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Most X-ray sources are inherently polychromatic. Polychromatic ("pink") X-rays provide an efficient way to conduct diffraction experiments as many more photons can be used and large regions of reciprocal space can be probed without sample rotation during exposure-ideal conditions for time-resolved applications. Analysis of such data is complicated, however, causing most X-ray facilities to discard >99% of X-ray photons to obtain monochromatic data. Key challenges in analyzing polychromatic diffraction data include lattice searching, indexing and wavelength assignment, correction of measured intensities for wavelength-dependent effects, and deconvolution of harmonics. We recently described an algorithm, Careless, that can perform harmonic deconvolution and correct measured intensities for variation in wavelength when presented with integrated diffraction intensities and assigned wavelengths. Here, we present Laue-DIALS, an open-source software pipeline that indexes and integrates polychromatic diffraction data. Laue-DIALS is based on the dxtbx toolbox, which supports the DIALS software commonly used to process monochromatic data. As such, Laue-DIALS provides many of the same advantages: an open-source, modular, and extensible architecture, providing a robust basis for future development. We present benchmark results showing that Laue-DIALS, together with Careless, provides a suitable approach to the analysis of polychromatic diffraction data, including for time-resolved applications.
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Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.
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DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.
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A major goal in biomedical science is to move beyond static images of proteins and other biological macromolecules to the internal dynamics underlying their function. This level of study is necessary to understand how these molecules work and to engineer new functions and modulators of function. Stemming from a visionary commitment to this problem by Keith Moffat decades ago, a community of structural biologists has now enabled a set of x-ray scattering technologies for observing intramolecular dynamics in biological macromolecules at atomic resolution and over the broad range of timescales over which motions are functionally relevant. Many of these techniques are provided by BioCARS, a cutting-edge synchrotron radiation facility built under Moffat leadership and located at the Advanced Photon Source at Argonne National Laboratory. BioCARS enables experimental studies of molecular dynamics with time resolutions spanning from 100 ps to seconds and provides both time-resolved x-ray crystallography and small- and wide-angle x-ray scattering. Structural changes can be initiated by several methods-UV/Vis pumping with tunable picosecond and nanosecond laser pulses, substrate diffusion, and global perturbations, such as electric field and temperature jumps. Studies of dynamics typically involve subtle perturbations to molecular structures, requiring specialized computational techniques for data processing and interpretation. In this review, we present the challenges in experimental macromolecular dynamics and describe the current state of experimental capabilities at this facility. As Moffat imagined years ago, BioCARS is now positioned to catalyze the scientific community to make fundamental advances in understanding proteins and other complex biological macromolecules.
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A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Time-resolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and co-flow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein.
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Proteínas , Síncrotrons , Raios X , Proteínas/química , Radiografia , Fótons , Difração de Raios XRESUMO
Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.
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Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.
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Fitocromo/química , Fitocromo/metabolismo , Stigmatella aurantiaca/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Phytochromes (PHYs) are photoreceptor proteins first discovered in plants, where they control a variety of photomorphogenesis events. PHYs as photochromic proteins can reversibly switch between two distinct states: a red light (Pr) and a far-red light (Pfr) absorbing form. The discovery of Bacteriophytochromes (BphPs) in nonphotosynthetic bacteria has opened new frontiers in our understanding of the mechanisms by which these natural photoswitches can control single cell development, although the role of BphPs in vivo remains largely unknown. BphPs are dimeric proteins that consist of a photosensory core module (PCM) and an enzymatic domain, often a histidine kinase. The PCM is composed of three domains (PAS, GAF, and PHY). It holds a covalently bound open-chain tetrapyrrole (biliverdin, BV) chromophore. Upon absorption of light, the double bond between BV rings C and D isomerizes and reversibly switches the protein between Pr and Pfr states. We report crystal structures of the wild-type and mutant (His275Thr) forms of the canonical BphP from the nonphotosynthetic myxobacterium Stigmatella aurantiaca (SaBphP2) in the Pr state. Structures were determined at 1.65 Å and 2.2 Å (respectively), the highest resolution of any PCM construct to date. We also report the room temperature wild-type structure of the same protein determined at 2.1 Å at the SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Our results not only highlight and confirm important amino acids near the chromophore that play a role in Pr-Pfr photoconversion but also describe the signal transduction into the PHY domain which moves across tens of angstroms after the light stimulus.
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Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron X-ray sources. An expansive database of more than 100,000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in action and determining protein kinetics and structures of intermediates (Schmidt et al., 2005a; Schmidt 2008; Neutze and Moffat, 2012; Srajer 2014). In this approach, short and intense X-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron X-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard X-rays (XFELs; 5-20 keV), which provide exceptionally intense, femtosecond X-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al., 2014; Barends et al., 2015; Pande et al., 2016). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs. We also outline challenges and further developments necessary to broaden the application of these methods to many important proteins and enzymes of biomedical relevance.
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The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2PDZ2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale. The induced motions are subtle, involve diverse physical mechanisms, and occur throughout the protein structure. The global pattern of electric-field-induced motions is consistent with both local and allosteric conformational changes naturally induced by ligand binding, including at conserved functional sites in the PDZ domain family. This work lays the foundation for comprehensive experimental study of the mechanical basis of protein function.
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Cristalografia por Raios X/métodos , Eletricidade , Movimento , Domínios PDZ , Proteínas/química , Proteínas/metabolismo , Regulação Alostérica , Fenômenos Biomecânicos , Humanos , Ligantes , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only â¼1 µm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 µs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.
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Cristalografia/instrumentação , Grafite/química , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Desenho de Equipamento , Microscopia de Força Atômica , Muramidase/química , Conformação Proteica , Estabilidade Proteica , Propriedades de Superfície , Difração de Raios X/instrumentaçãoRESUMO
A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.
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Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efeitos da radiação , Cristalografia , Isomerismo , Luz , Fótons , Conformação Proteica/efeitos da radiação , Fatores de TempoRESUMO
Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR(1)/pR(E46Q) and pR(2)/pR(CW) states at 10â µs and the pB1 intermediate at 1â ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses.
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Proteínas de Bactérias/química , Dispositivos Lab-On-A-Chip , Fotorreceptores Microbianos/química , Difração de Raios X/métodos , Proteínas de Bactérias/análise , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/análise , Fatores de TempoRESUMO
The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright "on" and dark "off" states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand ß8 containing Thr159 as well as an altered A-C dimer interface involving strands ß7, ß8, ß10, and ß11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences.
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Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animais , Antozoários/genética , Cristalografia por Raios X , Proteínas Luminescentes/genética , Simulação de Dinâmica Molecular , Mutação , Proteínas Recombinantes/genética , TemperaturaRESUMO
Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.
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Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/ultraestrutura , Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Conformação Proteica , Fatores de TempoRESUMO
Renewed interest in room-temperature diffraction has been prompted by the desire to observe structural dynamics of proteins as they function. Serial crystallography, an experimental strategy that aggregates small pieces of data from a large uniform pool of crystals, has been demonstrated at synchrotrons and X-ray free-electron lasers. This work utilizes a microfluidic crystallization platform for serial Laue diffraction from macroscopic crystals and proposes that a collection of small slices of Laue data from many individual crystals is a realistic solution to the difficulties in dynamic studies of irreversible biochemical reactions.
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Femtosecond time resolved pump-probe protein X-ray crystallography requires highly accurate measurements of the photoinduced structure factor amplitude differences. In the case of femtosecond photolysis of single P63 crystals of the Photoactive Yellow Protein, it is shown that photochemical dynamics place a considerable restraint on the achievable time resolution due to the requirement to stretch and add second order dispersion in order to generate threshold concentration levels in the interaction region. Here, we report on using a 'quasi-cw' approach to use the rotation method with monochromatic radiation and 2 eV bandwidth at 9.465 keV at the Linac Coherent Light Source operated in SASE mode. A source of significant Bragg reflection intensity noise is identified from the combination of mode structure and jitter with very small mosaic spread of the crystals and very low convergence of the XFEL source. The accuracy with which the three dimensional reflection is approximated by the 'quasi-cw' rotation method with the pulsed source is modelled from the experimentally collected X-ray pulse intensities together with the measured rocking curves. This model is extended to predict merging statistics for recently demonstrated self seeded mode generated pulse train with improved stability, in addition to extrapolating to single crystal experiments with increased mosaic spread. The results show that the noise level can be adequately modelled in this manner, indicating that the large intensity fluctuations dominate the merged signal-to-noise (I/σI) value. Furthermore, these results predict that using the self seeded mode together with more mosaic crystals, sufficient accuracy may be obtained in order to resolve typical photoinduced structure factor amplitude differences, as taken from representative synchrotron results.
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Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Razão Sinal-RuídoRESUMO
Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001), Chem. Rev. 101, 1569-1581; Schmidt et al. (2005), Methods Mol. Biol. 305, 115-154; Schmidt (2008), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallography, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010), Acta Cryst. A66, 198-206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallographic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.
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Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Termodinâmica , Bactérias/química , Cristalografia por Raios X/métodos , Cinética , Conformação ProteicaRESUMO
Using time-resolved X-ray crystallography, we contrast a bifunctional dehaloperoxidase-hemoglobin (DHP) with previously studied examples of myoglobin and hemoglobin to understand the functional role of the distal pocket of globins. One key functional difference between DHP and other globins is the requirement that H2O2 enter the distal pocket of oxyferrous DHP to displace O2 from the heme Fe atom and thereby activate the heme for the peroxidase function. The open architecture of DHP permits more than one molecule to simultaneously enter the distal pocket of the protein above the heme to facilitate the unique peroxidase cycle starting from the oxyferrous state. The time-resolved X-ray data show that the distal pocket of DHP lacks a protein valve found in the two other globins that have been studied previously. The photolyzed CO ligand trajectory in DHP does not have a docking site; rather, the CO moves immediately to the Xe-binding site. From there, CO can escape but can also recombine an order of magnitude more rapidly than in other globins. The contrast with DHP dynamics and function more precisely defines the functional role of the multiple conformational states of myoglobin. Taken together with the high reduction potential of DHP, the open distal site helps to explain how a globin can also function as a peroxidase.