the myosin filaments which lie on a triangular lattice. The myosin filament profiles are variable and noisy, and the images exhibit a variable contrast and background. Filament positions are determined by filtering with a point spread function that incorporates the local symmetry of the lattice. Filament orientations are determined by correlation with a template that incorporates the salient filament characteristics, and the orientations are classified using a Gaussian mixture model. The precision of the technique is assessed by application to a variety of micrographs and comparison with manual classification of the orientations. The system provides a convenient, robust, and rapid means of analysing micrographs containing many filaments to study the distribution of filament orientations.

Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1.

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Šresolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.