Numerical calculation of strong-field laser-atom interaction: An approach with perfect reflection-free radiation boundary conditions

The time-dependent, single-particle Schrodinger equation with a finite-range potential is solved numerically on a three-dimensional spherical domain. In order to correctly account for outgoing waves, perfect reflection-free radiation boundary conditions are used on the surface of a sphere. These are computationally most effective if the particle wavefunction is expanded in the set of spherical harmonics and computations are performed in the Kramers-Henneberger accelerated frame. The method allows one to solve the full ionization dynamics in intense laser fields within a small region of atomic dimensions

Structure determination of an integral membrane protein at room temperature from crystals in situ

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

Serial Femtosecond Crystallography Reveals the Role of Water in the One- or Two-Electron Redox Chemistry of Compound i in the Catalytic Cycle of the B-Type Dye-Decolorizing Peroxidase DtpB

Controlling the reactivity of high-valent Fe(IV)-O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)-OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites

Serial femtosecond zero dose crystallography captures a water‐free distal heme site in a dye‐decolourising peroxidase to reveal a catalytic role for an arginine in FeIV=O formation

Obtaining structures of intact redox states of metal centres derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolourising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues, aspartate and arginine, in the heterolysis of peroxide to form the catalytic intermediate compound I (Fe IV =O and a porphyrin cation radical). Using serial femtosecond X‐ray (SFX) crystallography, we have determined the pristine structures of the Fe III and Fe IV =O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site, which together with the presence of an asparagine, infer the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis

Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein

Autotaxin, a four-domain ∼100 kDa mammalian glycoprotein, was expressed in stably transfected mammalian cells, purified from the medium and crystallized. Diffraction data from micrometre-thick crystal plates were collected on various European synchrotron beamlines and are presented and analysed

The design of macromolecular crystallography diffraction experiments

Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented

Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins

An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner

High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX

Non-contact universal sample presentation for room temperature macromolecular crystallography using acoustic levitation

Macromolecular Crystallography is a powerful and valuable technique to assess protein structures. Samples are commonly cryogenically cooled to minimise radiation damage effects from the X-ray beam, but low temperatures hinder normal protein functions and this procedure can introduce structural artefacts. Previous experiments utilising acoustic levitation for beamline science have focused on Langevin horns which deliver significant power to the confined droplet and are complex to set up accurately. In this work, the low power, portable TinyLev acoustic levitation system is used in combination with an approach to dispense and contain droplets, free of physical sample support to aid protein crystallography experiments. This method facilitates efficient X-ray data acquisition in ambient conditions compatible with dynamic studies. Levitated samples remain free of interference from fixed sample mounts, receive negligible heating, do not suffer significant evaporation and since the system occupies a small volume, can be readily installed at other light sources