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Bacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light–absorbing (Pr)–to–far-red light–absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of Pseudomonas aeruginosa . Kinetic analysis of the TRXSS data identifies three distinct structural species, which are attributed to lumi-F, meta-F, and Pr, connected by time constants of 95 μs and 21 ms. Structural analysis based on molecular dynamics simulations shows that the light activation of PaBphP accompanies quaternary structural rearrangements from an “II”-framed close form of the Pfr state to an “O”-framed open form of the Pr state in terms of the helical backbones. This study provides mechanistic insights into how modular signaling proteins such as BphPs transmit structural signals over long distances and regulate their downstream biological responses.

Phasor Based Control is a novel approach to controlling Distributed Energy Resources that aims at relieving various constraints that arise in the distribution grid. It is a two-layer control system with a supervisory control that coordinates distributed controllers to reach voltage phasor targets. The distributed controllers use local synchrophasor measurements and operate as feedback controllers. This control method is currently under development with several algorithms being under consideration for both the central and ditributed components. In this paper, we present the experimental setup that was prepared to prototype a hardware implementation and validate the control method in Hardware-In-the-Loop.

This paper describes how to use voltage phasor measurements to produce a sensitivity matrix that describes how real and reactive power injections at a node on a distribution network affect the local voltage magnitude and angle. Rather than estimating the sensitivity directly, the voltage phasor measurements and power commands/measurements are used to estimate the unbalanced, three-phase Thevenin impedance. The Thevenin impedance estimation is conducted using recursive least squares on temporal difference measurements. The Thevenin impedance and voltage phasor measurement are then used to build the local power-voltage sensitivity matrix with the closed form expression for the Jacobian of the power flow manifold. Hardware-in-the-loop simulations with phasor measurement units providing real phasor measurements are used to evaluate the recursive temporal difference Thevenin impedance estimation and Thevenin-based power-voltage sensitivity methods.

This paper investigates the feedback control challenge of tracking a voltage phasor target at a given node on an unbalanced three phase distribution network. We derive a quasi-steady state system model, modeling the progression of phasors as a martingale and describing the input sensitivity with the linearization of the unbalanced three phase power flow manifold for the Thevenin equivalent circuit. We establish the convexity of the stable linear feedback matrix region, and investigate the stability of Single Input Single Output Phasor Control (SISOPC) with gains chosen based on the reactance of the Thevenin equivalent circuit. To improve upon SISOPC, we propose the Linear Quadratic Phasor Controller (LQPC), which generates a multiple input multiple output linear feedback matrix which takes into account both the network resistance and the mutual impedances that couple the unbalanced three phase injections. We demonstrate the performance of the LQPC using OpenDSS simulations on an unbalanced three phase distribution network, demonstrating the effectiveness of the LQPC.

BACKGROUND Electronic health records (EHRs) provide potential for more efficient patient recruitment into clinical studies. In recent years, machine learning techniques have gained increasing popularity in EHR-based research, with several studies reporting that machine learning methods can be used for disease diagnosis, outcome prediction, and treatment personalization. OBJECTIVE To explore which machine learning methods have been utilized for recruiting patients through EHRs and to compare their characteristics and outcomes. METHODS Search was conducted in MEDLINE, Embase, Scopus and OpenGrey for papers published before June 20th, 2019. Both relevant subject headings and relevant terms in title, abstract and keywords were incorporated in the search strategy. Two reviewers screened papers and decided on which ones to include and then extracted data and assessed bias for each paper included independently. Conflicts were resolved through a third reviewer. Included studies were compared in terms of year of publication, study location, study type, EHRs setting, study aim, clinical trial domain, size of the dataset, models or methods, data types, data processing and feature selection, evaluation, outcomes. RESULTS 11 papers were included for synthesis. Ten were in-silico studies which simulated prediction of participant recruitment on computers. One study evaluated the machine learning assisted recruitment procedure in a real clinical setting. The in-silico studies covered diverse empirical frameworks in terms of the number of trials they used, trial domains, analysis unit, the size of the dataset, outcome definition, methods of data pre-processing, model building and evaluation, and performance measures. Different machine learning methods seem to have similar performance when evaluated under the same circumstances. NLP-incorporated similarity comparison appears more likely to generate better performance than similarity comparison applied to structured data alone in a similar experiment set-up. A single performance measure is not sufficient to fully evaluate a method. All the in-silico studies were judged at high risk of bias. The sole interventional study reported that a significant smaller percentage of time was spent on electronic screening using the system compared with not using it (P < 0.001), but a critical risk of bias was assigned to this study in the bias assessment phase. CONCLUSIONS Natural language processing techniques might be able to boost the accuracy of identifying eligible participants. However, complex methods impose more requirements on the availability of patient data and require customization of the methods to the target EHR system due to high risk of overfitting. It would be valuable if future in-silico studies in this domain could provide more details on data pre-processing, modelling and evaluation and could report different types of performance measures. More interventional studies are needed to generate higher quality evidence. CLINICALTRIAL Review registration: PROSPERO CRD42018103355

This paper introduces a novel Distributed Extensible Grid Control (DEGC) software and communication platform to facilitate the control of distributed energy resources on electric grids. The DEGC software platform leverages state-of-the-art advances in secure, distributed communication and decentralized authorization and authentication. We discuss how these advances enable the kind of robust and secure communication required for a distributed grid control platform, and show how DEGC applies these technologies to the agile development and deployment of grid software through an extensible and flexible API. We describe how DEGC can implement both Volt-VAR voltage magnitude control and Phasor-Based Control as sample applications and demonstrate the DEGC platform in hardware with the demanding Phasor-Based Control test case, and provide performance metrics.

This paper introduces two tools-the Effective Impedance State Estimator (EISE), and the Real Time Effective Impedance Estimator (RTEIE). The EISE is novel in that it is an entirely data-driven state estimator, and does not require a network model. Thus, the EISE is well suited for applications in which an accurate network model is not available. Instead of using a network impedance model, the EISE uses an effective impedance network model that is learned online by the RTEIE. The RTEIE recursively estimates the effective impedance network model using phasor measurement unit (PMU) measurements. The mathematical reasoning for using an effective impedance model, rather than a full network impedance model, is given. The recursive estimation framework allows the RTEIE to take the entire history of phasor measurements into account to produce a model estimate, while still adjusting to changes in the network impedance due to external factors such as topology changes and temperature. The merits of the RTEIE and EISE are demonstrated by comparing the accuracy of the EISE with a conventional state estimator on a toy network.

Electrochemical energy storage is critical for a range of applications spanning electrified transportation and grid energy storage, and there is a need to further improve both the active management and diagnostic capability of current battery management systems. Lithium-based battery chemistries have been favored for their high energy and power densities but require precise management to prevent premature degradation and failure. This work presents an efficient power converter (based on a switched-inductor ladder topology), instrumentation, and an embedded control platform that can provide both active balancing and real-time diagnostic capability through electrochemical impedance spectroscopy (EIS). A digital proportional-integral controller enforces sinusoidal reference signals from a direct digital synthesizer, enabling the power converter to perturb the cells and extract their impedance. Cell-level diagnostics allow for noninvasive measurement of physical electrochemical battery properties that can be used to assess the state of charge and state of health of a battery. A ladder converter prototype was implemented on a printed circuit board to perform EIS on two Panasonic 18650 cells in series. Experimental results showed balancing converter efficiency of 95%, and the accuracy of the prototype was validated through comparison to a state-of-the-art commercial benchtop system.

ObjectiveTo synthesise international evidence for demand, use and outcomes of primary care out-of-hours health services (OOHS).DesignSystematic scoping review.Data sourcesCINAHL; Medline; PsyARTICLES; PsycINFO; SocINDEX; and Embase from 1995 to 2019.Study selectionEnglish language studies in UK or similar international settings, focused on services in or directly impacting primary care.Results105 studies included: 54% from mainland Europe/Republic of Ireland; 37% from UK. Most focused on general practitioner-led out-of-hours cooperatives. Evidence for increasing patient demand over time was weak due to data heterogeneity, infrequent reporting of population denominators and little adjustment for population sociodemographics. There was consistent evidence of higher OOHS use in the evening compared with overnight, at weekends and by certain groups (children aged 65, women, those from socioeconomically deprived areas, with chronic diseases or mental health problems). Contact with OOHS was driven by problems perceived as urgent by patients. Respiratory, musculoskeletal, skin and abdominal symptoms were the most common reasons for contact in adults; fever and gastrointestinal symptoms were the most common in the under-5s. Frequent users of daytime services were also frequent OOHS users; difficulty accessing daytime services was also associated with OOHS use. There is some evidence to suggest that OOHS colocated in emergency departments (ED) can reduce demand in EDs.ConclusionsPolicy changes have impacted on OOHS over the past two decades. While there are generalisable lessons, a lack of comparable data makes it difficult to judge how demand has changed over time. Agreement on collection of OOHS data would allow robust comparisons within and across countries and across new models of care. Future developments in OOHS should also pay more attention to the relationship with daytime primary care and other services.PROSPERO registration numberCRD42015029741.

Background People with multimorbidity experience impaired quality of life, poor health and a burden from treatment. Their care is often disease-focused rather than patient-centred and tailored to their individual needs. Objective To implement and evaluate a patient-centred intervention to improve the management of patients with multimorbidity in general practice. Design Pragmatic, cluster randomised controlled trial with parallel process and economic evaluations. Practices were centrally randomised by a statistician blind to practice identifiers, using a computer-generated algorithm. Setting Thirty-three general practices in three areas of England and Scotland. Participants Practices had at least 4500 patients and two general practitioners (GPs) and used the EMIS (Egton Medical Information Systems) computer system. Patients were aged ≥ 18 years with three or more long-term conditions. Interventions The 3D (Dimensions of health, Depression and Drugs) intervention was designed to offer patients continuity of care with a named GP, replacing separate reviews of each long-term condition with comprehensive reviews every 6 months. These focused on individualising care to address patients’ main problems, attention to quality of life, depression and polypharmacy and on disease control and agreeing treatment plans. Control practices provided usual care. Outcome measures Primary outcome – health-related quality of life (assessed using the EuroQol-5 Dimensions, five-level version) after 15 months. Secondary outcomes – measures of illness burden, treatment burden and patient-centred care. We assessed cost-effectiveness from a NHS and a social care perspective. Results Thirty-three practices (1546 patients) were randomised from May to December 2015 [16 practices (797 patients) to the 3D intervention, 17 practices (749 patients) to usual care]. All participants were included in the primary outcome analysis by imputing missing data. There was no evidence of difference between trial arms in health-related quality of life {adjusted difference in means 0.00 [95% confidence interval (CI) –0.02 to 0.02]; p = 0.93}, illness burden or treatment burden. However, patients reported significant benefits from the 3D intervention in all measures of patient-centred care. Qualitative data suggested that both patients and staff welcomed having more time, continuity of care and the patient-centred approach. The economic analysis found no meaningful differences between the intervention and usual care in either quality-adjusted life-years [(QALYs) adjusted mean QALY difference 0.007, 95% CI –0.009 to 0.023] or costs (adjusted mean difference £126, 95% CI –£739 to £991), with wide uncertainty around point estimates. The cost-effectiveness acceptability curve suggested that the intervention was unlikely to be either more or less cost-effective than usual care. Seventy-eight patients died (46 in the intervention arm and 32 in the usual-care arm), with no evidence of difference between trial arms; no deaths appeared to be associated with the intervention. Limitations In this pragmatic trial, the implementation of the intervention was incomplete: 49% of patients received two 3D reviews over 15 months, whereas 75% received at least one review. Conclusions The 3D approach reflected international consensus about how to improve care for multimorbidity. Although it achieved the aim of providing more patient-centred care, this was not associated with benefits in quality of life, illness burden or treatment burden. The intervention was no more or less cost-effective than usual care. Modifications to the 3D approach might improve its effectiveness. Evaluation is needed based on whole-system change over a longer period of time. Trial registration Current Controlled Trials ISRCTN06180958. Funding This project was funded by the National Institute for Health Research (NIHR) Health Services and Delivery Research programme and will be published in full in Health Services and Delivery Research; Vol. 7, No. 5. See the NIHR Journals Library website for further project information.

Phytochromes are red/far-red photoreceptors that are widely distributed in plants and prokaryotes. Ultrafast photoisomerization of a double bond in a biliverdin cofactor or other linear tetrapyrrole drives their photoactivity, but their photodynamics are only partially understood. Multiexponential dynamics were observed in previous ultrafast spectroscopic studies and were attributed to heterogeneous populations of the pigment-protein complex. In this work, two-dimensional photon echo spectroscopy was applied to study dynamics of the bacteriophytochromes RpBphP2 and PaBphP. Two-dimensional photon echo spectroscopy can simultaneously resolve inhomogeneity in ensembles and fast dynamics by correlating pump wavelength with the emitted signal wavelength. The distribution of absorption and emission energies within the same state indicates an ensemble of heterogeneous protein environments that are spectroscopically distinct. However, the lifetimes of the dynamics are uniform across the ensemble, suggesting a homogeneous model involving sequential intermediates for the initial photodynamics of isomerization.

We investigate photocycle excitation of crystalline photoactive yellow protein using femtosecond laser pulses. This work establishes the feasibility and suitable optical excitation conditions to perform femtosecond time resolved X-ray crystallographic measurements using an X-ray free electron laser. Flash photolysis experiments demonstrated photocycle yields of the long-lived ‘pB’ signalling state of PYP of up to 10% with pulse durations of 130, 500 and 850 fs at 450 nm wavelength. The power density dependence of the transient pB concentration depends strongly on the pulse duration primarily because photobleaching is prominent at the GW/mm 2 level.

Background: \ud The management of people with multiple chronic conditions challenges health-care systems designed around single conditions. There is international consensus that care for multimorbidity should be patient-centred, focus on quality of life, and promote self-management towards agreed goals. However, there is little evidence about the effectiveness of this approach. Our hypothesis was that the patient-centred, so-called 3D approach (based on dimensions of health, depression, and drugs) for patients with multimorbidity would improve their health-related quality of life, which is the ultimate aim of the 3D intervention.\ud \ud Methods: \ud We did this pragmatic cluster-randomised trial in general practices in England and Scotland. Practices were randomly allocated to continue usual care (17 practices) or to provide 6-monthly comprehensive 3D reviews, incorporating patient-centred strategies that reflected international consensus on best care (16 practices). Randomisation was computer-generated, stratified by area, and minimised by practice deprivation and list size. Adults with three or more chronic conditions were recruited. The primary outcome was quality of life (assessed with EQ-5D-5L) after 15 months' follow-up. Participants were not masked to group assignment, but analysis of outcomes was blinded. We analysed the primary outcome in the intention-to-treat population, with missing data being multiply imputed. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN06180958.\ud \ud Findings: \ud Between May 20, 2015, and Dec 31, 2015, we recruited 1546 patients from 33 practices and randomly assigned them to receive the intervention (n=797) or usual care (n=749). In our intention-to-treat analysis, there was no difference between trial groups in the primary outcome of quality of life (adjusted difference in mean EQ-5D-5L 0·00, 95% CI −0·02 to 0·02; p=0·93). 78 patients died, and the deaths were not considered as related to the intervention.\ud \ud Interpretation: \ud To our knowledge, this trial is the largest investigation of the international consensus about optimal management of multimorbidity. The 3D intervention did not improve patients' quality of life.\ud \ud Funding: \ud National Institute for Health Research.

This publication presents an unsupervised network impedance estimation method for AC microgrid electrical networks using only voltage and current synchrophasor measurements. The proposed method combines the use of two techniques: the Kron reduction and a novel common mode elimination technique to improve estimation robustness to measurement noise. We motivate Kron reduction as an instrument for addressing the rank-deficiency of electrical network voltage measurement data. The Kron reduced model can be used in optimal power flow and other centralized and decentralized control algorithms. The proposed common mode elimination technique improves the robustness of the impedance estimate to sensor noise by improving the eigenstructure of the voltage measurement data matrix, without discarding any useful information. We present results demonstrating improved admittance matrix estimation accuracy on realistic data over a range of signal to noise (SNR) ratios. The proposed method is data-driven and will update the network impedance estimate based on sensor measurements alone, without any external input, making it is well suited for time-varying microgrid applications.

Synchrotron radiation (SR) research at Cornell University is presently carried out at the Cornell High Energy Synchrotron Source (CHESS), a National X-ray Facility on the Cornell Electron/Positron Storage Ring (CESR), a machine mainly dedicated to X-ray production and supported by the National Science Foundation (NSF). Each year, over a thousand scientists and students use a dozen X-ray stations for multidisciplinary research in physics, chemistry, biology, environmental and materials sciences, and engineering on a machine typically operating with compact undulators at 5.3 GeV and 100 to 200 mA.

SummaryRpBphP2 and RpBphP3, two tandem bacteriophytochromes from the photosynthetic bacterium Rhodopseudomonas palustris, share high sequence identity but exhibit distinct photoconversion behavior. Unlike the canonical RpBphP2, RpBphP3 photoconverts to an unusual near-red-absorbing (Pnr) state; both are required for synthesis of light-harvesting complexes under low-light conditions. Here we report the crystal structures of the photosensory core modules of RpBphP2 and RpBphP3. Despite different quaternary structures, RpBphP2 and RpBphP3 adopt nearly identical tertiary structures. The RpBphP3 structure reveals tongue-and-groove interactions at the interface between the GAF and PHY domains. A single mutation in the PRxSF motif at the GAF-PHY interface abolishes light-induced formation of the Pnr state in RpBphP3, possibly due to altered structural rigidity of the chromophore-binding pocket. Structural comparisons suggest that long-range signaling involves structural rearrangement of the helical spine at the dimer interface. These structures, together with mutational studies, provide insights into photoconversion and the long-range signaling mechanism in phytochromes.

When the molecules in a single crystal or a concentrated solution are driven far from equilibrium, the structural course of their return to equilibrium can be monitored by dynamic X-ray scattering. Light-sensitive systems such as signaling photoreceptors can readily be displaced from equilibrium by absorption of a photon of visible light by their non-protein chromophore. Return to their dark, equilibrium state often requires many decades in time from femtoseconds to seconds, and is accompanied by structural changes in the chromophore itself and the protein in which it is embedded. Although slower structural changes can be followed by synchrotron-based techniques that isolate single X-ray pulses of around 100 ps duration, the fastest, femtosecond structural processes, such as isomerization and electron transfer, require the highly intense, femtosecond, hard X-ray pulses from free electron laser sources. The principles of dynamic, time-resolved X-ray scattering and its application at synchrotron and free electron laser sources are presented and illustrated here by recent successful examples.

Absorption of a photon in the visible region of the solar spectrum is the fundamental step in the photochemical reactions that drive biological processes such as imaging, photosynthesis, and sensory perception. All known photoreceptors are proteins that contain a chromophore: a small organic molecule that absorbs in the visible region of the spectrum ( 1 ). The bacterial integral membrane protein bacteriorhodopsin (bR) uses retinal as its chromophore (see the figure), capturing the energy in a photon to drive retinal isomerization and pump protons across the membrane. bR is a readily accessible analog to the visual rhodopsins in, for example, the human eye. On page 145 of this issue, Nogly et al. ( 2 ) used time-resolved x-ray crystallography ( 3 , 4 ) to determine intermediate structures of bR at near-atomic resolution on the femtosecond to picosecond time scales at which isomerization occurs.

A personal, historical view is presented of Laue X-ray diffraction and its application to time-resolved studies of dynamic processes, largely in light-sensitive biological systems. In Laue diffraction, a stationary crystal is illuminated by a polychromatic X-ray source. Laue diffraction has inherent complications largely absent in monochromatic diffraction, and consequently fell into disuse for quantitative structure determination. However, the advent of naturally polychromatic, intense, pulsed storage ring X-ray sources in the 1970s led to re-examination at Daresbury and elsewhere of its underlying principles. Laue diffraction has been successfully applied at storage ring sources to time-resolved, pump–probe crystallographic studies, whose exposure time and time resolution were progressively reduced from minutes to seconds, milliseconds, nanoseconds and 100 ps. Most recently, hard X-ray free electron laser sources have been used to generate narrow bandpass Laue diffraction patterns. The femtosecond X-ray pulses from such sources are completely destructive, generate only one diffraction pattern per tiny crystal and have unusual properties. However, they too are being applied to time-resolved crystallography to explore, for example, isomerization and rapid tertiary structural changes on the chemical, femtosecond timescale. This article is part of the theme issue ‘Fifty years of synchrotron science: achievements and opportunities’.

Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods.

Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 A. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway. Time-resolved X-ray crystallography on photoactive yellow protein shows the existence of a short-lived, highly distorted intermediate whose reaction trajectory bifurcates along ‘bicycle-pedal’ and ‘hula-twist’ pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue, which switches off the bicycle-pedal pathway.

Background To identify, summarise and synthesise the evidence for using interactive digital interventions to support patient self-management of asthma, and determine their impact. Methods Systematic review with meta-analysis. We searched MEDLINE, EMBASE, CINAHL, PsycINFO, ERIC, Cochrane Library, DoPHER, TROPHI, Social Science Citation Index and Science Citation Index. The selection criteria requirement was studies of adults (16 years and over) with asthma, interventions that were interactive digital interventions and the comparator was usual care. Outcomes were change in clinical outcomes, cost effectiveness and patient-reported measures of wellbeing or quality of life. Only Randomised Controlled Trials published in peer-reviewed journals in English were eligible. Potential studies were screened and study characteristics and outcomes were extracted from eligible papers independently by two researchers. Where data allowed, meta-analysis was performed using a random effects model. Results Eight papers describing 5 trials with 593 participants were included, but only three studies were eligible for inclusion for meta-analysis. Of these, two aimed to improve asthma control and the third aimed to reduce the total dose of oral prednisolone without worsening control. Analyses with data from all three studies showed no significant differences and extremely high heterogeneity for both Asthma Quality of Life (AQLQ) (Standardised Mean Difference (SMD) 0.05; 95 % Confidence Interval (CI) 0.32 to −0.22: I2 96.8) and asthma control (SMD 0.21; 95 % CI −0.05 to .42; I2 = 87.4). The removal of the third study reduced heterogeneity and indicated significant improvement for both AQLQ (SMD 0.45; 95 % CI 0.13 to 0.77: I2 = 0.34) and asthma control (SMD 0.54; 95 % CI 0.22 to 0.86: I2 = 0.11). No evidence of harm was identified. Conclusion Digital self-management interventions for adults with asthma show promise, with some evidence of small beneficial effects on asthma control. Overall, the evidence base remains weak due to the lack of large, robust trials. Electronic supplementary material The online version of this article (doi:10.1186/s12890-016-0248-7) contains supplementary material, which is available to authorized users.

Time-resolved crystallography uses the brief, intense X-ray pulses emitted by synchrotron or hard X-ray free electron laser (FEL) sources to probe the time course of structural changes as they occur in the molecules in a crystal, via pump - probe experiments. Synchrotron sources such as BioCARS sector 14 at the Advanced Photon Source, Argonne National Laboratory, can access the time scale from seconds to 100 picoseconds, where the lower limit is set by the duration of a single X-ray pulse. The new FEL sources such as the Linac Coherent Light Source at Stanford extend this limit to femtoseconds. The characteristics of the X-ray pulses emitted by synchrotron and FEL sources are radically different which necessitates new approaches to the pump - probe experiments and data analysis. For both synchrotron and FEL sources, reaction initiation typically requires light-sensitive systems in which reaction can be initiated by a visible laser pulse - but clearly, not all interesting biological systems are light-sensitive. This raises the question: how can sensitivity to light be conferred on otherwise light-inert systems, by optogenetic approaches?Ultrafast time-resolved crystallography will be illustrated by experiments conducted at synchrotron and FEL sources, that probe the structure of short-lived structural intermediates in the photocycle of the naturally-occurring bacterial blue light photoreceptor known as photoactive yellow protein, PYP. The principles that can be used to confer sensitivity to light on light-inert systems are illustrated by the design and characterization of a blue-light-sensitive histidine kinase. Finally I ask: are these principles equally applicable to experiments in the femtosecond time range at FEL sources.This research has been supported by NIH grants GM111072 and EY024363.

Summary Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.

The Advanced Photon Source (APS) operates with a timing structure advantageous for ultrafast dynamics experiments and, as a result, X-ray time-resolved studies on the 100-picosecond timescale have flourished. The standard operating mode, 24-bunch mode, runs 65% of the time and a hybrid singlet mode runs 15% of the time, yielding a total of 80% of beamtime easily accessible for studies of ultrafast dynamics using laser-pump/X-ray probe techniques. This article highlights recent enhanced capabilities for time-resolved studies at the APS.

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C(15) = C(16) double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C(15) methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ∼4 × 1010photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.

The phototropins are blue-light receptors that base their lightdependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine in light, oxygen or voltage (LOV) domains. The primary reactions of the Avena sativa phototropin 1 LOV2 domain were investigated by means of time-resolved and lowtemperature fluorescence spectroscopy. Synchroscan streak camera experiments revealed a fluorescence lifetime of 2.2 ns in LOV2. A weak long-lived component with emission intensity from 600 to 650 nm was assigned to phosphorescence from the reactive FMN triplet state. This observation allowed determination of the LOV2 triplet state energy level at physiological temperature at 16600 cm )1 . FMN dissolved in aqueous solution showed pH-dependent fluorescence lifetimes of 2.7 ns at pH 2 and 3.9)4.1 ns at pH 3–8. Here, too, a weak phosphorescence band was observed. The fluorescence quantum yield of LOV2 increased from 0.13 to 0.41 upon cooling the sample from 293 to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K in the steady-state emission.

The initial output of a time-resolved macromolecular crystallography experiment is a time-dependent series of difference electron density maps that displays the time-dependent changes in underlying structure as a reaction progresses. The goal is to interpret such data in terms of a small number of crystallographically refinable, time-independent structures, each associated with a reaction intermediate; to establish the pathways and rate coefficients by which these intermediates interconvert; and thereby to elucidate a chemical kinetic mechanism. One strategy toward achieving this goal is to use cluster analysis, a statistical method that groups objects based on their similarity. If the difference electron density at a particular voxel in the time-dependent difference electron density (TDED) maps is sensitive to the presence of one and only one intermediate, then its temporal evolution will exactly parallel the concentration profile of that intermediate with time. The rationale is therefore to cluster voxels with respect to the shapes of their TDEDs, so that each group or cluster of voxels corresponds to one structural intermediate. Clusters of voxels whose TDEDs reflect the presence of two or more specific intermediates can also be identified. From such groupings one can then infer the number of intermediates, obtain their time-independent difference density characteristics, and refine the structure of each intermediate. We review the principles of cluster analysis and clustering algorithms in a crystallographic context, and describe the application of the method to simulated and experimental time-resolved crystallographic data for the photocycle of photoactive yellow protein.