Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Teres minor muscle hypertrophy is a negative predictor of outcomes after reverse total shoulder arthroplasty: an evaluation of preoperative magnetic resonance imaging and postoperative implant position.

BACKGROUND: Predictors of outcomes after reverse total shoulder arthroplasty (rTSA) remain unclear. The purpose of this study was to analyze the impact of preoperative muscle quality and postoperative implant positioning on patient-reported outcomes following rTSA. METHODS: We evaluated 88 shoulders treated with rTSA in which preoperative magnetic resonance imaging was available. Preoperative muscle quality was evaluated, including fatty infiltration, rotator cuff muscle volume, and total tear size. Postoperative implant position was determined radiographically. The correlation between imaging parameters and the 2-year postoperative American Shoulder and Elbow Surgeons (ASES) score was examined. Multivariate analyses were performed to adjust for confounding factors including patient demographic characteristics and implant position. RESULTS: Univariate analysis showed that the ASES score was significantly lower in patients with teres minor muscle hypertrophy relative to those with normal muscle (73.3 ± 22.8 vs. 84.2 ± 16.9, P = .02). The functional subscore was significantly lower in patients with grade 2 fatty infiltration of the deltoid muscle relative to those with grade 0 fatty infiltration (26.1 ± 14.6 vs. 34.8 ± 11.6, P = .03). Older age was associated with a higher pain subscore (ρ = 0.32, P = .002). Multivariate analysis demonstrated that teres minor muscle hypertrophy remained a significant independent predictor of the ASES score (ß coefficient = 91.3, P = .03). CONCLUSION: Teres minor muscle hypertrophy is an independent negative predictor of patient-reported outcomes after rTSA.

Changes in night sky brightness after a countywide LED retrofit.

The US National Park Service (NPS) Night Skies Program measured changes in sky brightness resulting from a countywide lighting retrofit project. The retrofit took place in Chelan County, a gateway community to North Cascades National Park and Lake Chelan National Recreation Area in Washington State. The county retrofitted all 3693 county-owned high pressure sodium (HPS) street lamps to full cutoff LEDs. This number is about 60% of the County's total outdoor street and area lights. About 80% of the newly installed lights were 3000 K in color temperature and 20% were 4000 K. The 4000 K LEDs were used to meet Washington State Department of Transportation guidelines. To measure sky brightness, we used the NPS night sky camera system before the retrofit started in 2018 and after its completion in 2019. These images were photometrically calibrated and mosaicked together to provide hemispherical images in V band. For comparison with our ground-based measurement, we obtained the satellite imagery taken by Visible Infrared Imaging Radiometer Suite (VIIRS) onboard the Suomi National Polar-orbiting Partnership satellite. Our measurements show that the post-retrofit skyglow became brighter and extended higher in the sky, but upward radiance, as measured by the day-night band radiometer, decreased. These divergent results are likely explained by a substantial increase in light emitted at wavelengths shorter than 500 nm, and a relative decrease in upward light emission due to better shielded luminaires. These results also demonstrate that earlier models relating VIIRS day-night band data to skyglow will - at a minimum - require substantial revision to account for the different characteristics of solid state luminaires.

Structural basis for DNA recognition by STAT6.

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6CF) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNA-binding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.

Outcomes of revision arthroplasty for shoulder periprosthetic joint infection: a three-stage revision protocol.

BACKGROUND: This study evaluated outcomes after treatment of shoulder periprosthetic joint infection (PJI) with a 3-stage revision protocol consisting of (1) débridement, explantation, and cement spacer placement, followed by parenteral antibiotics; (2) open biopsy and débridement; and (3) reimplantation if cultures were negative. We hypothesized this protocol would eradicate persistent infection while producing excellent functional and subjective outcomes, and there would be no difference in these parameters for patients with shoulder PJI compared with patients with revision for aseptic indications. METHODS: We retrospectively analyzed a prospectively collected revision shoulder arthroplasty cohort to identify shoulder PJI patients treated with a 3-stage protocol. Demographics, culture data, range of motion, and patient-reported outcomes were collected. Outcomes for patients with shoulder PJI and revision to RTSA were compared with patients revised to RTSA for noninfectious indications. Significance was defined as P < .05. RESULTS: There were 28 cases of shoulder PJI in 27 patients (age, 66.4 ± 11.2 years,); of these, 21 shoulders were revised to RTSA, and 7 shoulders were revised to hemiarthroplasty. There was no recurrent infection at a mean 32-month follow-up. One year after surgery, mean forward flexion was 110° ± 41° and abduction was 106° ± 42°. Mean final American Shoulder and Elbow Surgeons subjective score was 66.5 ± 23.3. The 21 shoulders with PJI revised to RTSA had no differences for functional and subjective outcomes compared with revised patients without shoulder PJI. CONCLUSIONS: A 3-stage revision protocol for shoulder PJI reliably eradicated infection. Patients with PJI revised to RTSA can have similar outcomes as patients with noninfectious revision to RTSA.

Functional workspace and patient-reported outcomes improve after reverse and total shoulder arthroplasty.

BACKGROUND: Low-cost motion analysis systems (LCMASs) have emerged as easy and practical methods to measure the functional workspace (FWS). Thus, we ventured to apply an LCMAS, the Kinect2 gaming camera, to evaluate the FWS in patients with shoulder osteoarthritis (OA) and patients who underwent total shoulder arthroplasty (TSA) or reverse total shoulder arthroplasty (RTSA). METHODS: A cross-sectional study of participants with OA (n = 53), TSA (n = 70), and RTSA (n = 34) was performed. The FWS as measured by an LCMAS, the American Shoulder and Elbow Surgeons (ASES) Standardized Shoulder Assessment Form score, and the Patient-Reported Outcomes Measurement Information System (PROMIS) score were collected. For participants who underwent TSA or RTSA, the FWS was evaluated at 6, 12, and 24 months postoperatively. The correlation of the FWS with the ASES score and PROMIS score was determined. Significance was set at P < .05. RESULTS: Patients who underwent TSA or RTSA had a significantly higher FWS than patients with shoulder OA at almost all time points. Patients who underwent TSA had a significantly higher FWS than patients who underwent RTSA at 24 months after surgery. PROMIS and ASES scores showed strong correlations with the FWS in patients who underwent TSA (R = 0.75 [P < .001] and R = 0.83 [P < .001], respectively) and RTSA (R = 0.84 [P < .001] and R = 0.73 [P < .001], respectively). CONCLUSION: The FWS measured by an LCMAS is an easy and low-cost method to quantify the reachable space of the hand in patients and shows strong correlations with patient-reported outcome measures. This may be a useful tool to assess upper-extremity range of motion before and after shoulder arthroplasty.

Functional Diversity of Cytotoxic tRNase/Immunity Protein Complexes from Burkholderia pseudomallei.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDI(+) bacteria deploy large CdiA effector proteins, which carry variable C-terminal toxin domains (CdiA-CT). CDI(+) cells also produce CdiI immunity proteins that specifically neutralize cognate CdiA-CT toxins to prevent auto-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(E479) toxin/immunity protein complex from Burkholderia pseudomallei isolate E479. The CdiA-CT(E479) tRNase domain contains a core α/ß-fold that is characteristic of PD(D/E)XK superfamily nucleases. Unexpectedly, the closest structural homolog of CdiA-CT(E479) is another CDI toxin domain from B. pseudomallei 1026b. Although unrelated in sequence, the two B. pseudomallei nuclease domains share similar folds and active-site architectures. By contrast, the CdiI(E479) and CdiI(1026b) immunity proteins share no significant sequence or structural homology. CdiA-CT(E479) and CdiA-CT(1026b) are both tRNases; however, each nuclease cleaves tRNA at a distinct position. We used a molecular docking approach to model each toxin bound to tRNA substrate. The resulting models fit into electron density envelopes generated by small-angle x-ray scattering analysis of catalytically inactive toxin domains bound stably to tRNA. CdiA-CT(E479) is the third CDI toxin found to have structural homology to the PD(D/E)XK superfamily. We propose that CDI systems exploit the inherent sequence variability and active-site plasticity of PD(D/E)XK nucleases to generate toxin diversity. These findings raise the possibility that many other uncharacterized CDI toxins may belong to the PD(D/E)XK superfamily.

High short-term and long-term excess mortality in geriatric patients after hip fracture: a prospective cohort study in Taiwan.

BACKGROUND: Hip fracture has a high mortality rate, but the actual level of long-term excess mortality and its impact on population-wide mortality remains controversial. The present prospective study investigated short- and long-term excess mortality after hip fractures with adjustment of other risk factors. We calculated the population attributable risk proportion (PARP) to assess the impact of each risk factor on excess mortality. METHODS: We recruited 217 elders with hip fractures and 215 age- and sex-matched patients without fractures from the geriatric department of the same hospital. The mean follow-up time was 46.1 months (range: 35 to 57 months). We recorded data on 55 covariates, including baseline details about health, function, and bone mineral density. We used the multivariate Cox proportional hazards model to analyze hazard ratios (HRs) of short-term (<12 months follow-up) and long-term (≧ 2 months follow-up) excess mortality for each covariate and calculated their PARP. RESULTS: Patients with hip fractures had a higher short-term mortality than non-fractured patients, and the long-term excess mortality associated with hip fracture remained high. The significant risk factors for short-term mortality were hip fracture, comorbidities, and lower (below cutoff) Mini Mental State Examination score with HRs of 2.4, 2.3, and 2.3, respectively. Their PARPs were 44.7%, 38.1%, and 34.3%, respectively. The significant risk factors for long-term mortality were hip fracture (HR: 2.7; PARP: 48.0%), lower T-score (HR: 3.3; PARP: 36.2%), lower body mass index (HR: 2.5; PARP: 42.8%), comorbidities (HR: 2.1; PARP: 34.8%), difficulty in activities of daily living (HR: 1.9; PARP: 31.8%), and smoking (HR: 2.5; PARP: 19.2%). CONCLUSIONS: After comprehensive adjustment, hip fracture was a significant risk factor and contributed the most to long-term as well as short-term excess mortality. Its adequate prevention and treatment should be targeted.

Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution.

AcrB is an inner membrane resistance-nodulation-cell division efflux pump and is part of the AcrAB-TolC tripartite efflux system. We have determined the crystal structure of AcrB with bound Linezolid at a resolution of 3.5 Å. The structure shows that Linezolid binds to the A385/F386 loops of the symmetric trimer of AcrB. A conformational change of a loop in the bottom of the periplasmic cleft is also observed.

Split green fluorescent protein as a modular binding partner for protein crystallization.

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP ß-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

Model morphing and sequence assignment after molecular replacement.

A procedure termed `morphing' for improving a model after it has been placed in the crystallographic cell by molecular replacement has recently been developed. Morphing consists of applying a smooth deformation to a model to make it match an electron-density map more closely. Morphing does not change the identities of the residues in the chain, only their coordinates. Consequently, if the true structure differs from the working model by containing different residues, these differences cannot be corrected by morphing. Here, a procedure that helps to address this limitation is described. The goal of the procedure is to obtain a relatively complete model that has accurate main-chain atomic positions and residues that are correctly assigned to the sequence. Residues in a morphed model that do not match the electron-density map are removed. Each segment of the resulting trimmed morphed model is then assigned to the sequence of the molecule using information about the connectivity of the chains from the working model and from connections that can be identified from the electron-density map. The procedure was tested by application to a recently determined structure at a resolution of 3.2 Å and was found to increase the number of correctly identified residues in this structure from the 88 obtained using phenix.resolve sequence assignment alone (Terwilliger, 2003) to 247 of a possible 359. Additionally, the procedure was tested by application to a series of templates with sequence identities to a target structure ranging between 7 and 36%. The mean fraction of correctly identified residues in these cases was increased from 33% using phenix.resolve sequence assignment to 47% using the current procedure. The procedure is simple to apply and is available in the Phenix software package.

Hip fracture risk assessment: artificial neural network outperforms conditional logistic regression in an age- and sex-matched case control study.

BACKGROUND: Osteoporotic hip fractures with a significant morbidity and excess mortality among the elderly have imposed huge health and economic burdens on societies worldwide. In this age- and sex-matched case control study, we examined the risk factors of hip fractures and assessed the fracture risk by conditional logistic regression (CLR) and ensemble artificial neural network (ANN). The performances of these two classifiers were compared. METHODS: The study population consisted of 217 pairs (149 women and 68 men) of fractures and controls with an age older than 60 years. All the participants were interviewed with the same standardized questionnaire including questions on 66 risk factors in 12 categories. Univariate CLR analysis was initially conducted to examine the unadjusted odds ratio of all potential risk factors. The significant risk factors were then tested by multivariate analyses. For fracture risk assessment, the participants were randomly divided into modeling and testing datasets for 10-fold cross validation analyses. The predicting models built by CLR and ANN in modeling datasets were applied to testing datasets for generalization study. The performances, including discrimination and calibration, were compared with non-parametric Wilcoxon tests. RESULTS: In univariate CLR analyses, 16 variables achieved significant level, and six of them remained significant in multivariate analyses, including low T score, low BMI, low MMSE score, milk intake, walking difficulty, and significant fall at home. For discrimination, ANN outperformed CLR in both 16- and 6-variable analyses in modeling and testing datasets (p?

Effects of lateral instability on ankle coupled motions in vivo using 3D fluoroscopy.

Lateral ankle instability (LAI) compromises the normal kinematics of the ankle, affecting activities of daily living. In vitro kinematics of ankles with LAI during single-plane motions are available, but the active control stability of these motions remains unclear. The current study measured the 3D ankle kinematics during unresisted single-plane motion tests using a bi-plane fluoroscope with a CT model-based 2D/3D registration method in 12 patients with LAI and 14 healthy peers. The coupling of the kinematic components at the talocrural and subtalar joints was quantified by the path difference between the forward and return paths of the coupled motion. Significantly increased path differences were found in the subtalar dorsiflexion/plantarflexion and inversion/eversion components during internal/external rotation tests (p < 0.05). During inversion/eversion, significantly reduced tibiocalcaneal ranges of motion and the path differences in the talocrural and subtalar dorsiflexion/plantarflexion components were noted (p < 0.05). The current results suggest that chronic LAI had compromised control stability at the subtalar joint during internal/external rotation tests and a conservative motion control strategy with significantly reduced ranges of motion to maintain good control of out-of-plane motion components in response to direct challenges of the anterior talofibular ligament during inversion/eversion tests. The current results also suggest that, compared to kinematic patterns of individual components, the path difference of the coupled motion may serve as a better measure of the motion control stability of the ankle in differentiating LAI from healthy controls.

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography.

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of atleast 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins' functions were obtained from their interactions with each of a panel of ligands.

Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement.

An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Šradius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Šr.m.s.d. representing 65-100% of the structure.

S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS.

A subset of tumour necrosis factor receptor (TNFR) superfamily members contain death domains in their cytoplasmic tails. Death receptor 6 (DR6) is one such member and can trigger apoptosis upon the binding of a ligand by its cysteine-rich domains (CRDs). The crystal structure of the ectodomain (amino acids 1-348) of human death receptor 6 (DR6) encompassing the CRD region was phased using the anomalous signal from S atoms. In order to explore the feasibility of S-SAD phasing at longer wavelengths (beyond 2.5 Å), a comparative study was performed on data collected at wavelengths of 2.0 and 2.7 Å. In spite of sub-optimal experimental conditions, the 2.7 Å wavelength used for data collection showed potential for S-SAD phasing. The results showed that the R(ano)/R(p.i.m.) ratio is a good indicator for monitoring the anomalous data quality when the anomalous signal is relatively strong, while d''/sig(d'') calculated by SHELXC is a more sensitive and stable indicator applicable for grading a wider range of anomalous data qualities. The use of the `parameter-space screening method' for S-SAD phasing resulted in solutions for data sets that failed during manual attempts. SAXS measurements on the ectodomain suggested that a dimer defines the minimal physical unit of an unliganded DR6 molecule in solution.

The Phenix software for automated determination of macromolecular structures.

X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.

Label-free affinity screening, design and synthesis of inhibitors targeting the Mycobacterium tuberculosis L-alanine dehydrogenase.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.