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1.  High resolution microtubule structures reveal the structural transitions in αβ–tubulin upon GTP hydrolysis 
Cell  2014;157(5):1117-1129.
Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice, and is inhibited by anticancer agents like Taxol. We provide new insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7–5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α–tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α– and β–tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.
PMCID: PMC4054694  PMID: 24855948
2.  Utilization of a Web-Based vs Integrated Phone/Web Cessation Program Among 140,000 Tobacco Users: An Evaluation Across 10 Free State Quitlines 
Phone-based tobacco cessation program effectiveness has been established and randomized controlled trials have provided some support for Web-based services. Relatively little is known about who selects different treatment modalities and how they engage with treatments in a real-world setting.
This paper describes the characteristics, Web utilization patterns, and return rates of tobacco users who self-selected into a Web-based (Web-Only) versus integrated phone/Web (Phone/Web) cessation program.
We examined the demographics, baseline tobacco use, Web utilization patterns, and return rates of 141,429 adult tobacco users who self-selected into a Web-Only or integrated Phone/Web cessation program through 1 of 10 state quitlines from August 2012 through July 2013. For each state, registrants were only included from the timeframe in which both programs were offered to all enrollees. Utilization data were limited to site interactions occurring within 6 months after registration.
Most participants selected the Phone/Web program (113,019/141,429, 79.91%). After enrollment in Web services, Web-Only were more likely to log in compared to Phone/Web (21,832/28,410, 76.85% vs 23,920/56,892, 42.04%; P<.001), but less likely to return after their initial log-in (8766/21,832, 40.15% vs 13,966/23,920, 58.39%; P<.001). In bivariate and multivariable analyses, those who chose Web-Only were younger, healthier, more highly educated, more likely to be uninsured or commercially insured, more likely to be white non-Hispanic and less likely to be black non-Hispanic, less likely to be highly nicotine-addicted, and more likely to have started their program enrollment online (all P<.001). Among both program populations, participants were more likely to return to Web services if they were women, older, more highly educated, or were sent nicotine replacement therapy (NRT) through their quitline (all P<.001). Phone/Web were also more likely to return if they had completed a coaching call, identified as white non-Hispanic or “other” race, or were commercially insured (all P<.001). Web-Only were less likely to return if they started their enrollment online versus via phone. The interactive Tobacco Tracker, Cost Savings Calculator, and Quitting Plan were the most widely used features overall. Web-Only were more likely than Phone/Web to use most key features (all P<.001), most notably the 5 Quitting Plan behaviors. Among quitlines that offered NRT to both Phone/Web and Web-Only, Web-Only were less likely to have received quitline NRT.
This paper adds to our understanding of who selects different cessation treatment modalities and how they engage with the program in a real-world setting. Web-Only were younger, healthier smokers of higher socioeconomic status who interacted more intensely with services in a single session, but were less likely to re-engage or access NRT benefits. Further research should examine the efficacy of different engagement techniques and services with different subpopulations of tobacco users.
PMCID: PMC4342619  PMID: 25673013
Internet-based intervention; tobacco cessation; smoking cessation; Internet; telephone; behavior; evaluation studies; online support
3.  Role of conformational sampling in computing mutation-induced changes in protein structure and stability 
Proteins  2010;79(3):830-838.
The prediction of changes in protein stability and structure resulting from single amino acid substitutions is both a fundamental test of macromolecular modeling methodology and an important current problem as high throughput sequencing reveals sequence polymorphisms at an increasing rate. In principle, given the structure of a wild-type protein and a point mutation whose effects are to be predicted, an accurate method should recapitulate both the structural changes and the change in the folding-free energy. Here, we explore the performance of protocols which sample an increasing diversity of conformations. We find that surprisingly similar performances in predicting changes in stability are achieved using protocols that involve very different amounts of conformational sampling, provided that the resolution of the force field is matched to the resolution of the sampling method. Methods involving backbone sampling can in some cases closely recapitulate the structural changes accompanying mutations but not surprisingly tend to do more harm than good in cases where structural changes are negligible. Analysis of the outliers in the stability change calculations suggests areas needing particular improvement; these include the balance between desolvation and the formation of favorable buried polar interactions, and unfolded state modeling.
PMCID: PMC3760476  PMID: 21287615
ΔΔG prediction; protein stability; backbone flexibility; free energy change
4.  Scientific Benchmarks for Guiding Macromolecular Energy Function Improvement 
Methods in enzymology  2013;523:109-143.
Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011).
PMCID: PMC3724755  PMID: 23422428
Rosetta; energy function; scientific benchmarking; parameter estimation; decoy discrimination
5.  Structure prediction for CASP8 with all-atom refinement using Rosetta 
Proteins  2009;77(0 9):89-99.
We describe predictions made using the Rosetta structure prediction methodology for the Eighth Critical Assessment of Techniques for Protein Structure Prediction. Aggressive sampling and all-atom refinement were carried out for nearly all targets. A combination of alignment methodologies was used to generate starting models from a range of templates, and the models were then subjected to Rosetta all atom refinement. For 50 targets with readily identified templates, the best submitted model was better than the best alignment to the best template in the Protein Data Bank for 24 domains, and improved over the best starting model for 43 domains. For 13 targets where only very distant sequence relationships to proteins of known structure were detected, models were generated using the Rosetta de novo structure prediction methodology followed by all-atom refinement; in several cases the submitted models were better than those based on the available templates. Of the 12 refinement challenges, the best submitted model improved on the starting model in 7 cases. These improvements over the starting template-based models and refinement tests demonstrate the power of Rosetta structure refinement in improving model accuracy.
PMCID: PMC3688471  PMID: 19701941
6.  Early inflorescence development in the grasses (Poaceae) 
The shoot apical meristem of grasses produces the primary branches of the inflorescence, controlling inflorescence architecture and hence seed production. Whereas leaves are produced in a distichous pattern, with the primordia separated from each other by an angle of 180°, inflorescence branches are produced in a spiral in most species. The morphology and developmental genetics of the shift in phyllotaxis have been studied extensively in maize and rice. However, in wheat, Brachypodium, and oats, all in the grass subfamily Pooideae, the change in phyllotaxis does not occur; primary inflorescence branches are produced distichously. It is unknown whether the distichous inflorescence originated at the base of Pooideae, or whether it appeared several times independently. In this study, we show that Brachyelytrum, the genus sister to all other Pooideae has spiral phyllotaxis in the inflorescence, but that in the remaining 3000+ species of Pooideae, the phyllotaxis is two-ranked. These two-ranked inflorescences are not perfectly symmetrical, and have a clear “front” and “back;” this developmental axis has never been described in the literature and it is unclear what establishes its polarity. Strictly distichous inflorescences appear somewhat later in the evolution of the subfamily. Two-ranked inflorescences also appear in a few grass outgroups and sporadically elsewhere in the family, but unlike in Pooideae do not generally correlate with a major radiation of species. After production of branches, the inflorescence meristem may be converted to a spikelet meristem or may simply abort; this developmental decision appears to be independent of the branching pattern.
PMCID: PMC3721031  PMID: 23898335
phyllotaxis; shoot apical meristem; phylogeny; branching; APO1
7.  Five Nuclear Loci Resolve the Polyploid History of Switchgrass (Panicum virgatum L.) and Relatives 
PLoS ONE  2012;7(6):e38702.
Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels.
PMCID: PMC3377691  PMID: 22719924
8.  Taking the First Steps towards a Standard for Reporting on Phylogenies: Minimal Information about a Phylogenetic Analysis (MIAPA) 
In the eight years since phylogenomics was introduced as the intersection of genomics and phylogenetics, the field has provided fundamental insights into gene function, genome history and organismal relationships. The utility of phylogenomics is growing with the increase in the number and diversity of taxa for which whole genome and large transcriptome sequence sets are being generated. We assert that the synergy between genomic and phylogenetic perspectives in comparative biology would be enhanced by the development and refinement of minimal reporting standards for phylogenetic analyses. Encouraged by the development of the Minimum Information About a Microarray Experiment (MIAME) standard, we propose a similar roadmap for the development of a Minimal Information About a Phylogenetic Analysis (MIAPA) standard. Key in the successful development and implementation of such a standard will be broad participation by developers of phylogenetic analysis software, phylogenetic database developers, practitioners of phylogenomics, and journal editors.
PMCID: PMC3167193  PMID: 16901231
9.  Phylogenetic studies favour the unification of Pennisetum, Cenchrus and Odontelytrum (Poaceae): a combined nuclear, plastid and morphological analysis, and nomenclatural combinations in Cenchrus 
Annals of Botany  2010;106(1):107-130.
Backgrounds and Aims
Twenty-five genera having sterile inflorescence branches were recognized as the bristle clade within the x = 9 Paniceae (Panicoideae). Within the bristle clade, taxonomic circumscription of Cenchrus (20–25 species), Pennisetum (80–140) and the monotypic Odontelytrum is still unclear. Several criteria have been applied to characterize Cenchrus and Pennisetum, but none of these has proved satisfactory as the diagnostic characters, such as fusion of bristles in the inflorescences, show continuous variation.
A phylogenetic analysis based on morphological, plastid (trnL-F, ndhF) and nuclear (knotted) data is presented for a representative species sampling of the genera. All analyses were conducted under parsimony, using heuristic searches with TBR branch swapping. Branch support was assessed with parsimony jackknifing.
Key Results
Based on plastid and morphological data, Pennisetum, Cenchrus and Odontelytrum were supported as a monophyletic group: the PCO clade. Only one section of Pennisetum (Brevivalvula) was supported as monophyletic. The position of P. lanatum differed among data partitions, although the combined plastid and morphology and nuclear analyses showed this species to be a member of the PCO clade. The basic chromosome number x = 9 was found to be plesiomorphic, and x = 5, 7, 8, 10 and 17 were derived states. The nuclear phylogenetic analysis revealed a reticulate pattern of relationships among Pennisetum and Cenchrus, suggesting that there are at least three different genomes. Because apomixis can be transferred among species through hybridization, its history most likely reflects crossing relationships, rather than multiple independent appearances.
Due to the consistency between the present results and different phylogenetic hypotheses (including morphological, developmental and multilocus approaches), and the high support found for the PCO clade, also including the type species of the three genera, we propose unification of Pennisetum, Cenchrus and Odontelytrum. Species of Pennisetum and Odontelytrum are here transferred into Cenchrus, which has priority. Sixty-six new combinations are made here.
PMCID: PMC2889798  PMID: 20570830
Pennisetum; Cenchrus; Odontelytrum; Poaceae; phylogenetic analyses; ndhF; trnL-trnF; kn1; apomixis
10.  High Resolution Mapping of Protein Sequence–Function Relationships 
Nature methods  2010;7(9):741-746.
We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity, and high throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position possessed unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.
PMCID: PMC2938879  PMID: 20711194
11.  Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport 
PLoS ONE  2010;5(7):e11688.
A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.
PMCID: PMC2908147  PMID: 20657737
12.  The Plant Ontology Database: a community resource for plant structure and developmental stages controlled vocabulary and annotations 
Nucleic Acids Research  2008;36(Database issue):D449-D454.
The Plant Ontology Consortium (POC, is a collaborative effort among model plant genome databases and plant researchers that aims to create, maintain and facilitate the use of a controlled vocabulary (ontology) for plants. The ontology allows users to ascribe attributes of plant structure (anatomy and morphology) and developmental stages to data types, such as genes and phenotypes, to provide a semantic framework to make meaningful cross-species and database comparisons. The POC builds upon groundbreaking work by the Gene Ontology Consortium (GOC) by adopting and extending the GOC's principles, existing software and database structure. Over the past year, POC has added hundreds of ontology terms to associate with thousands of genes and gene products from Arabidopsis, rice and maize, which are available through a newly updated web-based browser ( for viewing, searching and querying. The Consortium has also implemented new functionalities to facilitate the application of PO in genomic research and updated the website to keep the contents current. In this report, we present a brief description of resources available from the website, changes to the interfaces, data updates, community activities and future enhancement.
PMCID: PMC2238838  PMID: 18194960
13.  Plant Ontology (PO): a Controlled Vocabulary of Plant Structures and Growth Stages 
Comparative and Functional Genomics  2005;6(7-8):388-397.
The Plant Ontology Consortium (POC) ( is a collaborative effort among several plant databases and experts in plant systematics, botany and genomics. A primary goal of the POC is to develop simple yet robust and extensible controlled vocabularies that accurately reflect the biology of plant structures and developmental stages. These provide a network of vocabularies linked by relationships (ontology) to facilitate queries that cut across datasets within a database or between multiple databases. The current version of the ontology integrates diverse vocabularies used to describe Arabidopsis, maize and rice (Oryza sp.) anatomy, morphology and growth stages. Using the ontology browser, over 3500 gene annotations from three species-specific databases, The Arabidopsis Information Resource (TAIR) for Arabidopsis, Gramene for rice and MaizeGDB for maize, can now be queried and retrieved.
PMCID: PMC2447502  PMID: 18629207

Results 1-13 (13)