Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre.

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.

Ultrafast structural changes within a photosynthetic reaction centre.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography using an X-ray free-electron laser to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light.

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RC).

Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.

Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba -type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography.

Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA.

A three-dimensional movie of structural changes in bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side.

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography.

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes.

Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement.

Bacteriorhodopsin: Would the real structural intermediates please stand up?

Background: Bacteriorhodopsin (bR) is the simplest known light driven proton pump and has been heavily studied using structural methods: eighty four X-ray diffraction, six electron diffraction and three NMR structures of bR are deposited within the protein data bank. Twenty one X-ray structures report light induced structural changes and changes induced by mutation, changes in pH, thermal annealing or X-ray induced photo-reduction have also been examined.

Scope Of Review: We argue that light-induced structural changes that are replicated across several studies by independent research groups are those most likely to represent what is happening in reality.

Acyl transfer from membrane lipids to peptides is a generic process.

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol.

The innate reactivity of a membrane associated peptide towards lipids: acyl transfer to melittin without enzyme catalysis.

The innate reactivity of the peptide melittin (H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH(2)) towards membrane lipids has been explored using LC-MS methods. The high sensitivity afforded by LC-MS analysis enabled acyl transfer to the peptide to be detected, within 4 h, from membranes composed of phosphocholines (PCs). Acyl transfer from PCs was also observed from mixtures of PC with phosphoserine (PS) or phosphoglycerol (PG).