A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Variability in X-ray induced effects in [Rh(COD)Cl]2 with changing experimental parameters.

X-ray characterisation methods have undoubtedly enabled cutting-edge advances in all aspects of materials research. Despite the enormous breadth of information that can be extracted from these techniques, the challenge of radiation-induced sample change and damage remains prevalent. This is largely due to the emergence of modern, high-intensity X-ray source technologies and the growing potential to carry out more complex, longer duration in situ or in operando studies. The tunability of synchrotron beamlines enables the routine application of photon energy-dependent experiments. This work explores the structural stability of [Rh(COD)Cl]2, a widely used catalyst and precursor in the chemical industry, across a range of beamline parameters that target X-ray energies of 8 keV, 15 keV, 18 keV and 25 keV, on a powder X-ray diffraction synchrotron beamline at room temperature. Structural changes are discussed with respect to absorbed X-ray dose at each experimental setting associated with the respective photon energy. In addition, the X-ray radiation hardness of the catalyst is discussed, by utilising the diffraction data collected at the different energies to determine a dose limit, which is often considered in protein crystallography and typically overlooked in small molecule crystallography. This work not only gives fundamental insight into how damage manifests in this organometallic catalyst, but will encourage careful consideration of experimental X-ray parameters before conducting diffraction on similar radiation-sensitive organometallic materials.

Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases.

Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

The HARE chip for efficient time-resolved serial synchrotron crystallography.

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV =O Formation.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising 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 (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply 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.

Direct measurement of X-ray-induced heating of microcrystals.

Temperature control is a key aspect of macromolecular crystallography, with the technique of cryocooling routinely being used to mitigate X-ray-induced damage. Beam-induced heating could cause the temperature of crystals to rise above the glass transition temperature, greatly increasing the rate of damage. X-ray-induced heating of ruby crystals of 20-40 µm in size has been quantified non-invasively by monitoring the emission wavelengths of X-ray-induced fluorescence during exposure to the X-ray beam. For the beam sizes and dose rates typically used in macromolecular crystallography, the temperature rises are of the order of 20 K. The temperature changes observed are compared with models in the literature and can be used as a validation tool for future models.

Dynamic adaptive X-ray optics. Part II. High-speed piezoelectric bimorph deformable Kirkpatrick-Baez mirrors for rapid variation of the 2D size and shape of X-ray beams.

The tangential curvature of actively bent X-ray mirrors at synchrotron radiation and X-ray free-electron laser (XFEL) facilities is typically only changed every few hours or even days. This operation can take tens of minutes for active optics with multiple bending actuators and often requires expert guidance using in situ monitoring devices. Hence, the dynamic performance of active X-ray optics for synchrotron beamlines has historically not been exploited. This is in stark contrast to many other scientific fields. However, many areas of synchrotron radiation and XFEL science, including macromolecular crystallography, could greatly benefit from the ability to change the size and shape of the X-ray beam rapidly and continuously. The advantages of this innovative approach are twofold: a large reduction in the dead time required to change the size of the X-ray beam for different-sized samples and the possibility of making multiple changes to the beam during the measurement of a single sample. In the preceding paper [Part I; Alcock, Nistea, Signorato & Sawhney (2019), J. Synchrotron Rad. 26, 36-44], which accompanies this article, high-speed visible-light Fizeau interferometry was used to identify the factors which influence the dynamic bending behaviour of piezoelectric bimorph deformable X-ray mirrors. Building upon this ex situ metrology study, provided here is the first synchrotron radiation beamline implementation of high-speed adaptive X-ray optics using two bimorphs operating as a Kirkpatrick-Baez pair. With optimized substrates, novel opto-mechanical holders and a next-generation high-voltage power supply, the size of an X-ray beam was rapidly and repeatedly switched in <10 s. Of equal importance, it is also shown that compensation of piezoelectric creep ensures that the X-ray beam size remains stable for more than 1 h after making a major change. The era of high-speed adaptive X-ray optics for synchrotron radiation and XFEL beamlines has begun.

Reducing sample consumption for serial crystallography using acoustic drop ejection.

Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.

Time-resolved crystallography using the Hadamard transform.

We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy.

Current advances in synchrotron radiation instrumentation for MX experiments.

Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualisation and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.

Exploiting Microbeams for Membrane Protein Structure Determination.

A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 - http://blanco.biomol.uci.edu/mpstruc ). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography.

A modular and compact portable mini-endstation for high-precision, high-speed fixed target serial crystallography at FEL and synchrotron sources.

The design and implementation of a compact and portable sample alignment system suitable for use at both synchrotron and free-electron laser (FEL) sources and its performance are described. The system provides the ability to quickly and reliably deliver large numbers of samples using the minimum amount of sample possible, through positioning of fixed target arrays into the X-ray beam. The combination of high-precision stages, high-quality sample viewing, a fast controller and a software layer overcome many of the challenges associated with sample alignment. A straightforward interface that minimizes setup and sample changeover time as well as simplifying communication with the stages during the experiment is also described, together with an intuitive naming convention for defining, tracking and locating sample positions. The setup allows the precise delivery of samples in predefined locations to a specific position in space and time, reliably and simply.

Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding.

Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.

Exploiting fast detectors to enter a new dimension in room-temperature crystallography.

A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.

Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.

Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin.

Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases.

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys â†’ T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.

The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.