Search

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 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

A crystal structure of a photolyase at room temperature confirms the structural information obtained from cryogenic crystallography and paves the way for time-resolved studies of the photolyase at an X-ray free-electron laser., (6–4) photolyases are flavoproteins that belong to the photolyase/cryptochrome family. Their function is to repair DNA lesions using visible light. Here, crystal structures of Drosophila melanogaster (6–4) photolyase [Dm(6–4)photolyase] at room and cryogenic temperatures are reported. The room-temperature structure was solved to 2.27 Å resolution and was obtained by serial femtosecond crystallography (SFX) using an X-ray free-electron laser. The crystallization and preparation conditions are also reported. The cryogenic structure was solved to 1.79 Å resolution using conventional X-ray crystallo­graphy. The structures agree with each other, indicating that the structural information obtained from crystallography at cryogenic temperature also applies at room temperature. Furthermore, UV–Vis absorption spectroscopy confirms that Dm(6–4)photolyase is photoactive in the crystals, giving a green light to time-resolved SFX studies on the protein, which can reveal the structural mechanism of the photoactivated protein in DNA repair.

Biliverdin IXα (BV) binds to several prokaryotic and eukaryotic proteins. How nature exploits the versatility of BV's properties is not fully understood. Unlike free BV, the Sandercyanin fluorescent protein bound to BV (SFP-BV) shows enhanced red fluorescence (675 nm) on excitation in the UV region (380 nm). Site-directed mutagenesis showed that the BV complex of two SFP variants, F55A and E79A, resulted in the loss of red fluorescence. Crystal structures of the complexes of these proteins with BV show the absence of stacking interactions of the F55 phenyl ring with BV. BV changes from ZZZssa conformation in the wild-type to ZZZsss conformation in the variants. In the nonfluorescent mutants, the lowest excited state is destabilized, resulting in nonradiative decay.

A novel method is presented to screen for suitable crystallization conditions and produce large amounts of microcrystals of membrane proteins in lipidic cubic phase for serial crystallography experiments., Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

The enzyme N-acetylmannosamine kinase (NanK) catalyzes the second step of the bacterial sialic acid catabolic pathway. Here, the structure of F. nucleatum NanK is presented at 2.23 Å resolution., Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5′-triphos­phate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.