Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Processing of X-ray snapshots from crystals in random orientations.

A functional expression is introduced that relates scattered X-ray intensities from a still or a rotation snapshot to the corresponding structure-factor amplitudes. The new approach was implemented in the program nXDS for processing monochromatic diffraction images recorded by a multi-segment detector where each exposure could come from a different crystal. For images containing indexable spots, the intensities of the expected reflections and their variances are obtained by profile fitting after mapping the contributing pixel contents to the Ewald sphere. The varying intensity decline owing to the angular distance of the reflection from the surface of the Ewald sphere is estimated using a Gaussian rocking curve. This decline is dubbed `Ewald offset correction', which is well defined even for still images. Together with an image-scaling factor and other corrections, an explicit expression is defined that predicts each recorded intensity from its corresponding structure-factor amplitude. All diffraction parameters, scaling and correction factors are improved by post-refinement. The ambiguous case of a lower point group than the lattice symmetry is resolved by a method reminiscent of the technique of `selective breeding'. It selects the indexing alternative for each image that yields, on average, the highest correlation with intensities from all other images. Processing a test set of rotation images by XDS and treating the same images by nXDS as snapshots of crystals in random orientations yields data of comparable quality, clearly indicating an anomalous signal from Se atoms.

XDS.

The usage and control of recent modifications of the program package XDS for the processing of rotation images are described in the context of previous versions. New features include automatic determination of spot size and reflecting range and recognition and assignment of crystal symmetry. Moreover, the limitations of earlier package versions on the number of correction/scaling factors and the representation of pixel contents have been removed. Large program parts have been restructured for parallel processing so that the quality and completeness of collected data can be assessed soon after measurement.

Integration, scaling, space-group assignment and post-refinement.

Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-reduction package XDS are explained, including the new features of automatic determination of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the determination of correction/scaling factors.

Viscous hydrophilic injection matrices for serial crystallography.

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates - gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro-gels as viscous injection matrices is described, namely sodium carb-oxy-methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.

Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C.

Glutarylamidase is an important enzyme employed in the commercial production of 7-aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7-aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two-step enzymatic process utilizing the enzymes D-amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7-aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 A resolution and with bound glycerol to 2.4 A. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side-chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the alpha-amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the O(gamma) through a hydrogen bond relay network involving the alpha-amino group of the serine and a bound water molecule. A plausible model of the enzyme-substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid.

Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser.

X-ray free-electron lasers deliver intense femtosecond pulses that promise to yield high resolution diffraction data of nanocrystals before the destruction of the sample by radiation damage. Diffraction intensities of lysozyme nanocrystals collected at the Linac Coherent Light Source using 2 keV photons were used for structure determination by molecular replacement and analyzed for radiation damage as a function of pulse length and fluence. Signatures of radiation damage are observed for pulses as short as 70 fs. Parametric scaling used in conventional crystallography does not account for the observed effects.

X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA. How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage.

The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA. With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage. The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration. The structure shows CoA in a syn conformation awaiting pyruvate cleavage. By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage.

A new view of the mechanisms of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) derived from X-ray structures of their tetrahedral reaction intermediate states.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate-3-phosphate synthase (AroA) constitute the small enzyme family of enolpyruvyl transferases, which catalyze the chemically unusual reaction of enolpyruvyl transfer. MurA catalyzes the first step in the biosynthesis of the bacterial cell wall; AroA is the sixth enzyme of the shikimate pathway leading to the synthesis of aromatic compounds in numerous microorganisms and plants. Because both metabolic pathways are absent from mammals but essential for the growth of microorganisms, MurA and AroA are attractive targets for the development of novel antimicrobial drugs. We have determined the x-ray structures of the D305A mutant of Enterobacter cloacae MurA and the D313A mutant of Escherichia coli AroA, both of which crystallized in the presence of their substrates. The structures depict the tetrahedral reaction intermediate states of the enzymes and prove that, without the aspartate side chain, the overall addition-elimination reaction in both enzymes is halted after the addition step. The presented structures lead to a new view of the catalytic mechanism and, moreover, provide an ideal starting point for the rational design of potent inhibitors of MurA and AroA.

X-ray structure of glutathione S-transferase from the malarial parasite Plasmodium falciparum.

GSTs catalyze the conjugation of glutathione with a wide variety of hydrophobic compounds, generally resulting in nontoxic products that can be readily eliminated. In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one GST isoenzyme (PfGST). This GST is highly abundant in the parasite, its activity was found to be increased in chloroquine-resistant cells, and it has been shown to act as a ligandin for parasitotoxic hemin. Thus, the enzyme represents a promising target for antimalarial drug development. We now have solved the crystal structure of PfGST at a resolution of 1.9 A. The homodimeric protein of 26 kDa per subunit represents a GST form that cannot be assigned to any of the known GST classes. In comparison to other GSTs, and, in particular, to the human isoforms, PfGST possesses a shorter C-terminal section resulting in a more solvent-accessible binding site for the hydrophobic and amphiphilic substrates. The structure furthermore reveals features in this region that could be exploited for the design of specific PfGST inhibitors.