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Microbial rhodopsins form a diverse family of light-sensitive seven-transmembrane helix retinal proteins that function as active proton or ion pumps, passive light-gated ion channels, and photosensors. To understand how light-sensing in archaea is initiated by sensory rhodopsins, we perform serial synchrotron X-ray crystallography (SSX) studies of light induced conformational changes in sensory rhodopsin II ( Np SRII) from the archaea Natronomonas pharaonis , both collecting time-resolved SSX data and collecting SSX data during continuous illumination. Comparing light-induced electron density changes in Np SRII with those reported for bacteriorhodopsin (bR) reveals several common light-induced structural perturbations. Unlike bR, however, helix G of Np SRII does not unwind near the conserved lysine residue to which retinal is covalently bound and therefore transient water molecule binding sites do not arise immediately to the cytoplasmic side of retinal. These structural differences prolong the duration of the Np SRII photocycle relative to bR, allowing time for the light-initiated sensory signal to be amplified. Time resolved serial X-ray crystallography reveals light-driven structural changes in sensory rhodopsin II. These observations explain why this light receptor has a long-lived signaling conformation.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light. Plants adapt to the availability of light throughout their lives because it regulates so many aspects of their growth and reproduction. To detect the level of light, plant cells use proteins called phytochromes, which are also found in some bacteria and fungi. Phytochrome proteins change shape when they are exposed to red light, and this change alters the behaviour of the cell. The red light is absorbed by a molecule known as chromophore, which is connected to a region of the phytochrome called the PHY-tongue. This region undergoes one of the key structural changes that occur when the phytochrome protein absorbs light, turning from a flat sheet into a helix. Claesson, Wahlgren, Takala et al. studied the structure of a bacterial phytochrome protein almost immediately after shining a very brief flash of red light using a laser. The experiments revealed that the structure of the protein begins to change within a trillionth of a second: specifically, the chromophore twists, which disrupts its attachment to the protein, freeing the protein to change shape. Claesson, Wahlgren, Takala et al. note that this structure is likely a very short-lived intermediate state, which however triggers more changes in the overall shape change of the protein. One feature of the rearrangement is the disappearance of a particular water molecule. This molecule can be found at the core of many different phytochrome structures and interacts with several parts of the chromophore and the phytochrome protein. It is unclear why the water molecule is lost, but given how quickly this happens after the red light is applied it is likely that this disappearance is an integral part of the reshaping process. Together these events disrupt the interactions between the chromophore and the PHY-tongue, enabling the PHY-tongue to change shape and alter the structure of the phytochrome protein. Understanding and controlling this process could allow scientists to alter growth patterns in plants, such as crops or weeds.

Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3 -type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3 Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3 -type and aa 3 -type cytochrome c oxidases around the proton-loading site are also described.

The aim of this study was to validate and compare the performance of the Acute Physiology and Chronic Health Evaluation (APACHE) IV in the Dutch intensive care unit (ICU) population to the APACHE II and Simplified Acute Physiology Score (SAPS) II. This is a prospective study based on data from a national quality registry between 2006 and 2009 from 59 Dutch ICUs. The validation set consisted of 62 737 patients; the 3 models were compared using 44 112 patients. Measures of discrimination, accuracy, and calibration (area under the receiver operating characteristic curve (AUC), Brier score, R 2, and Ĉ-statistic) were calculated using bootstrapping. In addition, the standardized mortality ratios were calculated. The original APACHE IV showed good discrimination and accuracy (AUC = 0.87, Brier score = 0.10, R 2 = 0.29) but poor calibration (Ĉ-statistic = 822.67). Customization significantly improved the performance of the APACHE IV. The overall discrimination and accuracy of the customized APACHE IV were statistically better, and the overall Ĉ-statistic was inferior to those of the customized APACHE II and SAPS II, but these differences were small in perspective of clinical use. The 3 models have comparable capabilities for benchmarking purposes after customization. Main advantage of APACHE IV is the large number of diagnoses that enable subgroup analysis. The APACHE IV coronary artery bypass grafting (CABG) model has a good performance in the Dutch ICU population and can be used to complement the 3 models.

Time-resolved x-ray solution scattering (TR-XSS) is a sub-field of structural biology, which observes secondary structural changes in proteins as they evolve along their functional pathways. While the number of distinct conformational states and their rise and decay can be extracted directly from TR-XSS experimental data recorded from light-sensitive systems, structural modeling is more challenging. This step often builds from complementary structural information, including secondary structural changes extracted from crystallographic studies or molecular dynamics simulations. When working with integral membrane proteins, another challenge arises because x-ray scattering from the protein and the surrounding detergent micelle interfere and these effects should be considered during structural modeling. Here, we utilize molecular dynamics simulations to explicitly incorporate the x-ray scattering cross term between a membrane protein and its surrounding detergent micelle when modeling TR-XSS data from photoactivated samples of detergent solubilized bacteriorhodopsin. This analysis provides theoretical foundations in support of our earlier approach to structural modeling that did not explicitly incorporate this cross term and improves agreement between experimental data and theoretical predictions at lower x-ray scattering angles.

Antibiotic resistance is a growing global health threat that risks the lives of millions. Among the resistance mechanisms, that mediated by metallo-β-lactamases is of particular concern as these bacterial enzymes dismantle most β-lactam antibiotics, which are our widest applied and cheapest to produce antibiotic agents. So far, no clinically applicable metallo-β-lactamase inhibitors are available. Aiming to adapt to structural variations, we introduce the inhibitor concept: dynamically chiral phosphonic acids. We demonstrate that they are straightforward to synthesize, penetrate bacterial membranes, inhibit the metallo-β-lactamase enzymes NDM-1, VIM-2 and GIM-1, and are non-toxic to human cells. Mimicking the transition state of β-lactam hydrolysis, they target the Zn ions of the metallo-β-lactamase active site. As a unique feature, both of their stereoisomers bind metallo-β-lactamases, which provides them unparalleled adaptability to the structural diversity of these enzymes, and may allow them to hamper bacteria’s ability for resistance development. Metallo-β-lactamases dismantle most β-lactam antibiotics and cause antibiotic resistance, however, no metallo-β-lactamase inhibitors are clinically available. Here, the authors report stereodynamically chiral phosphonic acids as potential metallo-β-lactamase inhibitors, showing unparalleled adaptability to the structural diversity of metallo-β-lactamases.

Organisms from bacteria to humans sense and react to light. Proteins that contain the light-sensitive molecule retinal couple absorption of light to conformational changes that produce a signal or move ions across a membrane. Nogly et al. used an x-ray laser to probe the earliest structural changes to the retinal chromophore within microcrystals of the ion pump bacteriorhodopsin (see the Perspective by Moffat). The excited-state retinal wiggles but is held in place so that only one double bond of retinal is capable of isomerizing. A water molecule adjacent to the proton-pumping Schiff base responds to changes in charge distribution in the chromophore even before the movement of atoms begins. Science , this issue p. eaat0094 ; see also p. 127 Ultrafast crystallography captures the response of the pigment of bacteriorhodopsin to absorption of light. Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

Nowadays, registration of patient data on paper is gradually being replaced by registration using an intensive care information system (ICIS). The aim of this study was to evaluate the effect of the use of an ICIS on nursing activity. Randomized controlled trial with a crossover design. An 18-bed medical-surgical ICU in a teaching hospital. PATIENTS, NURSES AND INTERVENTIONS: During a 6week period 145 consecutive adult patients admitted to the ICU after uncomplicated cardiothoracic surgery were randomized into two groups: for one group the documentation was carried out using a paper-based registration (Paper), in the second group an ICIS was used for documentation. The nursing activities for these patients were studied during two separate periods: the admission period and the registration phase (the period directly following the admission procedure). The duration of the admission procedure was measured by time-motion analysis and the nursing activities in the registration phase were studied by work sampling methodology. All nursing activities during the registration phase were grouped in four main categories: patient care, documentation, unit-related and personal time. The duration of the admission procedure was longer in the ICIS group (18.1+/-4.1 versus 16.8+/-3.1 min, p<0.05). In the registration phase, a 30% reduction in documentation time (Paper 20.5% of total nursing time versus ICIS 14.4%, p<0.001), corresponding to 29 min (per 8h nursing shift) was achieved. This time was completely re-allocated to patient care. The use of the present ICIS in patients after cardiothoracic surgery alters nursing activity; it reduces the time for documentation and increases the time devoted to patient care. is available if you access this article at http://dx.org/10.1007/s00134-002-1542-9. On that page (frame on the left side), a link takes you directly to the supplementary material.

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography 1 using an X-ray free-electron laser 2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions. Time-resolved serial femtosecond crystallography is used to reveal the structural changes that stabilize the charge-separation steps of electron-transfer reactions in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds.