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1.  Automating crystallographic structure solution and refinement of protein–ligand complexes 
A software system for automated protein–ligand crystallography has been implemented in the Phenix suite. This significantly reduces the manual effort required in high-throughput crystallographic studies.
High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.
doi:10.1107/S139900471302748X
PMCID: PMC3919266  PMID: 24419387
protein–ligand complexes; automation; crystallographic structure solution and refinement
2.  Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis 
PLoS ONE  2013;8(12):e84669.
Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.
doi:10.1371/journal.pone.0084669
PMCID: PMC3887118  PMID: 24416096
3.  Ligand placement based on prior structures: the guided ligand-replacement method 
A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available.
The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure.
doi:10.1107/S1399004713030071
PMCID: PMC3919265  PMID: 24419386
ligand placement; guided ligand-replacement method; GLR
4.  High throughput nanostructure-initiator mass spectrometry screening of microbial growth conditions for maximal β-glucosidase production 
Production of biofuels via enzymatic hydrolysis of complex plant polysaccharides is a subject of intense global interest. Microbial communities are known to express a wide range of enzymes necessary for the saccharification of lignocellulosic feedstocks and serve as a powerful reservoir for enzyme discovery. However, the growth temperature and conditions that yield high cellulase activity vary widely, and the throughput to identify optimal conditions has been limited by the slow handling and conventional analysis. A rapid method that uses small volumes of isolate culture to resolve specific enzyme activity is needed. In this work, a high throughput nanostructure-initiator mass spectrometry (NIMS)-based approach was developed for screening a thermophilic cellulolytic actinomycete, Thermobispora bispora, for β-glucosidase production under various growth conditions. Media that produced high β-glucosidase activity were found to be I/S + glucose or microcrystalline cellulose (MCC), Medium 84 + rolled oats, and M9TE + MCC at 45°C. Supernatants of cell cultures grown in M9TE + 1% MCC cleaved 2.5 times more substrate at 45°C than at all other temperatures. While T. bispora is reported to grow optimally at 60°C in Medium 84 + rolled oats and M9TE + 1% MCC, approximately 40% more conversion was observed at 45°C. This high throughput NIMS approach may provide an important tool in discovery and characterization of enzymes from environmental microbes for industrial and biofuel applications.
doi:10.3389/fmicb.2013.00365
PMCID: PMC3854461  PMID: 24367356
NIMS; high throughput; β-glucosidase; enzymatic activity screening; microbial communities
5.  Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass 
PLoS ONE  2013;8(11):e79725.
Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.
doi:10.1371/journal.pone.0079725
PMCID: PMC3828181  PMID: 24244549
6.  Model morphing and sequence assignment after molecular replacement 
A procedure for model building is described that combines morphing a model to match a density map, trimming the morphed model and aligning the model to a sequence.
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.
doi:10.1107/S0907444913017770
PMCID: PMC3817698  PMID: 24189236
morphing; model building; sequence assignment; model–map correlation; loop-building
7.  Phaser.MRage: automated molecular replacement 
The functionality of the molecular-replacement pipeline phaser.MRage is introduced and illustrated with examples.
Phaser.MRage is a molecular-replacement automation framework that implements a full model-generation workflow and provides several layers of model exploration to the user. It is designed to handle a large number of models and can distribute calculations efficiently onto parallel hardware. In addition, phaser.MRage can identify correct solutions and use this information to accelerate the search. Firstly, it can quickly score all alternative models of a component once a correct solution has been found. Secondly, it can perform extensive analysis of identified solutions to find protein assemblies and can employ assembled models for subsequent searches. Thirdly, it is able to use a priori assembly information (derived from, for example, homologues) to speculatively place and score molecules, thereby customizing the search procedure to a certain class of protein molecule (for example, antibodies) and incorporating additional biological information into molecular replacement.
doi:10.1107/S0907444913022750
PMCID: PMC3817702  PMID: 24189240
molecular replacement; pipeline; automation; phaser.MRage
8.  Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus  
In an effort to better understand the control of the formation of branched fatty acids in Micrococcus luteus, the structure of β-ketoacyl-ACP synthase III, which catalyzes the initial step of fatty-acid biosynthesis, has been determined.
Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-­head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.
doi:10.1107/S0907444912028351
PMCID: PMC3447401  PMID: 22993086
biofuels; β-ketoacyl-ACP synthase III; iso- and anteiso-branched alkenes; microarray
9.  Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates 
Introduction
Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules.
Method
We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements.
Results
Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film.
Conclusion
Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.
doi:10.1186/1754-6834-6-93
PMCID: PMC3716932  PMID: 23819686
Cellulases; Endoglucanases; Carbohydrate-Binding modules; Cellulose model films; Neutron reflectometry
10.  The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: A Structural Biology Web Resource 
The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB (http://sbkb.org), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a Functional Sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.
doi:10.1007/s10969-012-9133-7
PMCID: PMC3588887  PMID: 22527514
Database; Protein; Protein Production; Structural Biology; Structural Genomics; Technology
11.  Survey of renewable chemicals produced from lignocellulosic biomass during ionic liquid pretreatment 
Background
Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. We present an approach to produce these chemicals based on the selective breakdown of lignin during ionic liquid pretreatment.
Results
The lignin breakdown products generated are found to be dependent on the starting biomass, and significant levels were generated on dissolution at 160°C for 6 hrs. Guaiacol was produced on dissolution of biomass and technical lignins. Vanillin was produced on dissolution of kraft lignin and eucalytpus. Syringol and allyl guaiacol were the major products observed on dissolution of switchgrass and pine, respectively, whereas syringol and allyl syringol were obtained by dissolution of eucalyptus. Furthermore, it was observed that different lignin-derived products could be generated by tuning the process conditions.
Conclusions
We have developed an ionic liquid based process that depolymerizes lignin and converts the low molecular weight lignin fractions into a variety of renewable chemicals from biomass. The generated chemicals (phenols, guaiacols, syringols, eugenol, catechols), their oxidized products (vanillin, vanillic acid, syringaldehyde) and their easily derivatized hydrocarbons (benzene, toluene, xylene, styrene, biphenyls and cyclohexane) already have relatively high market value as commodity and specialty chemicals, green building materials, nylons, and resins.
doi:10.1186/1754-6834-6-14
PMCID: PMC3579681  PMID: 23356589
Lignin valorization; Ionic liquid pretreatment; Renewable chemicals; Biofuels
12.  Intensity statistics in the presence of translational noncrystallographic symmetry 
The statistical effects of translational noncrystallographic symmetry can be characterized by maximizing parameters describing the noncrystallographic symmetry in a likelihood function, thereby unmasking the competing statistical effects of twinning.
In the case of translational noncrystallographic symmetry (tNCS), two or more copies of a component in the asymmetric unit of the crystal are present in a similar orientation. This causes systematic modulations of the reflection intensities in the diffraction pattern, leading to problems with structure determination and refinement methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm to estimate these parameters and refine their values against a likelihood function is presented, and it is shown that by accounting for the statistical effects of tNCS it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning.
doi:10.1107/S0907444912045374
PMCID: PMC3565438  PMID: 23385454
translational noncrystallographic symmetry; intensity statistics; twinning; maximum likelihood
13.  Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis 
Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength.
doi:10.3389/fpls.2013.00083
PMCID: PMC3622038  PMID: 23596448
xylan; irregular xylan mutants; cell walls; type II cell walls; xylosyltransferase
14.  Modelling dynamics in protein crystal structures by ensemble refinement 
eLife  2012;1:e00311.
Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships.
DOI: http://dx.doi.org/10.7554/eLife.00311.001
eLife digest
It has been clear since the early days of structural biology in the late 1950s that proteins and other biomolecules are continually changing shape, and that these changes have an important influence on both the structure and function of the molecules. X-ray diffraction can provide detailed information about the structure of a protein, but only limited information about how its structure fluctuates over time. Detailed information about the dynamic behaviour of proteins is essential for a proper understanding of a variety of processes, including catalysis, ligand binding and protein–protein interactions, and could also prove useful in drug design.
Currently most of the X-ray crystal structures in the Protein Data Bank are ‘snap-shots’ with limited or no information about protein dynamics. However, X-ray diffraction patterns are affected by the dynamics of the protein, and also by distortions of the crystal lattice, so three-dimensional (3D) models of proteins ought to take these phenomena into account. Molecular-dynamics (MD) computer simulations transform 3D structures into 4D ‘molecular movies’ by predicting the movement of individual atoms.
Combining MD simulations with crystallographic data has the potential to produce more realistic ensemble models of proteins in which the atomic fluctuations are represented by multiple structures within the ensemble. Moreover, in addition to improved structural information, this process—which is called ensemble refinement—can provide dynamical information about the protein. Earlier attempts to do this ran into problems because the number of model parameters needed was greater than the number of observed data points. Burnley et al. now overcome this problem by modelling local molecular vibrations with MD simulations and, at the same time, using a course-grain model to describe global disorder of longer length scales.
Ensemble refinement of high-resolution X-ray diffraction datasets for 20 different proteins from the Protein Data Bank produced a better fit to the data than single structures for all 20 proteins. Ensemble refinement also revealed that 3 of the 20 proteins had a ‘molten core’, rather than the well-ordered residues core found in most proteins: this is likely to be important in various biological functions including ligand binding, filament formation and enzymatic function. Burnley et al. also showed that a HIV enzyme underwent an order–disorder transition that is likely to influence how this enzyme works, and that similar transitions might influence the interactions between the small-molecule drug Imatinib (also known as Gleevec) and the enzymes it targets. Ensemble refinement could be applied to the majority of crystallography data currently being collected, or collected in the past, so further insights into the properties and interactions of a variety of proteins and other biomolecules can be expected.
DOI: http://dx.doi.org/10.7554/eLife.00311.002
doi:10.7554/eLife.00311
PMCID: PMC3524795  PMID: 23251785
protein; crystallography; structure; function; dynamics; None
15.  Improving the accuracy of macromolecular structure refinement at 7 Å resolution 
SUMMARY
In X-ray crystallography, molecular replacement and subsequent refinement is challenging at low resolution. We compared refinement methods using synchrotron diffraction data of photosystem I at 7.4 Å resolution, starting from different initial models with increasing deviations from the known high-resolution structure. Standard refinement spoiled the initial models moving them further away from the true structure and leading to high Rfree-values. In contrast, DEN-refinement improved even the most distant starting model as judged by Rfree, atomic root-mean-square differences to the true structure, significance of features not included in the initial model, and connectivity of electron density. The best protocol was DEN-refinement with initial segmented rigid-body refinement. For the most distant initial model, the fraction of atoms within 2 Å of the true structure improved from 24% to 60%. We also found a significant correlation between Rfree-values and the accuracy of the model, suggesting that Rfree is useful even at low resolution.
doi:10.1016/j.str.2012.04.020
PMCID: PMC3380535  PMID: 22681901
DEN refinement; membrane protein; low-resolution refinement; simulated annealing; free R value
16.  Supplementation of Intracellular XylR Leads to Coutilization of Hemicellulose Sugars 
Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy of xylR was inserted into the araBAD operon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of an E. coli biocatalyst that can simultaneously convert all biomass sugars.
doi:10.1128/AEM.06761-11
PMCID: PMC3302627  PMID: 22286982
17.  The Phenix Software for Automated Determination of Macromolecular Structures 
Methods (San Diego, Calif.)  2011;55(1):94-106.
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 favour 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.
doi:10.1016/j.ymeth.2011.07.005
PMCID: PMC3193589  PMID: 21821126
Macromolecular Crystallography; Automation; Phenix; X-ray; Diffraction; Python
18.  Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions 
Background
Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents.
Results
Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails.
Conclusions
T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.
doi:10.1186/1754-6834-5-54
PMCID: PMC3507748  PMID: 22839529
Thermoascus aurantiacus; Thielavia terrestris; GH 61; Polysaccharide monooxygenases; Fungal secretome; Ammonia fiber expansion; Ionic liquid; 1-ethyl-3-methylimidazolium acetate; Switchgrass (Panicum virgatum)
19.  A switch I mutant of Cdc42 exhibits decreased conformational freedom 
Biochemistry  2011;50(28):6196-6207.
Cdc42 is a Ras-related small G-protein, and functions as a molecular switch in signal transduction pathways linked with cell growth and differentiation. It is controlled by cycling between GTP-bound (active) and GDP-bound (inactive) forms. Nucleotide binding and hydrolysis are modulated by interactions with effectors and/or regulatory proteins. These interactions are centralized in two relatively flexible “Switch” regions as characterized by internal dynamics on multiple timescales (Loh et al., (2001) Biochemistry 40, 4590–4600), and this flexibility may be essential for protein interactions. In the Switch I region, Thr35 seems critical for function, as it is completely invariant in Ras-related proteins. To investigate the importance of conformational flexibility in Switch I of Cdc42, we mutated threonine to alanine, determined the solution structure and characterized the backbone dynamics of the single-point mutant protein, Cdc42(T35A). Backbone dynamics data suggests that the mutation changes the timescale of the internal motions of several residues, with several resonances appearing not discernable in Cdc42 wild type (Adams and Oswald (2007) Biomolecular NMR Assignments 1, 225–227). The mutation does not appear to affect the thermal stability of Cdc42, and chymotrypsin digestion data further suggests that changes in conformational flexibility in Switch I slow proteolytic cleavage relative to wild type. In-vitro binding assays show reduced binding of Cdc42(T35A), relative to wild type, to a GTPase binding protein that inhibits GTP hydrolysis in Cdc42. These results suggest that the mutation of T35 leads to the loss of conformational freedom in Switch I that could affect effector/regulatory protein interactions.
doi:10.1021/bi2004284
PMCID: PMC3134622  PMID: 21667996
Ras GTPase; Signal transduction; Cdc42; Threonine; Alanine; Switch 1 mutant; conformational flexibility; backbone dynamics
20.  Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization 
BMC Biotechnology  2012;12:38.
Background
Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals.
Results
Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C.
Conclusions
Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.
doi:10.1186/1472-6750-12-38
PMCID: PMC3477009  PMID: 22759983
21.  Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement 
A density-based procedure is described for improving a homology model that is locally accurate but differs globally. The model is deformed to match the map and refined, yielding an improved starting point for density modification and further model-building.
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 Å radius 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 Å r.m.s.d. representing 65–100% of the structure.
doi:10.1107/S0907444912015636
PMCID: PMC3388814  PMID: 22751672
molecular replacement; automation; macromolecular crystallography; structure similarity; modeling; Phenix; morphing
22.  Automatic Fortran to C++ conversion with FABLE 
Background
In scientific computing, Fortran was the dominant implementation language throughout most of the second part of the 20th century. The many tools accumulated during this time have been difficult to integrate with modern software, which is now dominated by object-oriented languages.
Results
Driven by the requirements of a large-scale scientific software project, we have developed a Fortran to C++ source-to-source conversion tool named FABLE. This enables the continued development of new methods even while switching languages. We report the application of FABLE in three major projects and present detailed comparisons of Fortran and C++ runtime performances.
Conclusions
Our experience suggests that most Fortran 77 codes can be converted with an effort that is minor (measured in days) compared to the original development time (often measured in years). With FABLE it is possible to reuse and evolve legacy work in modern object-oriented environments, in a portable and maintainable way. FABLE is available under a nonrestrictive open source license. In FABLE the analysis of the Fortran sources is separated from the generation of the C++ sources. Therefore parts of FABLE could be reused for other target languages.
doi:10.1186/1751-0473-7-5
PMCID: PMC3448510  PMID: 22640868
Fortran; C++; Source-to-source conversion; Python; Test-driven development
23.  Graphical tools for macromolecular crystallography in PHENIX  
Journal of Applied Crystallography  2012;45(Pt 3):581-586.
The foundations and current features of a widely used graphical user interface for macromolecular crystallography are described.
A new Python-based graphical user interface for the PHENIX suite of crystallography software is described. This interface unifies the command-line programs and their graphical displays, simplifying the development of new interfaces and avoiding duplication of function. With careful design, graphical interfaces can be displayed automatically, instead of being manually constructed. The resulting package is easily maintained and extended as new programs are added or modified.
doi:10.1107/S0021889812017293
PMCID: PMC3359726  PMID: 22675231
macromolecular crystallography; graphical user interfaces; PHENIX
24.  Towards automated crystallographic structure refinement with phenix.refine  
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.
doi:10.1107/S0907444912001308
PMCID: PMC3322595  PMID: 22505256
structure refinement; PHENIX; joint X-ray/neutron refinement; maximum likelihood; TLS; simulated annealing; subatomic resolution; real-space refinement; twinning; NCS
25.  Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution 
Recent developments in PHENIX are reported that allow the use of reference-model torsion restraints, secondary-structure hydrogen-bond restraints and Ramachandran restraints for improved macromolecular refinement in phenix.refine at low resolution.
Traditional methods for macromolecular refinement often have limited success at low resolution (3.0–3.5 Å or worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a ‘reference-model’ method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from non­redundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered R free and a decreased gap between R work and R free.
doi:10.1107/S0907444911047834
PMCID: PMC3322597  PMID: 22505258
macromolecular crystallography; low resolution; refinement; automation

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