Structure of the Angiotensin receptor revealed by serial femtosecond crystallography.

Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.

Examining the Biological Impacts of Parent-Child Relationship Dynamics on Preschool-Aged Children who have Experienced Adversity.

Parent-child relationship dynamics have been shown to predict socioemotional and behavioral outcomes for children, but little is known about how they may affect biological development. The aim of this study was to test if observational assessments of parent-child relationship dynamics (cohesion, enmeshment, and disengagement) were associated with three biological indices of early life adversity and downstream health risk: (1) methylation of the glucocorticoid receptor gene (NR3C1), (2) telomere attrition, and (3) mitochondrial biogenesis, indexed by mitochondrial DNA copy number (mtDNAcn), all of which were measured in children's saliva. We tested hypotheses using a sample of 254 preschool-aged children (M age = 51.04 months) with and without child welfare-substantiated maltreatment (52% with documented case of moderate-severe maltreatment) who were racially and ethnically diverse (17% Black, 40% White, 23% biracial, and 20% other races; 45% Hispanic) and from primarily low-income backgrounds (91% qualified for public assistance). Results of path analyses revealed that: (1) higher parent-child cohesion was associated with lower levels of methylation of NR3C1 exon 1D and longer telomeres, and (2) higher parent-child disengagement was associated with higher levels of methylation of NR3C1 exon 1D and shorter telomeres. Results suggest that parent-child relationship dynamics may have distinct biological effects on children.

Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

Macromolecular diffractive imaging using imperfect crystals.

The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed--and are of interest as a source of information about the dynamics of proteins--they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

Maternal and paternal unsupportive parenting and children's externalizing symptoms: The mediational role of children's attention biases to negative emotion.

This study examined children's duration of attention to negative emotions (i.e., anger, sadness, fear) as a mediator of associations among maternal and paternal unsupportive parenting and children's externalizing symptoms in a sample of 240 mothers, fathers, and their preschool children (Mage = 4.64 years). The multimethod, multi-informant design consisted of three annual measurement occasions. Analysis of maternal and paternal unsupportive parenting as predictors in latent difference changes in children's affect-biased attention and behavior problems indicated that children's attention to negative emotions mediated the specific association between maternal unsupportive parenting and children's subsequent increases in externalizing symptoms. Maternal unsupportive parenting at Wave 1 predicted decreases in children's attention to negative facial expressions of adults from Wave 1 to 2. Reductions in children's attention to negative emotion, in turn, predicted increases in their externalizing symptoms from Wave 1 to 3. Additional tests of children's fearful distress and hostile responses to parental conflict as explanatory mechanisms revealed that increases in children's fearful distress reactivity from Wave 1 to 2 accounted for the association between maternal unsupportive parenting and concomitant decreases in their attention to negative emotions. Results are discussed in the context of information processing models of family adversity and developmental psychopathology.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Understanding links between maternal perinatal posttraumatic stress symptoms and infant socioemotional and physical health.

Maternal posttraumatic stress symptoms (PTSS) are associated with adverse consequences for older children, but very few studies have examined links between perinatal maternal PTSS and infant outcomes. Trauma exposure and psychopathology, including PTSS, is often heightened for women during pregnancy through 1 year postpartum. Therefore, the perinatal period may be a critical time for understanding the risk maternal PTSS and other mental health factors pose to the socioemotional and physical health of infants. The present study explored the relation between maternal PTSS and infant socioemotional and physical health problems in a sample of racially and ethnically diverse mother-infant dyads (N = 295) assessed prenatally and at 12 months postpartum. This study also examined whether there are: (1) moderating effects of maternal depressive symptoms and parenting stress on these associations and (2) indirect effects of PTSS on infant outcomes through observed maternal sensitivity. Results indicated that postpartum depressive symptoms and parenting stress, rather than PTSS, were associated with greater infant socioemotional health problems. However, prenatal PTSS were associated with greater infant physical health problems when mothers also reported clinically significant levels of postpartum depressive symptoms. Maternal sensitivity was not associated with maternal PTSS, depressive symptoms, or parenting stress, nor was it related to infant socioemotional and physical health; thus, maternal sensitivity was not tested as an intermediary mechanism linking maternal mental health with infant outcomes. Implications for promoting maternal mental health in the perinatal period to bolster socioemotional and physical health of infants are discussed.

Los síntomas de estrés materno postraumático (PTSS) se asocian con consecuencias adversas para niños de mayor edad, pero pocos estudios han examinado las conexiones entre PTSS materno perinatal y los resultados en el infante. El estar expuesta al trauma y la sicopatología de este, incluyendo PTSS, a menudo se intensifican más en el caso de mujeres durante el embarazo y hasta un año después del parto. Por tanto, el período perinatal pudiera ser un omento crítico para comprender el riesgo que el PTSS materno y otros factores de salud mental presentan para la salud socioemocional y física de los infantes. Este estudio exploró la relación entre el PTSS materno y los problemas de salud socioemocional y física del infante en un grupo muestra de díadas madre-infante racial y étnicamente diversas (N = 295), evaluadas prenatalmente y a los 12 meses después del parto. Este estudio también examinó si hay: (1) efectos moderadores de los síntomas depresivos maternos y el estrés de crianza sobre estas asociaciones y (2) efectos indirectos de PTSS sobre los resultados en el infante a través de la observada sensibilidad materna. Los resultados indican que los síntomas depresivos y el estrés de crianza, en vez de PTSS, estaban asociados con mayores problemas de salud socioemocional del infante. Sin embargo, el PTSS se asoció con mayores problemas de salud física en el infante cuando las madres también reportaron niveles clínicamente significativos de síntomas depresivos. No se corroboró el que la sensibilidad materna fuera un mecanismo intermediario de conexión entre PTSS y otros factores de salud mental materna y resultados en el infante.

Les symptômes des stress posttraumatique (SPT) maternel sont liés à des conséquences adverses pour les enfants plus âgés mais peu d'études ont examiné les liens entre le SPT périnatal maternel et les résultats sur les nourrissons. L'exposition au trauma et la psychopathologie, y compris le SPT, est souvent accrues pour les femmes durant la grossesse jusqu'à un an après la naissance. Par conséquent la période périnatale peut être un moment critique pour la compréhension du risque que posent le SPT maternel et d'autres facteurs de santé mentale à la santé socio-émotionnelle et physique des nourrissons. Cette étude a exploré la relation entre le SPT maternel et les problèmes socio-émotionnels et physiques des nourrissons chez un échantillon de dyades mère-nourrisson racialement et ethniquement diverses (N = 295) évaluées au stade prénatal et à 12 mois après la naissance. Cette étude a aussi examiné s'il existait : (1) des effets modérateurs de symptômes dépressifs maternels et du stress de parentage sur ces associations et (2) des effets indirects de SPT sur les résultats du nourrisson à travers une sensibilité maternelle observée. Les résultats ont indiqué que les symptômes dépressifs postpartum et le stress de parentage, plus que le SPT, étaient liés à des problèmes plus élevés de santé socio-émotionnels du nourrisson. Cependant le SPT prénatal était lié à de plus grands problèmes de santé physique du nourrisson quand les mères faisaient aussi état de niveaux cliniquement élevés de symptômes dépressifs postpartum. La sensibilité maternelle n'était pas liée au SPT maternel, aux symptômes dépressifs ou au stress de parentage, et n'était pas non plus liée à la santé socio-émotionnelle et physique du nourrisson. Donc la sensibilité maternelle n'a pas testée comme mécanisme intermédiaire liant la santé mentale maternelle aux résultats du nourrisson. Les implications pour la promotion de la santé mentale maternelle dans la période périnatale afin de soutenir la santé socio-émotionnelle et physique des nourrissons sont discutées.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Temperamental Emotionality Attributes as Antecedents of Children's Social Information Processing.

This study examined temperament dimensions of emotion as precursors of children's social information processing (SIP) of stressful peer events. Two hundred and forty-three preschool children (M = 4.60 years) and their primary caregivers participated in two measurement occasions spaced 2 years apart. Observations of temperamental anger, fearful distress, positive affect, and effortful control were assessed in multiple laboratory tasks across two visits at Wave 1. SIP assessments from vignettes of peer challenges were repeated across two waves and included: eye tracking measures of attention to peer emotion displays, hostile attribution bias, hostile solutions, and subjective distress. Findings from structural equation models with inclusion of autoregressive controls indicated that effortful control, fear, and anger predicted subsequent changes in specific SIP dimensions.

Understanding the nature of associations between family instability, unsupportive parenting, and children's externalizing symptoms.

This study examined the mediating role of maternal unsupportive parenting in explaining associations between family instability and children's externalizing symptoms during the transition to formal schooling in early childhood. Participants included 243 preschool children (M age = 4.60 years) and their parents. Findings from cross-lagged autoregressive models conducted with multimethod (survey and observations), multi-informant (parent, teacher, and observer), longitudinal (three annual waves of data collection) data indicated that experiences with heightened family instability predicted decreases in supportive parenting, which in turn predicted increases in children's externalizing symptoms. Analyses also revealed a bidirectional association between parenting and family instability over time, such that higher levels of instability predicted decreases in supportive parenting, which in turn predicted increases in family instability.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Family Instability and Young Children's School Adjustment: Callousness and Negative Internal Representations as Mediators.

This study examined the mediating roles of children's callousness and negative internal representations of family relationships in associations between family instability and children's adjustment to school in early childhood. Participants in this multimethod (i.e., survey, observations), multiinformant (i.e., parent, teacher, observer), longitudinal study included 243 preschool children (Mage  = 4.60 years) and their families. Findings from the lagged, autoregressive tests of the mediational paths indicated that both children's callousness and negative internal representations of family relationships mediated longitudinal associations between family instability and children's school adjustment problems over a 2-year period (i.e., the transition from preschool to first grade). Findings are discussed in relation to the attenuation hypothesis (E. J. Susman, 2006) and emotional security theory (EST; P. T. Davies, M. A. Winter, & D. Cicchetti, 2006).

The Interplay Among Children's Negative Family Representations, Visual Processing of Negative Emotions, and Externalizing Symptoms.

This study examined the transactional interplay among children's negative family representations, visual processing of negative emotions, and externalizing symptoms in a sample of 243 preschool children (Mage  = 4.60 years). Children participated in three annual measurement occasions. Cross-lagged autoregressive models were conducted with multimethod, multi-informant data to identify mediational pathways. Consistent with schema-based top-down models, negative family representations were associated with attention to negative faces in an eye-tracking task and their externalizing symptoms. Children's negative representations of family relationships specifically predicted decreases in their attention to negative emotions, which, in turn, was associated with subsequent increases in their externalizing symptoms. Follow-up analyses indicated that the mediational role of diminished attention to negative emotions was particularly pronounced for angry faces.

Family instability and children's effortful control in the context of poverty: Sometimes a bird in the hand is worth two in the bush.

Effortful control has been demonstrated to have important ramifications for children's self-regulation and social-emotional adjustment. However, there are wide socioeconomic disparities in children's effortful control, with impoverished children displaying heightened difficulties. The current study was designed to demonstrate how instability within the proximal rearing context of young children may serve as a key operant on the development of children's effortful control in the context of poverty. Two separate studies were conducted that included samples of children living within homes characterized by heightened economic risk. In Study 1, we tested the differential prediction of family instability on two domains of children's effortful control: cool effortful control and delay control. Consistent with hypotheses, elevated instability was associated with decreased hot effortful control but not cool effortful control over the span of 2 years. In Study 2, we examined how children's basal cortisol activity may account for associations between heightened instability and effortful control in reward tasks. The results were consistent with sensitization models, suggesting that elevated cortisol activity arising from increased uncertainty and unpredictability in rearing contexts may influence children's hot effortful control. The findings are interpreted within emerging evolutionary-developmental frameworks of child development.

Serial femtosecond crystallography: A revolution in structural biology.

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein.

Transactional cascades of destructive interparental conflict, children's emotional insecurity, and psychological problems across childhood and adolescence.

This study examined the transactional interplay among dimensions of destructive interparental conflict (i.e., hostility and dysphoria), children's emotional insecurity, and their psychological problems from middle childhood and adolescence. Participants were 232 families, with the first of five measurement occasions occurring when children were in first grade (M age = 7 years). Cross-lagged, autoregressive models were conducted with a multiple-method, multiple-informant measurement approach to identify developmental cascades of interparental and child cascades. Results indicated that emotional insecurity was a particularly powerful mediator of prospective associations between interparental conflict (i.e., dysphoria and hostility) and child adjustment during adolescence rather than childhood. In reflecting bidirectionality in relationships between interparental and child functioning, children's psychological problems predicted increases in interparental dysphoria during childhood and adolescence. Although emotional insecurity was not identified as a proximal predictor of interparental difficulties, an indirect cascade was identified whereby insecurity in early adolescence was associated with increases in teen psychological problems, which in turn predicted greater interparental dysphoria over time. Results are interpreted in the context of how they advance transactional formulation of emotional security theory and its resulting translational implications for clinical initiatives.

High throughput protein nanocrystal fractionation in a microfluidic sorter.

Protein crystallography is transitioning into a new generation with the introduction of the X-ray free electron laser, which can be used to solve the structures of complex proteins via serial femtosecond crystallography. Sample characteristics play a critical role in successful implementation of this new technology, whereby a small, narrow protein crystal size distribution is desired to provide high quality diffraction data. To provide such a sample, we developed a microfluidic device that facilitates dielectrophoretic sorting of heterogeneous particle mixtures into various size fractions. The first generation device demonstrated great potential and success toward this endeavor; thus, in this work, we present a comprehensive optimization study to improve throughput and control over sorting outcomes. First, device geometry was designed considering a variety of criteria, and applied potentials were modeled to determine the scheme achieving the largest sorting efficiency for isolating nanoparticles from microparticles. Further, to investigate sorting efficiency within the nanoparticle regime, critical geometrical dimensions and input parameters were optimized to achieve high sorting efficiencies. Experiments revealed fractionation of nanobeads from microbeads in the optimized device with high sorting efficiencies, and protein crystals were sorted into submicrometer size fractions as desired for future serial femtosecond crystallography experiments.

Evaluating the implementation of family-centered substance use treatment for pregnant and postpartum people: A mixed-methods study.

INTRODUCTION: Family-centered substance use treatment (FCSUT) approaches for pregnant and postpartum people have the potential to prevent intergenerational transmission of adverse childhood experiences (ACEs). Guided by two theoretical frameworks drawn from implementation science (the Consolidated Framework for Implementation Research [CFIR] and the Reach, Effectiveness, Adoption, Implementation and Maintenance [RE-AIM] framework), this study used a mixed methods approach to answer: (1) What is the extent to which FCSUT approaches are offered for pregnant and postpartum people seeking substance use disorder (SUD) treatment? and (2) How are FCSUT approaches for pregnant and postpartum people implemented? METHODS: This study utilized a sequential mixed methods design that began with quantitative data collection followed by qualitative data collection. The quantitative component consisted of service provision surveys of facilities that provided FCSUT to pregnant and postpartum people (n = 118). The qualitative component consisted of semi-structured in-depth interviews with administrators and providers working at FCSUT facilities (n = 26) and pregnant and postpartum people who were currently receiving or had previously received services in the last two years from FCSUT facilities (n = 27). The qualitative findings were used to deepen understanding of the quantitative findings. RESULTS: Findings from the quantitative survey of treatment facilities' FCSUT provision revealed that while most facilities offered services related to substance use treatment, behavioral health, and parenting skills development or parent training, a smaller proportion offered services related to prenatal and postpartum health, sexual and reproductive health, and family-related services. Qualitative in-depth interviews with program administrators and providers and pregnant and postpartum people who had participated in FCSUT programs revealed major themes around expanding reach of facilities by maintaining participants' familial connections, resources for implementation and maintenance of FCSUT, the importance of program adaptation, and gaps in service delivery. CONCLUSIONS: Results indicated there is a wide range of FCSUT services offered at treatment facilities across the United States. Furthermore, while many pregnant and postpartum people expressed positive experiences with FCSUT, there are some areas that should be considered for future progress to be made.

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu B ) and a heme a 3 iron, where O 2 binds and is reduced to water. CO is a versatile O 2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (Cu B -CO), where CO is bound to Cu B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the Cu B and the heme a 3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.