Rounds Of Refinement Research Articles

A New Stereo Matching Function by a Hybrid Convolutional Neural Network

This research introduces a novel method for creating stereo matching algorithms, combining elements of learned and custom methods. The purpose of this strategy is to provide more reliable outcomes than conventional approaches. The primary goal of the technique for stereo corresponding remains to harvest a difference map. Three-dimensional (3D) reconstruction is only one of many uses for this map. Convolutional neural network (CNN) produced raw disparity maps may still contain mistakes in low-resolution textures. The programme will utilise a hybrid CNN-based approach and a faster directional intensity computation to enhance the accuracy of the matching cost calculation phase. Use of the modified truncated value of directional intensity has the potential to greatly reduce radiometric errors. To further enhance the accuracy attained by the approach, a cost aggregation phase is used in which the bilateral filter (BF) is applied to the raw matching cost. Building a gap map with the whole cost of costs is the first step in the WTA optimisation process. The final disparity map is an improvement above the basic map thanks to several rounds of refinement. By means of the Middlebury Online Stereophonic Benchmarking Scheme, the article verified the algorithm efficacy.

Performance of EHR classifiers for patient eligibility in a clinical trial of precision screening

BackgroundValidated computable eligibility criteria use real-world data and facilitate the conduct of clinical trials. The Genomic Medicine at VA (GenoVA) Study is a pragmatic trial of polygenic risk score testing enrolling patients without known diagnoses of 6 common diseases: atrial fibrillation, coronary artery disease, type 2 diabetes, breast cancer, colorectal cancer, and prostate cancer. We describe the validation of computable disease classifiers as eligibility criteria and their performance in the first 16 months of trial enrollment. MethodsWe identified well-performing published computable classifiers for the 6 target diseases and validated these in the target population using blinded physician review. If needed, classifiers were refined and then underwent a subsequent round of blinded review until true positive and true negative rates ≥80% were achieved. The optimized classifiers were then implemented as pre-screening exclusion criteria; telephone screens enabled an assessment of their real-world negative predictive value (NPV-RW). ResultsPublished classifiers for type 2 diabetes and breast and prostate cancer achieved desired performance in blinded chart review without modification; the classifier for atrial fibrillation required two rounds of refinement before achieving desired performance. Among the 1077 potential participants screened in the first 16 months of enrollment, NPV-RW of the classifiers ranged from 98.4% for coronary artery disease to 99.9% for colorectal cancer. Performance did not differ by gender or race/ethnicity. ConclusionsComputable disease classifiers can serve as efficient and accurate pre-screening classifiers for clinical trials, although performance will depend on the trial objectives and diseases under study.

Vistas of the field: examining quality indicators of health and physical education journals

ABSTRACT In this paper, we use the field of Health and Physical Education (H/PE) to explain the limitations, nuances, and inconsistencies of three global, digitally available, and regularly updated systems: library holdings, metrics, and altmetrics. We understand these systems through the analogy of seeing different vistas of a landscape when driving a car: looking in the rear-view mirror (i.e. library holdings as past indicators of quality) and out the side windows (i.e. metrics and altmetrics as current indicators). Before examining these measures, we acknowledge the neoliberal audit culture and outline our understanding of the H/PE ‘field’. To identify H/PE journals, we searched databases, websites, and literature, generating a list of 202 potentially relevant journals. After three rounds of refinement, 13 journals remained that: (1) focused on health and/or physical education as a school subject (including H/PE teacher education), (2) had official metrics, and (3) self-identified as ‘Social Sciences – Education’ and had more than 20% of the papers in the last two volumes focus on H/PE. For each of these journals, we provide the WorldCat library holdings, metrics (i.e. Journal Impact Factor, Journal h-index, CiteScore, SCImago Journal Rank, and Source Normalized Impact per Paper), and altmetrics. We explain how to understand these measures, as well as their contested, problematic and, often, confusing dimensions. Overall, we argue that it is crucial for scholars to understand these measures so that they can critically reflect on how measurement shapes their research and professional lives.

Acetyl‐CoA Electron Density: Acyl‐CoA Reactivity and Crystallographic Data

Acetyl‐CoA and other acyl‐CoAs are the building blocks used in carbon‐carbon bond forming reactions underlying fatty acid and polyketide biosynthesis and many other biological activities. Since many enzymes use acetyl‐CoA, understanding how the substrate binds in the active site is key to determining interactions leading to enzymatic catalysis and substrate selectivity. We examined all of the PDB deposited crystal structures that report acetyl‐CoA bound and report stats for the reactive acetyl‐thioester moiety. We quantified this data by generating the real‐space R‐factor (RSR) and real‐space correlation coefficient (RSCC) by running a few rounds of refinement with Refmac to normalize the data. We examined the modeled thioester ends in comparison to the rest of CoA by creating a Linux script that splits acetyl‐CoA into the nucleic acid, pantetheine, and acetyl‐thioester moieties. We also analyzed the 3’phosphate of acetyl‐CoA to examine how splitting the molecule affects the RSR/RSCC values of small portions of the molecules along with the ribonucleotide containing substrates NAD+, NADH, NADP, and NADPH. Preliminary analysis of the output data reveals the nucleic acid and pantetheine RSR/RSCC values indicate good fits of models to electron density, but there is a portion of structures where RSR/RSCC values indicate the acetyl‐thioester moiety is poorly fit. We also find that the RSR method of calculation shows a significant difference between the acetyl‐CoA thioester and the 3’phosphate where the 3’phosphate is better than the average of the entire structure while the thioester is poorly fit. This is also consistent with the RSR values of NADP and NADPH 3’ phosphates. Conversely, the RSCC values of the acetyl‐CoA thioester end and the 3’phosphate were both equally lower than the entire structure. It appears there is an overfitting of the acetyl‐thioester fitting in crystal structures, likely due to a pressure to claim mechanistic inference from the structures.

An Interactive Voice Response System to Increase Physical Activity and Prevent Cancer in the Rural Alabama Black Belt: Design and Usability Study.

BackgroundIncreased physical activity (PA) levels are associated with reduced risk and improved survival for several cancers; however, most Americans engage in less than the recommended levels of PA. Using interactive voice response (IVR) systems to provide personalized health education and counseling may represent a high-reach, low-cost strategy for addressing physical inactivity and cancer disparities in disproportionately burdened rural regions. However, there has been a paucity of research conducted in this area to date.ObjectiveThe aim of this study is to design, develop, and test the usability of an IVR system aimed at increasing PA levels in the rural Alabama Black Belt.MethodsA pilot version of the IVR system was used to assess initial feasibility and acceptability. Detailed exit interviews were conducted to elicit participant feedback, which helped inform the development of a substantially upgraded in-house IVR system. This refined IVR system was then subjected to a sequential explanatory mixed methods evaluation. Participating rural county coordinators and research staff (N=10) tested the usability of the IVR system features for 2 weeks and then completed the System Usability Scale and qualitative semistructured interviews.ResultsThe study sample comprised mostly African American people, women, rural county coordinators, and research staff (N=10). Participants rated the IVR system with a mean score of 81 (SD 5) on the System Usability Scale, implying excellent usability. In total, 5 overarching themes emerged from the qualitative interviews: likes or dislikes of the intervention, barriers to or facilitators of PA, technical difficulties, quality of calls, and suggestions for intervention improvement. Message framing on step feedback, call completion incentives, and incremental goal-setting challenges were areas identified for improvement. The positive areas highlighted in the interviews included the personalized call schedules, flexibility to call in or receive a call, ability to make up for missed calls, narration, and PA tips.ConclusionsThe usability testing and feedback received from the rural county coordinators and research staff helped inform a final round of refinement to the IVR system before use in a large randomized controlled trial. This study stresses the importance of usability testing of all digital health interventions and the benefits it can offer to the intervention.

Co-design and qualitative validation of animated assessment item content for a child-reported digital distress screener

PurposeThe Interactive Child Distress Screener (ICDS) is a novel, digital screening tool that is currently under development and aims to broadly assess self-reported emotional and behavioural distress in children aged five to 11 years. This study implemented a generative participatory codesign and iterative refinement process to qualitatively validate the content of 30 animated assessment items developed for the ICDS by assessing their acceptability and accuracy from the child's perspective. MethodsThe participants (N = 62) were five to 11-year-old children. Individual interviews were conducted with each child to determine acceptability and validity of animated items and facilitate the co-design refinement process of the animated assessment items. ResultsTwenty-two out of 30 (73%) items met ≥80% satisfaction and accuracy consensus in their original format, six items (20%) required one round of refinement before meeting consensus, and two items (7%) required two rounds of refinements. Combined acceptability of animated items was high, ranging from 4.1 to 5 out of 5 across all items. ConclusionParticipants were able to accurately identify and understand socio-emotional and behavioural constructs when depicted as animated items. Acceptability was high, even in first iterations when accuracy of understanding required refinement. This study highlighted the importance and benefits of iterative participatory design methodology in ensuring assessment items developed for children are understood, accepted and likely to be effective in obtaining accurate self-report.

DosePredict: A Shiny Application for Generalized Pharmacokinetics-Based Dose Predictions.

In drug development, dose prediction is a routinely conducted quantitative data analysis. It involves combining available relevant preclinical and/or clinical data to conduct modeling and simulation analyses where various dose prediction scenarios are usually considered. As further data emerges during drug development, dose prediction may undergo several rounds of refinement. In this article, I present DosePredict, a Shiny-based graphical user interface software that can be used for the conduct of dose predictions. DosePredict is built based on pharmacokinetic (PK) models, including 1-, 2-, and 3-compartment models. The user specifies the desired PK model and then provides input parameter values and variables associated with the evaluated dose prediction scenario. On that basis, DosePredict conducts stochastic simulations for a specified number of subjects and follows that by providing detailed PK and dose prediction outputs. An HTML report can then be generated in which all the input parameters and variables and all the output plots and tables are captured. DosePredict is deployed through the Shiny server. A Shiny dose prediction software, DosePredict, was developed to assist scientists involved in the drug discovery and development process in their dose prediction analyses. This software comes as a step forward in developing individualized solutions as it provides a comprehensive dose prediction analysis. The software allows for expediting the analysis process in a reproducible manner, therefore allowing exploration of various scenarios in a timely fashion.

Enhancing the SCIROCCO tool using quantitative and qualitative methods

Background: The SCIROCCO tool, developed as part of the SCIROCCO project, incorporates the Maturity Model for the adoption of Integrated Care developed by the European Innovation Partnership on Active and Healthy Ageing (B3 Action Group on Integrated Care). The online self-assessment tool can be used to facilitate scaling-up of integrated care across different health and care sectors, at different levels in health or care systems, and in a range of communities. Currently, more than 30 European regions are involved in testing the SCIROCCO tool. Since the project launch in April 2016, the tool underwent two iterations. Each of the iterations of the tool was followed by a round of refinement to inform its subsequent development so that the tool increases its usability and usefulness. Moreover, several focus group sessions were conducted to gather end-users’ experiences and perspectives on the tool. Aims and Objectives: The three-fold objective of this workshop is to present the SCIROCCO tool and the learnings that have taken place around it: Use of the tool to facilitate knowledge transfer and scaling-up of integrated care. Quantitative and qualitative evaluation methodologies used for the SCIROCCO tool construction, refinement, and improvement. Future directions for the use of the tool. The workshop is also intended to gather feedback from the workshop participants on their opinions and perspectives about the SCIROCCO tool and its potential (policy) impact. Format (timing, speakers, discussion, group work, etc.): A workshop session of 90 minutes in length will be conducted. Four individual members of the SCIROCCO project will be responsible for conducting the workshop (the chair, and three people making two presentations). The session will be chaired by a SCIROCCO project leader, who will introduce the workshop. A set of presentations will follow, covering the different session objectives. First, a presentation on the tool construction and use will be given. This will be followed by a second presentation on the users’ experiences and perspectives on the SCIROCCO tool collected through focus group sessions in four European regions. This second presentation will include the learnings about the future directions of the SCIROCCO tool and its policy implications. Participants will be asked to actively join in the discussion on the further enhancement of SCIROCCO tool. Target audience: This workshop targets all stakeholders interested in the scaling-up of integrated care and the development of new tools that help to understand this complex process. Learnings/Take aways: Workshop participants will learn about: The development of the SCIROCCO online self-assessment tool. The experiences in using the SCIROCCO tool to facilitate the learning and scaling-up of integrated care in more than 30 European regions. Policy impact of the SCIROCCO tool. The project itself will benefit from this workshop by: Promoting the SCIROCCO tool and sharing the insights of users of the SCIROCCO tool. Collecting feedback on the SCIROCCO tool’s usability and potential policy implications.

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Error bounds for Loop subdivision surfaces

In this paper, we obtain the error bounds on the distance between a Loop subdivision surface and its control mesh. Both local and global bounds are derived by means of computing and analysing the control meshes with two rounds of refinement directly. The bounds can be expressed with the maximum edge length of all triangles in the initial control mesh. Our results can be used as posterior estimates and also can be used to predict the subdivision depth for any given tolerance.

The Open Conformation of the Mg2+ Channel CorA from Solvent Accessibility and Distance Constraints

Using a combination of electrophysiology and spectroscopic approaches we have recently shown that CorA acts as a Mg2+-gated Mg2+-selective channel. NiEddA/O2 accessibility together with intersubunit distances were measured in two conditions: 1) Saturating Mg2+, which stabilizes CorA in the closed conformation and 2) The nominal absence of divalent ions, which stabilizes the open state. Here, we use a computational approach for incorporating both solvent accessibility data and distance constraints through restrained molecular dynamics (MD) simulations using CorA in a closed conformation as the starting structure. The accessibility restraints were enforced through interactions between a pseudoatom representation of the spin-label and environmental probe-surrounding particles. Intersubunit Cβ-Cβ distances estimated from DEER experiments, together with the accessibility data were used to generate hundreds of models by varying the upper and lower bound of distance constraints and increasing the number of MD refining cycles. The top 25 models show structural convergence especially around the stalk and inner helices, where the EPR restraints were imposed. After a second round of refinement, the stability of the top ranked structure was evaluated by an all-atom MD simulation in a fully hydrated phospholipid bilayer. After 2ns of the simulation, the RMSD of the stalk and inner helices is stable around 4-5A. Based on the pore-radius profile, the permeation pathway has dilated enough to allow a hydrated Mg2+, as expected if this conformation is conductive. A linear interpolation between the closed and open conformations suggests a gating mechanism for CorA, where the tips of the stalk helix come together like the ribs of an umbrella. After a kink, this motion translates into an expansion of the cavity and the mouth of the pore opens up with a motion reminiscent of an iris of a camera.

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.

Transference of responsibility model goals to the school environment: exploring the impact of a coaching club program

Background: The Teaching Personal and Social Responsibility Model (TPSR) has been used throughout the USA and in several other countries to integrate systematically life skill development within physical activity-based programs. While TPSR is widely used in practice and has a growing empirical base, few studies have examined the degree of transference of its four primary goals beyond the scope of a given program. In addition, the field of youth development has charged programs to not only integrate life skill development within program content, but to provide the transference of these skills beyond the program. Aims: To examine the degree of transference of the four primary TPSR goals from a Coaching Club program to the participants' school environment. Method: Thirteen African American and Pacific Islander students (11 boys and 2 girls) at an underserved urban K–5 elementary school. Adult participants included two classroom teachers and the extended day program director. The Coaching Club used team sports as a vehicle for teaching life skills and promoting transference. The program took place once a week for two consecutive years (45 sessions), and adhered to TPSR format and strategies. An interpretivist/constructivist approach to program evaluation was used. Multiple qualitative data sources were used to understand experiences, perceptions, and individualized outcomes associated with the program. Data were organized around the following a priori themes related to the four TPSR goals: (a) respecting the rights and feelings of others; (b) effort and teamwork; (c) self-direction and goal setting; (d) and leadership and helping. Semi-structured interviews were conducted with youth and adult participants. Other data sources included documents and artifacts from the program such as attendance records, lesson plans, field notes, and participant journals. A combination of inductive and deductive analysis strategies was used to form and refine initial interpretations throughout the data collection process. Through several rounds of refinement and data reduction, prominent patterns were extracted that characterized the data. Strategies to establish trustworthiness included member checks, peer debriefing, data triangulation, checks for disconforming evidence, and audit trail. Results: This study provided sufficient evidence from both youth participants and adult participants to support transference of the four primary TPSR goals to the school environment. Additional strategies for enhancing TPSR transference are provided. Conclusions: Establishing meaningful pedagogical relationships is a cornerstone in youth development programs. TPSR cultivates a positive environment that creates self-growth and concern for others. In this regard, TPSR is representative of the broader field of youth development. Findings may inform policy and practice related not only to TPSR, but also to the broader field of youth development through physical activity.

Result Refinement in Web Services Retrieval Based on Multiple Instances Learning

Web services retrieval is a critical step for reusing existing services in the SOA paradigm. In the UDDI registry, traditional category-based approaches have been used to locate candidate services. However, these approaches usually achieve relatively low precision because some candidate Web Services in the result set cannot provide actually suitable operations for users. In this article, we present a new approach to improve this kind of category-based Web Services retrieval process that can refine the coarse matching results step by step. The refinement is based on the idea that operation specification is very important to service reuse. Therefore, a Web Service is investigated via multiple instances view in our approach, which indicates that a service is labeled as positive if and only if at least one operation provided by this service is usable to the user. Otherwise, it is labeled as negative. Experimental results demonstrate that our approach can increase the retrieval precision to a certain extent after one or two rounds of refinement.

Three-dimensional Structure of the EphB2 Receptor in Complex with an Antagonistic Peptide Reveals a Novel Mode of Inhibition

The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of approximately 15 microm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-A resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.

Structure of Synaptophysin: A Hexameric MARVEL-Domain Channel Protein

Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 Aå Resolution

The structure of bovine F(1)-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9A resolution. This structure has been compared with the previously described structure of bovine F(1)-ATPase determined at 1.95A resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the beta(DP)-subunit. In the present structure, the nucleotide binding sites in the beta(DP)- and beta(TP)-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the beta(TP) site was occupied by AMP-PNP and the beta(DP) site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, alphaArg-373 differs in the beta(DP)- and beta(TP)-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.

Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca(2+)-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65 A resolution and a calcium-free state with bound CPA and ADP at 3.4A resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1-M4. Inhibition of Ca(2+)-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln(56), Leu(61), Val(62), and Asn(101). We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg(489) of the nucleotide-binding domain and Arg(678) of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.