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We determine the number of authentic reaction intermediates in the later stages of the photocycle of photoactive yellow protein at room temperature, their atomic structures, and a consistent set of chemical kinetic mechanisms, by analysis of a set of time-dependent difference electron density maps spanning the time range from 5 micros to 100 ms. The(More)
Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of(More)
Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a(More)
Singular value decomposition (SVD) separates time-dependent crystallographic data into time-independent and time-dependent components. Procedures for the effective application of SVD to time-resolved macromolecular crystallographic data have yet to be explored systematically. Here, the applicability of SVD to experimental crystallographic data is tested by(More)
Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not(More)
In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic(More)
We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high(More)
The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein(More)
Time-resolved (TR) crystallography is a unique method for determining the structures of intermediates in biomolecular reactions. The technique reached its mature stage with the development of the powerful third-generation synchrotron X-ray sources, and the advances in data processing and analysis of time-resolved Laue crystallographic data. A time(More)
The first experimental data from single-particle scattering experiments from free electron lasers (FELs) are now becoming available. The first such experiments are being performed on relatively large objects such as viruses, which produce relatively low-resolution, low-noise diffraction patterns in so-called "diffract-and-destroy" experiments. We describe a(More)