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research papers
High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB_REDO strategies
aDivision of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, and bDepartment of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
*Correspondence e-mail: firstname.lastname@example.com
A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.
Keywords: carboxypeptidases; metalloproteins; refinement; specificity; zinc enzymes.
3D view: 4b6z
PDB reference: carboxypeptidase, 4b6z
1. Introduction
Carboxypeptidases (CPs) hydrolyse peptide bonds in target proteins to release C-terminal amino acids. These enzymes have been classified into different families according to sequence similarity, mechanism and function (Gomis-Rüth, 2008; Rawlings et al., 2012). One of the largest groups, carrying a single catalytic Zn2+ in the active site, is the M14 family. This is divided into four subfamilies known as M14A, M14B, M14C and M14D. One of the earliest and best studied family members is bovine carboxypeptidase A (CP-A; Christianson & Lipscomb, 1989). In general, M14A and M14B carboxypeptidases either function within the secretory pathway or are themselves secreted. The members of the M14A subfamily are produced as pro-enzymes that are activated following the cleavage of N-terminal segments. The M14B subfamily are produced as active enzymes with a transthyretin-like domain at the C-terminus. The M14C group consists of bacterial enzymes related to γ-D-glutamyl-(L)-meso-diaminopimelate peptidase I (http://merops.sanger.ac.uk/ ) and process components of the bacterial cell wall. Like their M14A relatives, they also carry an N-terminal extension.
The M14D subfamily has only recently been described and consequently there is only limited information regarding their structure and activity. They are referred to as cytosolic carboxypeptidases (CCPs; http://merops.sanger.ac.uk/ ) to reflect their cellular location. A search of available genomic data identified CCPs in many organisms (Otero et al., 2012) and the number of CCP paralogues varies between species (Rodriguez de la Vega et al., 2007). Four mammalian CCP proteins and a homologue from Caenorhabditis elegans called CeCCPP-6 have been identified as deglutamylases (Kimura et al., 2010; Rogowski et al., 2010). In addition to this activity, CCPs might process other C-terminal amino acids. For example, one member of the family has recently been reported to cleave the C-terminal tyrosine from α-tubulin (Sahab et al., 2010). Less is known about the functional activity of the bacterial CCPs. A recent study of Pseudomonas aeruginosa CCP (PaCCP) failed to detect any activity towards known CP substrates, and the possibility has been raised that structural changes might be required to generate an active enzyme (Otero et al., 2012).
Our interest in the bacterial homologues is twofold. It has proven difficult to obtain a recombinant source of mammalian CCP for characterization and therefore the prokaryotic proteins offer a surrogate system to inform on the post-translational modification of tubulin. We also note that in the important Gram-negative pathogen P. aeruginosa the gene encoding the CCP homologue (PA2831) has been identified as essential for the establishment of a lung infection in a murine model (Winstanley et al., 2009), and as such the CCP might represent a potential drug target for an important group of Gram-negative bacteria. As part of a wide-ranging project to advance early-stage drug discovery of novel antibacterials, we have adopted a structure-based approach to drive target assessment (Eadsforth et al., 2012; Moynie et al., 2013). Key to this process is to acquire material for ligand-binding studies: an accurate structural model which can be exploited to inform on aspects of druggability and crystals that might assist in fragment screening (Hunter, 2009). We initiated work on P. aeruginosa CCP (PaCCP; UniProt entry Q9I012) and on Burkholderia cenocepacia CCP (BcCCP; UniProt entry B4EEQ5). We made no progress with PaCCP, but a study has been published by others (Otero et al., 2012). Our work with BcCCP was more successful and here we report the preparation of an efficient recombinant protein-production system and protocols for purification and crystallization together with the high-resolution structure. With only small crystals available, it was necessary to exploit a microfocus beamline to obtain the diffraction data. BcCCP consists of 384 amino acids and forms a tetramer. The asymmetric unit comprises four subunits comprising in excess of 1500 amino acids. With a sizable model and high-resolution diffraction data with which to refine it, we sought an efficient route through the refinement process and incorporated the strategies employed in the PDB_REDO protocols (Joosten et al., 2011, 2012). We compare BcCCP with PaCCP (Otero et al., 2012), with two other bacterial CCP structures in the Protein Data Bank (PDB) and with mammalian carboxypeptidases of defined substrate specificity. These structures identify the key residues and motifs that are important for the stability of the CCP fold and the architecture of the catalytic centre, reveal the presence of a flexible loop at the periphery of the active site and suggest that the substrate of the bacterial proteins is acidic. Moreover, they support a druggability analysis of the active site.
2. Materials and methods
2.1. Reagents, preparation of a recombinant expression system and protein purification
Chemicals and materials were sourced from Sigma–Aldrich and VWR International, except where stated otherwise. Genomic DNA from B. cenocepacia J2315 [Laboratory for Microbiology, Faculty of Sciences, Ghent University (LMG) strain 16656] was the template for PCR with the following primers designed to amplify the BcCCP open reading frame using NdeI and XhoI restriction sites (bold), respectively: 5′-CAT-ATG-ACC-CTT-TCG-ATC-ACC-AG-3′ and 5′-CTC-GAG-TTA-CGC-GAA-CGT-GTC-3′. The PCR product was ligated into pCR-Blunt II-TOPO (Invitrogen) prior to being subcloned into a modified pET-15b vector (Novagen), which produces a histidine-tagged protein with a Tobacco etch virus (TEV) protease site. The hexa-His tag, TEV cleavage site and additional residues from the choice of cloning sites added a total of 24 extra amino acids at the N-terminus. The recombinant plasmid was amplified in Escherichia coli XL-1 Blue cells and the integrity of the gene sequence was verified by DNA Sequencing & Services (Dundee University) before being transformed into E. coli ArcticExpress (DE3) competent cells for protein production. Cells were grown in 1 l Luria–Bertani medium supplemented with 50 µg ml−1 carbenicillin. Gene expression was induced at 310 K using 1 mM isopropyl β-D-1-thiogalactopyranoside and growth continued for 72 h at 286 K.
The cells were harvested by centrifugation (30 min at 3500g and 277 K), resuspended in lysis buffer (50 mM Tris–HCl pH 7.5, 250 mM NaCl) containing DNase I (0.1 mg) and a single tablet of a cocktail of EDTA-free protease inhibitors (Roche) and lysed using a French press at 110 MPa. Insoluble debris was separated by centrifugation at 37 500g for 30 min at 277 K. The soluble fraction was loaded onto a 5 ml HisTrap HP column (GE Healthcare) and a linear imidazole concentration gradient was applied to elute BcCCP. Fractions were analyzed using SDS–PAGE and those containing BcCCP were pooled. The hexa-His tag could not be cleaved, so protein still carrying the affinity tag was further purified using a Superdex 200 26/60 size-exclusion column (GE Healthcare) equilibrated with 50 mM Tris–HCl, 250 mM NaCl pH 7.5. This column had previously been calibrated using the molecular-mass standards thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa) (Bio-Rad; Fig. 1). Selected fractions were pooled, dialyzed into 100 mM sodium acetate, 150 mM NaCl pH 5 and concentrated to 10 mg ml−1 using Amicon Ultra devices (Millipore). The protein concentration was determined spectrophotometrically using a theoretical extinction coefficient of 73 590 M−1 cm−1 at 280 nm calculated using ProtParam (Gasteiger et al., 2005). The sample purity was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS–PAGE (data not shown), with typical protein yields in excess of 20 mg per litre of culture. Native PAGE and size-exclusion chromatography with multi-angle light scattering (SEC–MALS) were used to establish the oligomeric state of the protein. SEC–MALS experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS multi-angle light-scattering detector and an Optilab T-rEX refractive-index detector. SEC–MALS analysis was performed on an Superdex 200 10/300 column (GE Healthcare) equilibrated with 50 mM Tris–HCl, 250 mM NaCl pH 7.5. Molar masses spanning the elution peaks were calculated with ASTRA v.18.104.22.168 (Wyatt).
2.2. Crystallization and data collection
Crystallization trials were carried out with a Phoenix liquid-handling system (Art Robbins Instruments/Rigaku) and commercially available screens (Hampton Research). 100 nl protein solution (10 mg ml−1 in 100 mM sodium acetate pH 5.0, 150 mM NaCl, 0.5 mM ZnSO4) was mixed in a 1:1 ratio with reservoir solution and equilibrated against 70 µl reservoir solution at 293 K. Orthorhombic rod-shaped crystals of approximately 30 × 10 × 10 µm in size were observed after 3 d using a reservoir consisting of 0.2 M Li2SO4, 25% PEG 3350, 0.1 M bis-tris pH 5.5. A single crystal was transferred into a cryosolution consisting of the original reservoir supplemented with 20% glycerol prior to flash-cooling at 100 K and was then characterized in-house using a MicroMax-007 rotating-anode generator and an R-AXIS IV++ dual image-plate detector (Rigaku) prior to storage in liquid nitrogen. X-ray diffraction data extending to 1.9 Å resolution were subsequently collected on the ID23-2 microfocus beamline at the European Synchrotron Radiation Facility (ESRF). Owing to the size of the crystal and its particularly long c axis, it was necessary to utilize a microfocus beam of approximately 10 µm and a helical data-collection strategy. Such a data-collection strategy, combined with a short exposure time of 0.6 s and a MAR Research Mosaic 225 charge-coupled device detector, allowed good separation of the reflections, appropriate sensitivity and low X-ray damage, allowing a complete data set to be collected. Integration and scaling of the data were carried out using MOSFLM (Leslie, 2006) and SCALA (Evans, 2006). The space group was P212121, with unit-cell parameters a = 62.9, b = 85.9, c = 289.0 Å. The asymmetric unit consists of four subunits each of mass 45.3 kDa, with approximately 1500 residues in total, a VM value of 2.17 Å3 Da−1 and a solvent content of approximately 45%.
2.3. Structure solution and refinement
The structure was solved by molecular replacement using a polyalanine model of the monomer of a CCP structure from B. mallei (BmCCP), which shares 85% sequence identity (PDB entry 3k2k ; Joint Center for Structural Genomics, unpublished work). The rotation and translation functions in Phaser (McCoy et al., 2007) revealed the positions of four polypeptide chains with a log-likelihood gain of 6469. Inspection of the molecular packing in Coot (Emsley et al., 2010) showed no steric clashes and suggested that a tetramer, consistent with the SEC–MALS results (discussed below), was present. Rigid-body refinement was carried out in REFMAC5 (Murshudov et al., 2011). The resolution range was set from 144.5 to 2.5 Å and the Rfree value was 33.3% following rigid-body refinement. Ten cycles of restrained refinement were then carried out with the resolution extended to 1.9 Å. The Rwork and Rfree values were 24.6 and 28.6%, respectively. Chain A was inspected with electron-density and difference density maps in Coot and some missing side chains were added, while those out of density were deleted. Chain A was rotated and translated to provide the other three chains in the asymmetric unit and the four Zn2+ ions were included. The model was subjected to several cycles of PDB_REDO refinement (Joosten et al., 2012) followed by electron-density and difference density map inspection, rebuilding and inclusion of water molecules and ligands. Translation/libration/screw analysis (TLS; Painter & Merritt, 2006) was applied to create different group descriptions.
The PDB_REDO process consisted of the following steps. (i) The B factor was reset to the Wilson B followed by ten cycles of TLS refinement using one TLS group per chain. (ii) Seven ten-cycle trial refinements were performed keeping the TLS model established in (i) fixed, using automatic geometric restraint weighting from REFMAC5 and trying increasingly loose B-factor restraint weights. The optimal weight was 0.30, which is lower than the default. (ii) Six 30-cycle refinements were performed using the fixed TLS model, the optimized B-factor restraint weight and increasingly tight geometric restraint weights. A restraint weight of 0.10 gave the best refinement results. Details of the selection algorithm are presented in Joosten et al. (2012). (iv) Model rebuilding consisting of water deletion, peptide flipping, side-chain rotamer searching and His/Asn/Gln flipping was performed to improve hydrogen bonding (Joosten et al., 2011). (v) A final refinement consisting of five cycles of TLS refinement starting from the previously refined model, followed by ten cycles of restrained refinement using a B-factor weight of 0.30 and trying three different geometric restraint weights (0.05, 0.10 and 0.15) was performed. Refinement with a slightly looser geometric restraint weight than before (0.15) gave the best results.
A further round of map inspections, model fitting and PDB_REDO refinement was carried out. The output model was again inspected with the relevant maps and adjusted, further waters were added and multiple conformers were now included at several positions. Further restrained refinement followed, using the B-factor and geometric weights established with PDB_REDO and a TLS description comprising 12 groups in total. Local noncrystallographic symmetry (NCS) restraints were imposed at the onset of the refinement and were maintained until the final round of calculations. The crystallographic statistics and model geometry are shown in Table 1. Coordinates and structure factors have been deposited in the PDB with accession code 4b6z . The model geometry was monitored with MolProbity (Chen et al., 2010). Structure superpositions were calculated using Secondary Structure Matching (Krissinel & Henrick, 2004) in Coot (using LSQKAB; Kabsch, 1976), and the Protein Interfaces, Surfaces and Assemblies (PISA) server (Krissinel & Henrick, 2007) was used to characterize the protein–protein oligomerization interface. Amino-acid sequence alignments were performed with Clustal Omega (Sievers et al., 2011). Active-site volumes were calculated using ICM PocketFinder (MolSoft Limited). Secondary structure was assigned using Coot and by inspection. Figures were prepared using PyMOL (DeLano, 2002) and ALINE (Bond & Schüttelkopf, 2009).
‡Rwork = , where Fobs is the observed structure factor and Fcalc is the calculated structure factor.
§Rfree is the same as Rwork except calculated with a subset (5%) of data that were excluded from refinement calculations.
¶Diffraction-component precision index (Cruickshank, 1999).
††Chen et al. (2010).
‡‡Engh & Huber (1991).
3. Results and discussion
3.1. General comments
Size-exclusion chromatography was used as the final purification step and the protein eluted as a single species with a mass of approximately 140 kDa; a native PAGE gel showed a band of around 146 kDa (Fig. 1). The theoretical mass of BcCCP is approximately 45.3 kDa; these data were therefore taken to infer the presence of an oligomer, but it was unclear what the quaternary structure was. However, SEC–MALS shows that the protein elutes with a mass of 174 kDa (Fig. 2) corresponding to a tetramer, and this is consistent with the assembly observed in the crystal structures of BcCCP (the subunits are labelled A–D; Figs. 3 and 4), BmCCP and the protein from Shewanella denitrificans (SdCCP; PDB entry 3l2n ; Joint Center for Structural Genomics, unpublished work). This is distinct from PaCCP, which was reported to behave as a monomer in solution (Otero et al., 2012). The tetramer has a gap or hole at the centre of the assembly and this may have influenced the elution properties, whereas the use of light scattering offers a better approximation of the assembly size.
Diffraction data were acquired by exploiting the use of a microfocus synchrotron-radiation beamline to accommodate the small size of the crystal and to help circumvent the issue of a long unit-cell edge. The structure was solved by molecular replacement and, in order to carry out the efficient refinement of a relatively large asymmetric unit with over 1500 residues and data to high resolution, we adopted a slightly unconventional route by using PDB_REDO. This procedure uses a variety of automated protocols to test different models for B factors using weighting restraints on geometry, optimization with respect to diffraction data terms and side-chain rebuilding, for example (Joosten et al., 2012). The approach has been developed to efficiently apply modern refinement techniques to update and improve the contents of the PDB. It has been our experience that the majority of structures that we have inspected have been improved in terms of agreement with the diffraction data and in many cases in chemical correctness. The approach worked well in this refinement and we suggest that the lessons encapsulated in PDB_REDO have much to offer especially if incorporated early in the refinement process. We suggest that near the end of the refinement and building process, when interactive rebuilding is limited to the final polishing of the model, it is no longer advantageous to perform subsequent PDB_REDO runs. Instead, the optimized refinement settings from the last PDB_REDO run can be used in REFMAC5 directly. To this end, PDB_REDO has been updated to provide a keyword file for REFMAC5 that can be used in the CCP4 graphical user interface (Winn et al., 2011) to quickly import these settings.
Continuous well defined electron density is observed for most of the structure. The only disordered regions include the N- and C-terminal residues, residues 157–158 in chains A and C and a number of residues in a loop (315–320) in subunits B, C and D. Superposition of Cα positions of all four chains gives root-mean-squared deviations (r.m.s.d.s) in the range 0.23–0.35 Å, indicating that the subunits are, within the errors associated with the structure, essentially identical. Note that the diffraction-component precision indicator (Cruickshank, 1999) was calculated to be 0.13 Å. We therefore detail only subunit A unless stated otherwise.
A strong feature in electron-density and difference density maps was observed in each active site near Arg226. These were modelled as acetates derived from the 50 mM sodium acetate present in the crystallization conditions. In the crystal structure of PaCCP complexed with guanidinoethylmercaptosuccinic acid (Otero et al., 2012; PDB entry 4a39 ) and in the structure of CP-A with benzylsuccinic acid (Christianson & Lipscomb, 1986; Mangani et al., 1992; PDB code 1cbx ) the carboxyl groups of the ligands are located at the same position as this acetate (data not shown).
3.2. Subunit structure and comparison with other CCPs
The structure of BcCCP consists of two domains, a common feature of M14 family members. The primary, secondary and tertiary structures are presented in Fig. 5. Residues Met1–Glu112 form the N-terminal domain, while the remaining 272 residues make up the CP domain. The N-terminal domain folds into a nine-stranded antiparallel β-sandwich. Strands β2, β3, β5, β8 and β7 form one sheet and strands β1, β4, β9 and β6 form the other. This domain is specific to CCP proteins and is absent in other carboxypeptidases. It has been hypothesized that the N-terminal domain might contribute to folding, might have a regulatory function and/or might be involved in binding other proteins (Kalinina et al., 2007; Rodriguez de la Vega et al., 2007). A comparison of the structure of the N-terminal domains of BcCCP and PaCCP gave an r.m.s.d of 0.87 Å over 109 Cα positions, indicating close similarity.
The CP domain of BcCCP displays a similar topology of secondary-structure elements, an α/β/α sandwich structure with a seven-stranded antiparallel β-sheet, as observed in other M14 family carboxypeptidases, for example bovine carboxypeptidase A (CP-A; Fig. 6). A superposition of BcCCP and bovine CP-A (Kim & Lipscomb, 1990; PDB entry 6cpa ), which share about 20% sequence identity, gives an r.m.s.d of 2.03 Å for 225 Cα positions. The cores of the structures fit well (Fig. 6), but on the periphery of the active site there are large differences that will be described later. Overlay of the CP domain of BcCCP with those of BmCCP (PDB code 3k2k ), SdCCP (PDB entry 3l2n ) and PaCCP (PDB entry 4a39 ; Otero et al., 2012) reveals a higher degree of conservation, with r.m.s.d values of 0.45 Å (374 Cα atoms), 0.85 Å (362 Cα atoms) and 1.07 Å (365 Cα atoms), respectively. These proteins share about 85, 50 and 40% sequence identity with BcCCP, respectively, and the degree of structural similarity correlates well with the level of sequence conservation.
BcCCP contains several motifs which are conserved in CCP family proteins. One of them, Asn216-Pro217-Asp218-Gly219, occurs between β13 and α4, with the proline at the start of the helix (Fig. 5b). This motif contributes to domain folding and the formation of part of the active site (Fig. 7). On one side of the motif Asp218 establishes hydrogen bonds to the side chains of Thr140 and Arg144 and to the main-chain amide of Glu142. These interactions serve to link β13 to β10 and β11, which are on the surface of the domain. On the other side of the motif Asn216 Oδ1 accepts a hydrogen bond from the amide of Gly219 to hold the side chain in place and Nδ2 is a donor in hydrogen-bond formation to the main-chain carbonyl of Gln170 in β12, thus linking different elements of secondary structure together. The side chain of Gln170 also participates in four hydrogen bonds that link β12 to β14 and the segment between α4 and α5. The β-strand link (not shown) involves the donation of a hydrogen bond from Gln170 N∊2 to Asp266 Oδ2. The other three hydrogen bonds, to the carbonyl of Leu234 and the side chains of Asn235 and Trp238, help to position β12 and the interhelical segment. The positioning of β12 and β14 contributes to the formation of the active site because they provide His171 and His268, respectively, to coordinate the catalytic Zn2+.
Mammalian CCP proteins are considerably larger than their bacterial homologues, with N- and C-terminal extensions plus numerous insertions. The sequences of the extended regions vary and their function is at present not known. However, all CCP members share three short sequence motifs within the N-terminal domain which are absent in other CP families (Kalinina et al., 2007). These conserved motifs are located in close proximity, even though they are distant in the primary amino-acid sequence (Figs. 5b and 8). Motif I, F(D,E)xGx(L,I), residues 9–14, links β1 with β2 and extends to form part of the second strand. Motif II, W(F,Y)(Y,N)(F,Y), residues 40–43, forms a major part of β4, a buried strand, and with the sequence Trp-Phe-Tyr-Tyr contributes significantly to the formation of the hydrophobic core of the domain together with other aromatic residues such as Phe9, Phe57, Trp69, Trp83, Tyr110 and Phe111 (data not shown). Residues 113–115, P(F,Y)(S,T), form motif III at the N-terminus of α1 on the surface of the domain. This segment forms part of the interface between the N-terminal and CP domains and contributes to interactions with the partner subunits in the dimers (A–B and C–D) forming a tetramer. It is noteworthy that these motifs and features are conserved in the PaCCP, BmCCP and SdCCP structures (Fig. 5b), and this observation supports the idea that together they are important for the correct folding of the domain and its position with respect to the CP domain.
3.3. Quaternary structure
BcCCP forms a tetramer arranged as a dimer of dimers (A–B and C–D in Fig. 3). The N-terminus of each BcCCP subunit is located close to the CP domain of the dimer partner (Fig. 4a). This might explain why the N-terminal hexa-His tag could not be cleaved with TEV protease. The average solvent-accessible surface area of the four subunits in the asymmetric unit is approximately 15 500 Å2. The total solvent-accessible area of the BcCCP tetramer is 52 600 Å2, with a buried area of 9400 Å2. The interface formed between two BcCCP dimers (A–B and C–D) occludes 2800 Å2, while the interface between subunits A–C and B–D equates to 1900 Å2. Therefore, about 9% of the surface area of each subunit is involved in dimer formation, with a further 6% involved in associations to assemble a tetramer. The free energy of dissociation is estimated to be 14.9 kcal mol−1, suggesting that the tetrameric assembly is thermodynamically stable. These values correlate well with those calculated for tetrameric BmCCP and SdCCP proteins. For example, the total surface areas of the BmCCP and SdCCP structures are 52 500 and 49 900 Å2, with buried areas of 11 900 and 9600 Å2, respectively. The secondary-structure elements forming the interface within a BcCCP tetramer are shown in Figs. 4(a) and 4(b). Individual BcCCP dimers are stabilized by around 24 hydrogen bonds, nine salt bridges and van der Waals interactions. Some of the residues contributing to dimer formation include Gln127 and Glu117 in α1, Asp104 in a loop between β8 and β9, Ser105 in β9, Arg80 in a loop between β6 and β7, Glu142 in a loop between β10 and β11, Val134 in β10 and Asn8 in β1. We note eight hydrogen bonds and two salt bridges between a pair of dimers in a tetramer. A few of the residues involved in these interactions include Asp193 in a loop between α2 and α3, Lys186 in α2, Arg201 in α3 and Pro354 in a loop between β16 and α8. These residues are not conserved in PaCCP, which is a monomer in solution. However, these residues are well conserved in BmCCP and SdCCP, in which similar subunit–subunit associations are noted in tetramer formation (data not shown).
3.4. The active site and specificity
The active site of BcCCP is an elongated cavity with a base formed by residues in β14, β15 and β16, walls formed by loops linking α4–α5, α6–α7, β3–β4 and β5–β6 and then the β14–β15 turn (Figs. 5 and 9). Here, the catalytic Zn2+ is coordinated by two histidines (His171 and His268), Glu174 in a chelating η2 interaction and a water molecule to give a pentacoordinated distorted tetrahedral geometry (Fig. 10). The active-site cleft is occupied by an acetate derived from the crystallization conditions. This anion, which marks the binding site for the substrate carboxylate, is held in place by electrostatic interactions with Arg226, Asn235 and Arg236 (Fig. 10).
The bacterial CCP active sites, and in particular the metal-ion environment (Fig. 11), display a high degree of structural conservation and this even extends to bovine CP-A, although here the overall sequence identity is only about 20% (see §3.2). The His171 and Glu174 residues occur in a His-Pro-Gly-Glu motif which is highly conserved in CCP proteins (Fig. 5b). In BcCCP and BmCCP a buried Arg169 side chain is positioned by a salt bridge to Glu178 and a hydrogen bond to the carbonyl of Tyr114, and in turn helps to stabilize the conformation of the His-Pro-Gly-Glu motif by making a hydrogen bond to the main-chain carbonyl of Pro172 (data not shown). In the other known structures different residues occupy the space near the motif and fulfil the role of stabilizing part of the metal ion-binding site. In bovine CP-A Phe116 provides steric bulk and Gln76 provides the hydrogen bond to the active-site proline to position this metal-binding motif (Christianson & Lipscomb, 1989).
A proposed mechanism for bovine CP-A involves the activation of a water molecule by the divalent cation assisted by Glu270 acting as a general base and interacting directly with substrate. This supports the generation of a potent nucleophile (Christianson & Lipscomb, 1989). Arg127 helps to stabilize an oxyanion hole by binding to the substrate carbonyl (Kim & Lipscomb, 1990). These key residues are conserved as Arg226 and Glu344 in BcCCP (Fig. 11). Other residues conserved in the active site throughout the M14 family include Asn144 and Arg145 of bovine CP-A, which bind to the C-terminal carboxylate of the substrate. In BcCCP they are conserved as Asn235 and Arg236.
Whilst most of the CCP polypeptide chains overlay well, as described, there is a noteworthy exception, part of the flexible α6–α7 loop along one side of the active site, which displays different conformations (Fig. 6). Similar observations about flexibility and different configurations of this part of the active site have been made when comparing bovine and rat carboxypeptidase structures (Faming et al., 1991).
The inherent flexibility of this loop is indicated by the thermal parameters. In BcCCP the thermal parameters for the Cα atoms in this section, residues His313–Tyr320, average at 27.4 Å2 compared with an overall average of 6.7 Å2 for all protein atoms. The structural difference is best exemplified by comparing the positions of Lys319 and Tyr320 of the two most conserved structures, BcCCP and BmCCP (Fig. 12). The BcCCP structure displays an open configuration and the BmCCP structure a closed configuration with Tyr320 directed into the active site. The Cα positions of this pair of residues differ by just over 7 Å.
An overlay of BmCCP (PDB entry 3k2k ) and CP-A (PDB entry 1cbx ), both displaying the closed configuration, places the hydroxyl groups of Tyr320 and Tyr248 within 1 Å of each other (data not shown). In BcCCP Tyr320 is directed out and away from the active site and a major conformational change of about 17 Å would be required to place the hydroxyl in the active site in a similar position to that of Tyr248 in CP-A. In CP-A the tyrosine adopts an outward configuration in the absence of ligand but swings in when a ligand, for example glycyl-tyrosine, is present. Site-directed mutagenesis suggests that it is not a critical determinant of the carboxypeptidase reactivity since kcat is unaffected for small substrates. However, a significant increase in Km indicates a role in ligand binding, so this tyrosine and the pliable loop in which it resides may assist in the recognition and binding of protein substrates (Gardell et al., 1985). Subsequent mutagenesis studies led to the proposal that the tyrosine hydroxyl contributes to the mechanism by helping to activate the catalytic Zn2+-bound water (Cho et al., 2001). We cannot find any structural data to support such a conclusion since the functional group on the tyrosine side chain is too distant to make a hydrogen bond to the catalytic water that is bound to Zn2+ and there is already a glutamate contributing such a function. Rather, we agree with Gardell et al. (1985) that this tyrosine is well positioned to bind substrates and to assist in fulfilling the hydrogen-bonding capacity of substrates, intermediates or products during the catalytic cycle. There is another tyrosine, Tyr315, positioned in the active site a little closer to the catalytic Zn2+ and the associated water molecule. This then provides a tyrosine pair that might contribute to substrate placement in BcCCP.
The substrate specificities of bovine CP-A and rat CP-A2 are well understood (Faming et al., 1991) and structural differences between CP-A and BcCCP suggest that these enzymes have distinctive specificities. Firstly, it is worth noting that we are comparing a ligand complex of CP-A with unliganded BcCCP. This is relevant in that the α6–α7 loop displays a closed configuration in CP-A, positioned over the active site. The residues in these loops placed in the vicinity of the active site might interact with substrates, but the key determinants of specificity reside in the side-chain binding pocket, the S1′ site itself (Figs. 9, 10 and 11), and are strongly influenced by only a few residues. In CP-A these are Ile243, Asn144, Ala250, Gly253, Ile255, Asp256 and Thr268. Ile243 and Ala250, together with Tyr248, are in the α6–α7 loop capping the S1′ pocket. Asn144 and Thr268 are conserved as Asn235 and Thr342 in BcCCP part-way down to the bottom of the S1′ site. Ile255 in CP-A corresponds to Ser329 in BcCCP and the hydroxyl group makes the bottom of the S1′ subsite polar. Nearby, two differences, Gly253 and Asp256 in CP-A but Leu327 and Lys330 in BcCCP, make the the S1′ subsite much more shallow in the bacterial enzyme and replace an acidic residue with a basic one (Fig. 10). In bovine CP-A Asp256 directly interacts with a side chain of substrate (Christianson & Lipscomb, 1986) and by providing an acidic hydrogen-bond accepting capability helps to define the specificity of the enzyme. The side chain of Lys330 in BcCCP extends into the space occupied by Ile243 and Asp256 in CP-A (data not shown). Lys330 of BcCCP is strictly conserved in BmCCP and therefore the S1′ subsite in the Burkholderia enzymes is perfectly matched to bind glutamic acid, consistent with the putative function as a polyglutamylase. The lysine is replaced by an asparagine (Asn321) in both SdCCP and PaCCP, so the S1′ subsite would still be cabable of binding glutamate.
To test BcCCP for deglutamylase activity, in vitro assays were performed using a furylacryloyl glutamate derivative (data not shown). Only low levels of this substrate were cleaved, indicating that BcCCP might not be a deglutamylase, that the tested `substrate' is not appropriate or that the enzyme might require activation, as is the case for bovine CP-A.
3.5. Issues regarding druggability
The concept of a druggable protein concerns the properties of a binding site on the target to bind molecules that possess the right physicochemical attributes to make them bioavailable and have a high affinity for the target and a low toxicity to humans (Hunter, 2009; Fauman et al., 2011). The optimal target would be a small, well defined and ordered cavity in the protein with pronounced hydrophobic components, although there are notable exceptions. The active sites of enzymes such as pteridine reductase (PDB entry 2x9g ; Dawson et al., 2010) and dihydrofolate reductase (PDB entry 3cl9 ; Schormann et al., 2008) would be described as `druggable'. The volumes of their active sites are estimated as 350 and 380 Å3, respectively. Based on the crystal structures of the proteins discussed here the substrate-binding cavity of bacterial CCP is estimated to occupy a volume of approximately 310–410 Å3, which would be judged of a sufficient size to bind drug-like molecules (An et al., 2005).
Although size matters, the chemical properties of the active site are also critical. In this respect, we note specific functional groups (e.g Lys330) and hydrophobic surfaces (tyrosines) that might interact with potential inhibitors. There is, in addition, the presence of the catalytic Zn2+ that provides opportunities to incorporate metal-ion affinity into inhibitory molecules.
4. Concluding remarks
The structure of BcCCP has been determined. Only small crystals with a relatively long unit-cell c parameter of about 290 Å could be obtained. However, by exploiting the advantages of a microfocus synchrotron beamline we were able to obtain high-resolution diffraction data. An unconventional approach to refinement was adopted by exploiting the strengths inherent in the automated protocols of PDB_REDO. This ensured efficient refinement of an asymmetric unit containing over 1500 residues and a large number of water molecules. Our experience was positive and points to a future in which such an approach might become standard. The structure is compared with those of other CCP proteins and with that of CP-A. The CP domain of BcCCP is similar to other M14 family CPs, while the N-terminal domain assembles into a fold unique to CCP subfamily proteins. Our data provide details of the active site and reveal the spatial conformation of residues involved in Zn2+ coordination and reaction catalysis. Specific differences localized to the S1′ pocket on one side of the active site suggest that the bacterial CCP proteins process substrates based on the recognition of an acidic residue. This would be commensurate with their assignment as polyglutamylases, although further experiments are required to address this issue.
Supporting information
3D view: 4b6z
PDB reference: carboxypeptidase, 4b6z
This research was supported by awards from the Biotechnology and Biological Sciences Research Council, The Wellcome Trust (grant Nos. 082596, 094090 and 100476) and the European Commission Seventh Framework Programme (grant agreement 223461, Aeropath) to WNH. RPJ is supported by Veni grant 722.011.011 from the Netherlands Organization for Scientific Research. We thank the European Synchrotron Radiation Facility, Grenoble for excellent support.
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text:
new update for GSII is available now via kies :)
just chk it by urself
AnonD-519, 23 May 2011I have got an official answer from Samsung (Switzerland). There won't be a 32 GB version of SG... moreI wouldn't believe that if I were you..
Infact don't. Maybe specifically where you are getting from will not order the 32GB, but it is coming weither they are getting it or not and trying to cover it or not. If Samsung said it someone lied or it was not really them.
Im now using it and it so wonderful with smoothing camera and high solution picture.but de main problem is battery cant stay longer than 1day if we using it too much,not base only talking but also in checking some applications or ... im hope they will consider about battery life more better than this product either it cant compete with Iphone4g.
As per Samsung co-website, SGS2 will Launched in June 2011.
[deleted post]Yes, its true that S II is coming to India in the May itself and launch date is 99.99% acurate.
Hi guys.
I have been using iphone 3GS for like 5months now and have always felt a feeling like something's missing.My friend recently got iphone 4 and belimme even he has this feeling of something missing. Iphone 3/3gs/4 are good phones no doubt but but...but Samsung Galaxy SII is the best phone out der..No comparisons. Ppl have been coming n talking bot Xperia Arc n HTC Sensation n N8 n what not but here is how the actual story goes...
SGSII 4.27' Super-AMOLED screen(bright,vibrant,saturated,vivid colors,power efficient(i dont understand y sme stupids r talking bot screen burn n some stupid crap)...only phones dat can come near are Xperia Arc(LED-backlit display: Saturated and nice display but colors overcooked)HTC Sensation(qHD display: good infact very good but battery hungry(ever knows what HTC rates in battery department)..overall winner SGSII
Processor: Dual Core 1.2Ghz(fastest and most efficient)..competitors: HTC Sensation(Fast and equally efficient but low benchmarks compared to SGSII)Motorola Atrix(any1 interestd in Motorola Still???) Overall winner: SGSII
Internal memory,weight factor,gaming(Xperia Gameplay is a winner here)and the specification lists goes on..where the only clear winner seems to be SGSII.EOS. So ppl who have bought other phones in hurry or due to lack of patience or are simply jealous(for nt having the best amongst the lot)stop complaining an cherish an Engineering marvel i.e. SGSII
U all my be asking what hapned to my iphone 3GS...well it has been sold already and my SGSII has been ordered :D
Mo, 23 May 2011Waited 2 months for this and finally got it this weekend gone. I have to say; it has certainl... moreHey I completely agree!!
i just got mine today guys and it is the greatest mobile i have ever had (Desire, iP4.. etc) by faaaar! So fast! Screen is just amazing. BLOWS the iP4 screen away! mail app is amazing.
I just sold my iP4 and got this today. What can I say, after maybe 3 iPhones this is the most amazing gadget I have had in years. Awesome build quality whilst being very light. the first thing that you notice is the screen. Believe the hype. It better than they say in the reviews! The actual UI is so silky smooth and the email is amazing!! it slike on a PC and even has auto rotate so you can read the message next to the message list like on the iPad! camera - amazing. Andorid - amazing. Sorry I just cant believe what i have been missing to good too strong SGS2 FTW big time.
never been happier..
Do ALL android phones have an issue with showing call waiting when you try to get through to someone if they are on a call? I've had several missed calls from my friends who use Android phones, who say they never get a call waiting notification when I'm on the phone!
Waited 2 months for this and finally got it this weekend gone. I have to say; it has certainly lived up to everything I expected and MORE!!! Can't get my hands off it; probably the best phone I have ever had. People don't bother going for the iPhone 4 (or dare I say, the iPhone 5 if and when it comes out) this is far far superior!!
AnonD-5595, 23 May 2011got GSII two weeks back on contract and yesterday went to unbox event of GSII at Covent Garden... moretime for u2 do some social work . Send it over to me ok not for free . . . Place ur bid :-p
I have got an official answer from Samsung (Switzerland). There won't be a 32 GB version of SGSII. I am extremely disappointed. Looks like Samsung was lying about their products when thy were announcing a 32 GB version.
Maybe I'll wait for iPhone 5. Very Very Very Disappointed.
got GSII two weeks back on contract and yesterday went to unbox event of GSII at Covent Garden and won GSII :)
now have two GSII's .
I couldn't believe this waited for one for 3 months now have 2 ....WoW
whats the battery like on this phone? everything else looks great :-)
Its the best Android phone in the market...In one word its the king of all the phones. It has awsome design and features for best price in the current market.. Turn on 2Marow.. Its Samsung..
Two Questions to regarding sound quality and reception. I am curious with the bass response on the music player and is there an EQ. Secondly Network plays a big part but have you noticed any change on the dropped more/less the same?? Thanks
npkganesh, 22 May 2011hi frnds i have a doubt, pls clear it. if i already installed gingerbread 2.3.3 xxjvk. now i ... morewithout bootlooder
AnonD-8026, 22 May 2011what review are u talking about... lag in arc... man there's a review for all of u to read i'l... moreYeah there is a difference between se and samsung and it is that samsung is sooooo much better..... period

subset: commoncrawl


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Antifa have been busy showing their true colours recently, especially in the US. In the last week, a group of these unwashed cock jockeys turned at UC Berkley to try to deny a group of Donald Trump supporters their right to free speech. Thereby proving Churchill correct when he said, "The Fascists of the future will call themselves Anti-Fascists". Although despite turning up with weapons, with which to beat the unarmed Trump supporters, they managed to turn victory into defeat.
It started with pepper spray, bricks and stones. Then someone had the bright idea of throwing a smoke grenade to cover an Antifa charge. From a military perspective, that's not a bad idea. Unfortunately for Antifa, they're not very bright, especially the clown who threw the grenade, because nobody thought to check which way the wind was blowing.
Anybody care to guess what happened next? Yeessss! The smoke from the grenade blew back into the Antifa ranks, allowing the Trump supporters the chance to defend themselves. Despite being unarmed, they turned an unprovoked attack by left wing scum into a rout.
One of the funniest things about this, has been the whingeing from the left after a video of a female Antifa dickhead getting punched squarely in the face by a male Trump supporter. Now, I don't normally support violence against women. However, this bitch had gone their specifically to fight, having boasted that she would get "100 right wing scalps".
Her getting punched isn't what amuses me. It's all the left wingers, who continually tell us that gender is irrelevant and that the concept of man and woman is a myth, are now whingeing that it's wrong to hit a woman. Well, welcome to the real world snowflakes. A world where, if you turn up to an otherwise peaceful gathering of people whose views differ from yours, with the intention of committing violence, you're going to find that people will not simply stand by and let you hit them. They will hit back.
The saddest thing about this, is that the police were apparently ordered to stay in their vehicles and do nothing. An order which they cravenly obeyed to the letter. Cunts.
Nominated by Quick Draw McGraw


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Berkeley Technology Review
I joined the staff of Berkeley Tech Review, because the goal of the publication (to start a conversation on technology and its interactions with society in one of the top technological institutes in the country ) spoke to me. I created these designs so as to brand BTR in a way that evokes professionalism, journalistic integrity, and modernity.
Home Page
Using some of the research I did at InternMatch to design this landing page, I emphasized the ability to curate the main articles above the fold and highlighted the most popular content. The look of the publication is focused. I wanted to avoid a flashy style that would distract from the content and also tried to stay away from imagery that would date the publication, as technology is constantly changing and the look of the site should accomodate any of these aesthetic changes.
article Page
Much of the design of the article pages is inspired by the Medium reading experience. There is no clutter competing for your attention while reading. The content is presented in an up-front and comfortable-to-read style. I decided to point the reader towards one of three further reading options: related, popular, and recent. These are meant to leave an appealing option for those exploring posts casually, those trying to catch up with the popular recent editorials, and those returning users who want the freshest content.
Topic Page
Instead of several different topic pages, I wanted to create a single flexible page which utilizes two filters to look at specific topics in a specific order (e.g. show me the most popular articles on Computing). This design was made to unify the reading experience, so that the boundaries between topics are more fluid and readings can jump from one to the next without feeling as much like they are being taken to another page.
Promotional materials
This was a flyer designed to recruit for multiple teams at BTR. The design goal I had here was to make something bold and simple that would focus on the key details, which are the teams looking to hire and the recruitment timeline.
The front of the flyer is Art Deco-inspired, because I wanted to channel another time in history when the rapid pace of technological change was affecting society - an idea that speaks to the mission of the publication to examine technology's impact on the social, political, and cultural spheres.


text:
How Much Freedom Will You Give Up to Fight International Cybercrime?
- Sep 12, 2011 3:47 PM EST
- Comments
An effective effort to stop cyber attacks against government and industry will inevitably require increased surveillance of users and conscription of industry in the effort. Is this OK with you? Read More »


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1991 Nissan Sentra Top Comparisons
Users ranked 1991 Nissan Sentra against other cars which they drove/owned. Each ranking was based on 9 categories. Here is the summary of top rankings.
User Reviews
Displaying 1 - 10 of 20 1991 Nissan Sentra reviews.
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE-R CoupeReview
I Would Buy This Car Again In A Heartbeat — I love this car. This is my daily driver and it has been fantastic. The mileage is at over 260,000 and it just now started to develop a rod knock. Currently I am tearing it down and putting a turbo on. It was fun enough before, but should be even more fun after the modification.
Primary Use: Commuting to work
Pros: I love everything about this car
Cons: Nothing
Looking for a Used Sentra in your area?
CarGurus has 52,594 nationwide Sentra listings starting at $1,243.
CarGurus https://www.cargurus.com
1991 Nissan Sentra E CoupeReview
The Most Surprising Car I Have Ever Owned. — I bought this car when I got rid of my Mustang that had ever option known to man. I nearly cried because this car was very strippo but it was all I could afford at the time. It was roomy with a large trunk, easy to park. The clear coat basically dissolved. It had two small repairs (water pump and clutch) over the time I owned it. I got 40 mpg which was great since my commute was 100 miles a day. It still ran good when I sold it at 239K miles.
Pros: I used to get 40 mpg on the freeway.
Cons: Ugly with no accessories.
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE CoupeReview
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE-R CoupeReview
Niks — good perfomance if it got a vvl in it,good brakes,not 2 bad handling. no repairs on car,rattles after parys,no squeaks. very nice interior,good styling heave on gas,cheap on spairs very nice at 160km when crusing on free way,
Primary Use: Sport/fun (spirited driving, track racing, off-roading, etc.)
Pros: one of a kind
Cons: niks
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE CoupeReview
Great Sport Compact, All Around. — In my opinion, one of the best FWD import cars ever made. Light, agile, and still has room for real humans. Comfortable as I've ever been in a sport compact. Beautifully neutral handling until you really push it. Takes a licking, and keeps on ticking.
Pros: Cheap to buy, cheap to maintain, loads of fun.
Cons: Auto trans finally gave out.
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE-R CoupeReview
Nice Car... Two Thumbs Up. — Fun little car thats ok on gas, comes turbo in japan so upgradeing a us version is easy, handling is great, as is braking but thats because of my upgrades. To sum it up "Best in class" for model year.
Primary Use: Commuting to work
Pros: Fast as a camero, but flys under the coppers radar.
Cons: Car overall looks like a grocery getter in stock from.
CarGurus https://www.cargurus.com
1991 Nissan Sentra EReview
They Dont Break — they may not look it but thoes little sentras are tanks. you cant break them and even if you do its like playing with tinker toys to fix it. Its like driving a go-cart. Its even good in the snow if your not an idiot :)
Primary Use: Commuting to work
Pros: good fuel economy
Cons: Its not very fast stock
CarGurus https://www.cargurus.com
1991 Nissan Sentra XEReview
My First Car — HATED IT! Very very slow. No acceleration...not alot of parts for it. but over all it was a good starter car...it also had very good mpg's. my sister had it first then my parents gave it to me for a year...now its in the back yard while i drive my 92 prelude around. :)
Primary Use: Commuting to work
Pros: GOOD MPG!!!
Cons: Everything
CarGurus https://www.cargurus.com
1991 Nissan Sentra SE-R CoupeReview
Best Band For The Buck Sport Compact — Very well balanced, one of the best handling fwd cars ever made. Extremely reliable, left me stranded twice in 5 years, once was previous owners fault, second time i explodered the clutch. Of course the sr20 engine needs no introduction.
Primary Use: Sport/fun (spirited driving, track racing, off-roading, etc.)
Pros: Revvy engine, handling
Cons: Weak transmissions, less-than-ideal McStrut suspension
CarGurus https://www.cargurus.com
1991 Nissan Sentra E CoupeReview
91 Nissan Sentra - Not Bad For A College Student — Had some major exhaust problems, driver's side key lock didn't work, paint was wearing off, and ceiling fabric started hanging down. All this was mostly due to age and normal wear & tear, I'm sure. Otherwise it was decent for my first car and got me where I needed to go.
Primary Use: Commuting to work
Pros: Fast, strong frame, no engine or transmission problems
Cons: Exhaust (noisy), paint/clearcoat wore off, interior fabric lost adhesiveness
Displaying 1 - 10 of 20 1991 Nissan Sentra reviews.
Reviews From Other Years
2014 Nissan Sentra Reviews
Not A Bad Family Vehicle Has Some Potential With A Few Mods.
I love the look and feel of the new sentra, the gas mileage is plus the surplus of trunk space is awesome and all around it has a comfortable feel to it, the thing i dislike most is that there is basi... Read More
Beautiful Car And Rides Great
I have already stated above what I like about this car, but on my model I have bose. It sounds great if you like base it could use a subwoofer other than that very loud. Havn't really used the navigat... Read More
2013 Nissan Sentra Reviews
The Amazing Sentra
Good braking,ok acceleration,nice design,nice interior,good leg room,good cost of maintenance.I get 33 mpg in city and 40 with eco.It is a fun car to drive.Good commuter.It is a very nice deal for... Read More
The Best Nissan Sentra So Far
1 year Update 2/28/14 - after 1 year I still love the Sentra and have recorded 46.5 MPG average on a long 3.5 hour trip in the early fall last year using 87 octane gas. I wish Nissan would replace th... Read More
2012 Nissan Sentra Reviews
Nice Overall Transportation. Nice Styling.
I like the simplicity of the cab. It is not over wrought with bells and whistles and it serves the purpose of comfortable and safe transportation. The exterior design is very sporty and good to look... Read More
Love The Look, And The Roomy Interior.
Very nice car, love the pearl white color and the look of the interior design. Handles great, very low miles, and has everything you need. Fun car to drive, also the 4 doors is nice for the kiddos. Wo... Read More
2011 Nissan Sentra Reviews
Great Bang For The Buck
It is probably one of my first choices for a 1st car driver that is looking for reliability and easy and safe to drive. I would say the 2.0s is kind of a car for the girls, but easily can go both ways... Read More
Fun, Quiet Ride, Many Journeys
I loved this car because of some of the outstanding features it had. The Bluetooth system, iPod adapter and key-fobs for key-less entry and start up were amazing. I love having those features in a veh... Read More
2010 Nissan Sentra Reviews
Sentra Sr
I love my car just bought it four months ago BUT.. sometimes when I dont use it for a day or two it wont turn on. I dislike this so much because my car is great but it gives me problems in starting at... Read More
Sentra 2.0sr Is A Great Car For The Payment
I leased the Sentra and rolled in some inequity from my previous loan, and still with nothing down my payment was in the mid $200's. Nothing could come close when I made my rounds and did my shopping.... Read More
Cars compared to 1991 Nissan Sentra
Looking for a Used Sentra in your area?
CarGurus has 52,594 nationwide Sentra listings starting at $1,243.


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I'm not entirely sure what my face is doing in the photograph below...but I like the way the outfit details look, so I'm using the picture anyway. What could possibly have been going through my head at the moment the camera went off? Anyone want to take a guess?
Shirt: Mossimo Scoop Neck Tee
Shoes: Lanvin Ankle Strap Wedge Sandals (on sale here; similar here and here)
Necklace: Tammy Spencer Designs
Has anyone else been following the Diamond Jubilee coverage? I feel like it's all over the place - which I don't mind all that much, because it's always fun to look at Royals in their outfits, isn't it? Especially Duchess Kate. But you know what? My absolute favorite one has to be Princess Anne in her uniform. I mean, who knew? Did you know that Princesses could have uniforms too, just like the boys? (And is she swaggering? Though I might too, if I was as wearing that.)
|image via
Could she be any cooler? I thought not. Carry on.
Hope you're all having a great week!


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There is more than one artist by the name of Strauss:
1) A stoner rock, heavy metal, sludge band from London, UK.
BIO: Mother of all the multicultural cities, London is the melting pot where five lads, their respective nationalities and single musical influences happened to collide and form Strauss. A project inspired and driven by the adverse powers of modern western society, this is also the repressed need to speak out for those wishing to break away from a far too rationalized routine.
Groovy, fierce yet soul-rocking, Strauss's sound is the musical produce of a desperate attempt to drag out what is left of their heavily besmirched creativity. A mold of old-school metal, stoner and sludge, this is the tuneful achievement of five inspirationally distinct musicians with no obligations but a mutual tendency to overcome sonic clichés and deliver awesomely exiting live performances.
Sure they know how to have and bring fun. Vocals and riffs are just as heavy as the aggressive drums bashing throughout each bloody song. What you hear is what you get – a blast of musical adrenaline, power and coordination.
Stef - voice
Charles - guitar
Nano - guitar
Bill - bass
Doc - drums
2) Strauss (Tatsuki Hashimoto) is a electronic indie musician from Japan.
3) Strauss may refer to any of at least five classical composers:
1. Johann Strauss II (October 25, 1825 – June 3, 1899), Austrian composer, the son of Johann Strauss I (who was actually against his son's career as a musician) and also known as "the Waltz King". He revolutionized the waltz, elevating it from a lowly peasant dance to entertainment fit for the imperial Habsburg court. Most famous works include "The Blue Danube", "Wein, Weib und Gesang", "Tales from the Vienna Woods", "Tritsch-Tratsch-Polka", the "Kaiser-Walzer", and the operetta "Die Fledermaus".
2. Richard Strauss (June 11, 1864 – September 8, 1949), German composer of the late romantic era, no relation to the Austrian Strausses, and best known for the composition "Also Sprach Zarathustra". Although not as well known as Johann Strauss II in popular culture, he is considered the greater composer by many serious students of classical music.
3. Johann Strauss I (March 14, 1804 – September 25, 1849), father of Johann Strauss II and Josef Strauss. His most famous piece is probably the Radetzky March (named after Joseph Radetzky von Radetz) whereas his most famous waltz is probably the Lorelei Rhine Klänge op. 154.
4. Josef Strauss (August 20, 1827 - July 22, 1870), son and brother respectively of Johann Strauss I and II.
5. Eduard Strauss (15 March 1835 – 28 December 1916) was an Austrian composer who, together with brothers Johann Strauss II and Josef Strauss formed the Strauss musical dynasty. The family dominated the Viennese light music world for decades, creating many waltzes and polkas for Austrian emperors and their courtiers. He was affectionately known in his family as 'Edi'.
Artist descriptions on Last.fm are editable by everyone. Feel free to contribute!
All user-contributed text on this page is available under the Creative Commons Attribution-ShareAlike License; additional terms may apply.


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article and photos by Habeeb Salloum
The luxury hotel, Barceló Cayo Santa Maria Beach Resort, located on the north coast of Cuba, resides on a small island only 13 km (8 mi) long and 2 km (1mi) wide. It is connected to the mainland by a 48 km (30 mi) man-made causeway on the sea and is ideally isolated for an intimate wedding or a honeymoon getaway.
Surrounded by 11 km (7 mi) of alluring beaches of fine soft white sands, lapped by turquoise waters on one side and lush greenery on the other, it is a hidden Eden in the heart of natural beauty. The islet, which was designated a "Biosphere Reserve" by UNESCO, is a dream place that newly weds will always remember.
Barceló Cayo Santa Maria is made up of two hotels set amid beautiful landscaped gardens. It boasts a stay at one hotel and play at both hotels concept, with a wide variety of restaurants, bars and activities to choose from. For honeymooners, it is a perfect setting for a romantic holiday.
Sitting atop one of the most beautiful islands in Cuba, the resort will eventually become the largest and most modern and luxurious hotel resorts in the country. The central focal point of the resort complex will be its own 'pueblo' (town) with a large variety of bars, restaurants, a beer garden, entertainment establishments, shops, a Grand Spa and much more. You will find this all within the resort's own 'town' – a place to lure the newly-weds.
Honeymooners will not have much to worry about if they choose to spend their days of bliss in this resort hotel beautifying the cayo's (small islands or keys) of Cuba. The charming Katia Yera Vidal, Guest Relations Manager, will arrange everything for a couple planning to take the plunge – from a simple or elaborate wedding to an enjoyable honeymoon stay, she will manage all of a couple's needs.
For a wedding at Barcelò Cayo Santa Maria Beach Resort, the entire process is handled by the hotel's wedding coordinator. The couple only has to check in at the hotel, which for the soon to be bride and groom is a private check in and settle into their guest room with a chilled bottle of champagne, compliments of the hotel. The wedding coordinator has arranged all wedding ceremony details for them – providing a notary, translator, and a marriage certificate, along with paying the legal fees.
The wedding area, selected by the couple, is beautifully decorated for the wedding ceremony. Also provided are flowers for the bridal members and reservation in advance at the a la carte restaurants for the bridal party. As well, there are bottles of champagne for toasting, a special breakfast, a romantic dinner, and a special chocolate bar at the day of the wedding. As one of the staff in the hotel told me, "Each wedding at the hotel is like living a fairy tale."
The hotel's honeymoon package includes a personal express check in and a welcome cocktail; an ocean view room with a bottle of champagne; advanced restaurant reservation, which includes a special breakfast; an organized romantic dinner, as well as many other special offerings and surprises that makes ones honeymoon an unforgettable event.
After the wedding and if the couple want to spend time enjoying the surrounding deep blue waters and glittering snow white sands, there are many organized water activities in the encircling sea. Further still on the mainland are the cities of Caibarién, home to the many workers in the cayo hotels; Remedios, a historic town and Santa Clara, the burial place of Ché Guevara, the famous Cuban revolutionary leader.
All in all, a couple experiencing the beginning of their wedded bliss in Barceló Cayo Santa Maria Beach Resort will no doubt always remember that 'Once upon a time, on an exotic island, they had a dream wedding.' In the words of Katia Vidal, "We will organize for our wedding and honeymoon guests a memorable episode in their lives."
For Wedding Arrangements and more Information, Contact:
- Katia Yera Vidal, Guest Relations Manager and Wedding Coordinator
Barceló Cayo Santa Maria Beach Resort
- E-Mail: email@example.com


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Welcome to En-A1 group!
The En-A1 group is a dynamic, ambitious and trustworthy group of engineering companies specialising in the development and management of real estate projects in Southeastern Europe. Combining within our partnership of independent companies from Slovenia, Serbia and Croatia the knowledge, experience, ideas and expert references from local areas, we are able to provide real estate investors, buyers and other users with a comprehensive array of services to guarantee successful execution of their investment projects.


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City leaders have approved a plan to merge the redevelopment and planning departments to create a one-stop shop for efforts to develop housing and generate economic growth.
The City Council on Tuesday voted unanimously to place the community redevelopment and planning departments under one umbrella, forming the Community Development Department. Current Planning Director Hassan Haghani will run the consolidated operation of nearly 50 staffers. Philip Lanzafame, now the director of community redevelopment and housing, will be assistant director of the new department.
City Manager Jim Starbird told the City Council on Tuesday that the move would help the city respond to "challenges we face on the horizon and, frankly, the opportunities we face as we recover from the economic downturn."


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As some of you know, the Capital Centre (The Summit) has indoor lacrosse games/league in the fall and winter (7 v 7). We encourage new players to get a feel for the game by playing or visiting during the fall. Returning players should consider both or at least the Winter Session to get prepared for the upcoming season in the spring.
We've found that no matter how good you are at lacrosse the Summit league helps you in some way. It could be finally learning to cradle correctly, getting to know your teammates better before the actual season starts, working on using your opposite hand, or simply getting in shape and making tryouts a whole lot easier for you in March!
Registration forms can be found on the Summit's website
Check out the Summit's website at: http://www.capitalcentre.com/lacrosse
Cost: $75 per player


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Wednesday, November 26, 2014
Full of Love Ear-flap Beanie
12 comments:
Great pattern, thanks for sharing. I have one question, I can't seem to get the ribbing to go up/down like in your picture. I am doing fpdc in all the stiches but it doesn't look the same as yours. Any ideas?ReplyDeleteReplies
I think I'm having the same problem, were you able to work it out, I really want to make this cute hat as it matches a dress I made so well!Delete
The last time someone trying to make this hat had this type of issue, it was because they thought "front post" meant "front loop" - front post stitches go around the stitch below it, not through it. :) Hope this helps!Delete
Your welcome!ReplyDelete
My email is firstname.lastname@example.com... Email me a picture and I'll see if u can help you figure it out.
Do you have a particular method you use to change colors and weave in ends after doing so?ReplyDeleteReplies
There are a couple of methods I use. Let me share them!Delete
1) If the color changes are close together, I carry the yarn behind.
2) If the color changes are far away I cut and rejoin.
3) I use bobbins. :)
Hope this helps!
Lovely and well written pattern... i was able to whip it up quickly for a friend's baby :)
I have uploaded it on pinterest and mentioned pattern credit to u. Will link it to ur site whe i sit wih the laptop... i dont have that option on my tablet.
Thank u for such a lovely pattern ♡
made this last night and it is beautiful,only thing wrong is the seam going down the back..are you you suppose to fpdc around the chain 2 at beginning of roundsReplyDeleteReplies
No. Essentially everything has a seam. You're welcome to try various methods with getting it to appear seamless though. :)Delete
made another one and was very careful joining the rounds and it turned out great..grand daughter gets the first one for baby doll..thanks for sharing pattern..great for a baby shower gift that i need.ReplyDelete
I love this!! How can I make this for a 5 yr old?!ReplyDelete
Hi. I purchased the Be Mine Graph Hat. It doesn't have the ear flaps so I thought I'd try print this pattern to see if it would work with it. I don't see a print button. How can I get it printed please? Thank you.ReplyDelete


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A.M. Best Company is an international credit rating agency centered around the insurance market, according to its official website. Insurance regulators, brokers, professionals and customers use the company's ratings for a perspective on the financial creditworthiness and strength of risky investment vehicles and entities.
Insurers and reinsurers use A.M. Best Company's ratings for fixed-instrument debt on securitization products, bonds, notes and other monetary instruments, explains the company's website. International capital-market professionals and issuers use its ratings of commercial paper and debt, while the company also issues financial vitality ratings of captives, insurers and reinsurers. A.M. Best Company publishes in-depth industry reports and insurance industry resources and references. Best's Insurance Reports, its flagship publication, comprises detailed analyses of thousands of United States, Canadian, British and other global insurers. Data from the financial statements of insurers are printed in A.M. Best's Financial Suite, while qualified lawyers, adjusters and related professionals are listed in Best's Directories.
In addition to these publications, A.M Best Company claims on its website to be a highly regarded source for original news in the insurance market. Its Best's Insurance News & Analysis provides the BestDay, Best's Review and BestWeek publications, along with statistical studies and special reports. The company was established in 1899 and has offices in the United States, Mexico, United Kingdom, Dubai, Singapore and Hong Kong. Washington, D.C., is also a news bureau location.


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Los Angeles Dodgers place reliever Paco Rodriguez on the 15-day disabled list with a left elbow strain
The Los Angeles Dodgers placed left-handed pitcher Paco Rodriguez on the 15-day disabled list with an elbow strain, the team announced.
The move is retroactive to May 30, and in a corresponding roster move the team recalled pitcher Matt West from Triple-A Oklahoma City. West was acquired from the Toronto Blue Jays earlier this month and had three appearances with the Texas Rangers last season.
Dodgers manager Don Mattingly said Rodriguez had trouble extending and finishing off pitches after he experienced a drop in velocity. He was sent back to Los Angeles for further evaluation and is set for an MRI to see if there is any further damage to the elbow.
"He's not really having any pain, per se, but we know he's having trouble getting extended and finishing pitches," Mattingly said. "So we're going to try and catch this before it gets to anything.
Mattingly said that West is a player that can go extended innings and give the bullpen some relief if needed.
• CORCORAN: Awards Watch: Pederson leading Bryant in NL Rookie of the Year race
"He's a guy with some length," Mattingly said. "We don't know a whole lot about him, just that the arm is live and knowing that he's got length and is a guy who is fresh and ready to go."
Rodriguez has a 2.61 ERA, 1.26 WHIP, with four holds in 18 appearances this season. Los Angeles (29-20) is in first place in the National League West.
- Scooby Axson


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These days it's all about speed, reliability and flexibility. More and more people work with large files such as DTP/graphical files and uncompressed video and audio. The need for an external hard drive with top speed and top reliability is more present then ever. The new Freecom Hard Drive Quattro provides it all. The new USB 3.0 interface provides you with the fastest data transfer speed to date for a single external hard drive. Connected to eSATA, the Hard Drive Quattro performs up to 6x times faster than a standard USB 2.0 hard drive. The FireWire800 & FireWire400 interfaces have been the standard for years in the Mac-world when it comes to external storage, providing a reliable and fast data transfer. Since USB 3.0 is fully downwards compatible with USB 2.0 you have the ultimate flexibility- you can connect the drive to any modern computer that does not have an eSATA or FireWire interface.
The Hard Drive Quattro comes in a stylish aluminium enclosure which will compliment the design of your PC or Mac, with the possibility to position it horizontally or vertically on your desk. The Freecom Hard Drive Quattro is pre-formatted so you're ready to use your drive seconds after connecting it to your PC or Mac. At Freecom we create storage solutions from a consumer's perspective, where convenience and quality are key factors.
- External, no nonsense hard drive for professional and home use
- 4 interfaces in one drive, provides optimal flexibility, connects to any computer!
- FireWire 800 and FireWire 400, ideally suited for Mac users, ideal for reliable external storage and Audio/Video editing
- eSATA – ideal for professional Audio/Video editing and authoring of large, uncompressed files
- USB 3.0 – the fastest available interface to date, and thanks to the downwards compatibility you can also connect it to USB 2.0, still the most commonly used interface, connects to any modern computer
- High quality aluminium enclosure, recyclable material, puts less strain on the environment
- Optimal internal airflow management
- No cooling fan – no noise!
- Compact design, only 5.5 x14.8 x 4.3 cm, saves space on your desk
- Includes 2 rubber footstands for positioning the drive upright, taking up even less space on your desk
- Two years manufacturer's warranty and unlimited free helpdesk support
INTERFACE: USB 3.0 (can also be connected to USB 2.0**), eSATA, FireWire 800/400
HARD DRIVE TYPE: 3.5" SATA, low noise, low power consumption
DATA TRANSFER SPEED: max. 5000Mbit/s (USB 3.0), max. 3000Mbit/s (eSATA), max. 800Mbit/s (Firewire800), max. 400Mbit/s (FireWire400), theoretical maximum data transfer speeds
POWER REQUIREMENTS: AC adapter 12V/2A
DIMENSIONS: 15.5 x 14.8 x 4.3 cm
WEIGHT: 1400 g
Operating temperature: 10º C - 35º C / Storage temperature: -20º C - 70º C
PC: Intel Pentium III / AMD Duron 900 MHz or higher, 256MB RAM (Vista: 512MB RAM) or higher, available USB 3.0, USB 2.0**, FireWire 800, FireWire 400 or eSATA port, Windows XP / Vista / 7
MAC: PowerPC G3/G4/G5, Power Book G3/G4 or Intel Mac, 256MB RAM or higher, available USB 2.0**, FireWire 800 or FireWire 400 port, Mac OS X v10.4.8 or higher
Drives that are 2TB or under are formatted in FAT32 allowing them to operate immediately on Windows and Mac OSx. Drives above 2TB are formatted in NTFS which is compatible for Windows systems but require Mac users to reformat the drive to HFS+ to make them fully compatible with Mac OSx operating systems. HFS+ formatting can be applied by using the Freecom VHD Formatter which is on the drive.
When connected to USB 2.0, the data transfer speed will be according to USB 2.0 specification
- Freecom Hard Drive Quattro (FAT-32 pre formatted*)
- Power adapter (EU, UK)
- eSATA connection cable
- FireWire 800 & FireWire 400 connection cables
- USB connection cable
- Manual & Software preloaded on the drive
- Quick Install Guide


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More refinements
We couldn't find anything matching your search criteria:
Realestate.com.au is Australia's #1 home for rental property, listing real estate for rent in Cape Barren Island, and other rental property listings by real estate agents in Cape Barren Island. If you're looking to rent a property in Cape Barren Island, we have house prices in Cape Barren Island. Realestate.com.au can help you find rentals open for inspection in Cape Barren Island, and houses to share in Cape Barren Island. If you're searching for Cape Barren Island property for sale or sold property in Cape Barren Island, we can help you with your real estate search.


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Doing a powertrain tear down and assembly? Tired of hunting through plastic bins looking for hardware? JIMS®, a premier manufacturer of performance parts and specialty tools for use on Harley-Davidson® Motorcycles, has released the new JIMS Hardware Organizer.
The patent pending JIMS hardware organizers will guarantee all hardware is organized during powertrain teardown, storage, and final assembly. The organizers include a specific stand for top end, cam chest, transmission, and primary. When combined, they all nest within each other taking up very little space and can easily fit in a deep drawer of a tool box.
When preparing for final assembly, the stands can be turned on end to expose threads for ease of loctite preparation. The surface is powder coated in JIMS durable blue and includes silkscreened hardware position art with torque specifications. For more information find JIMS® on the web at www.jimsusa.com, become a JIMS Facebook fan, or call 805-482-6913.


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I can write out as many evaluations of my students that I would like and have my students do the same, but the expectations of grades still looms.
I need a fast reference point to see how an individual is doing on an assignment, a project, a unit, or even a skill.
Students need the same thing.
Parents need the same thing.
School administration needs the same thing.
Their future teachers need the same thing.
What I would like to change is how assignments and what assignments are graded.
I believe that students should be graded on their individual merit and effort. They are not being judged head to head like horses about to race. They are learners who should be encouraged to accept mistakes as learning opportunities. As an English teacher I need them to see that they spelled a word wrong or misplaced a comma, not so they can be punished with a grade for it, but so they can correct that mistake and similar ones in the future.
What I want to grade are the major assignments. I have no interest in pouring my time into entering grades for piles of grammar sheets or having students take notes on a skill they are going to need for a project and then delving out points for turning in the notes. Whether or not they took notes well and paid attention will be determined by their project. Then, if they didn't do that, or they don't learn that way, it will give me opportunities to re-teach them.
I am not interested in cutting corners, though my time, like I'm sure most teachers', is precious to me. What I'm trying to do is to emphasize and focus on what really matters. My students will demonstrate their knowledge and skills through the completion of projects and units of study. Those need my time and attention. If their work is not what it needs to be, then their grade - yes grade - will be lowered while I give them feedback and guidance as to how they can improve their work. Once that happens, the grade goes up.
So, how do you feel about grading? My beliefs and practices in this area have pretty much been a rollercoaster ride. From grade every little thing to grade nothing. What do you believe are our responsibilities and how does our grading factor into learning?
Comments, as always, are appreciated.


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Nick Stellino recipes are on his website, NickStellino.com. Several recipes are also on the Today Show website. Many of Stellino's recipes are from his two cooking shows on PBS, "Nick Stellino Cooking With Friends" and "Nick Stellino's Family Kitchen."
Stellino's website lists his recipes in categories such as entrées, sandwiches and desserts. He has a whole category for breakfast and brunch recipes as well as pizza and pasta recipes. Additionally, users have the option to search for recipes that include videos.
Stellino also provides One-Minute Cooking School videos on his website. These short videos include quick recipes with tips for meals such as French toast, sautéed halibut and steak. He also has video recipes for almond rice pudding, bread pudding and chocolate pudding. Under the section for all recipes, categories include soups, salads, appetizers, fish and side dishes. Another option users have is searching by recipe name in the search bar.
The Today Show website provides two Nick Stellino recipes as of July 2015, and both are for the summer season. The first recipe is called "Tomatoes and Bread Salad." This recipe makes four servings and uses ingredients such as arugula, capers and feta cheese. The second recipe is called "Pasta With Cherry Tomatoes, Basil and Shrimp." It serves four to six people and involves sautéing shrimp with a number of spices, making a shrimp sauce that gets tossed with pasta.


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☎ Order by phone on 0844 326 2200
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An aptly named variety, super fast growing, early maturing and poducing many well-filled pods approximately 20cm (8") long, each containing 5 or 6 plump, greenish white seeds of very good flavour. RHS Award of Garden Merit winner.
Hardiness:0 degrees
Aftercare - Easy:True
Aftercare - Moderate:False
Aftercare - High:False
Edible Flower:False
RHS Award of Garden Merit:True
Likes Acidic Soil:False
Beds & Borders:True
Prefers Full Sun:True
Grows in Sun or Shade:True
F1 Variety:False
Sow direct into soil prepared by digging and raking at a depth of 5cm (2"), spacing the seeds 20cm (8") apart. Best sown alternately in double rows allowing 23cm (9") between the two lines forming each row. Allow 60-90cm (24-36") between further rows. Germination 7-21 days. Can also be sown in pots or trays of compost on a windowsill or in a greenhouse for eventual planting outside. Grows best in a sheltered site, but not shaded, in well drained soil. Pinching out tips helps to prevent attacks of blackfly. Water well during dry weather to aid flower setting and bean formation. Sowing to Cropping: 14 weeks.
Ideal for beds and borders. Prefers full sun but grows well in sun or shade.
All Suttons products should reach you in perfect condition, just as they left us. If you are dissatisfied in any way with their condition on arrival, please let us know within 14 days. We are only liable for the cost of the goods as quoted on the website plus the cost of delivery if the goods are faulty.
SuttonsOrder Online or call 0844 326 2200 - 24hrs a day, 7 days a week Product or other query Email Customer Services Customer Services call 0333 043 0700 - 8:30am -5pm, Mon-Fri Charges to 0844 numbers cost 5p per minute plus your telephone company's access chargeCharges to 03 numbers are the same as standard UK landline numbers starting in 01 or 02 and mobile providers may vary
Suttons, Woodview Road, Paignton, Devon TQ4 7NG. Registered in England and Wales No. 284448. VAT No 158 9318 27.


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Colours (brown, green, red, blue and yellow)
Little hands
|Emma LOVED doing this!
** Jan 4, 2011: Just a small added update since we've made quite a few of these as Christmas gifts. We also glued mini pompoms in addition to finger prints to the card. Its the blue and white ones in this pic.


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Broadway Tickets
In the past, if you wanted discount tickets to Broadway theater in NYC, you had to stand in line on the day of the show, often for hours, at the TKTS booth in Times Square. When you finally got up to the ticket booth, you might find after all that waiting that the show you wanted to see was sold out. Now you can get discounted tickets to the *best broadway shows* from the comfort of your own home with the help of Headline Tickets.
Why Buy Broadway Theater Tickets Online?
Buying Broadway theater tickets online gives you the ability to shop around for the best prices. We encourage you to search for the Broadway ticket prices on other websites and compare them to ours to find who offers the best deals. However, be careful when you shop, because many sites post a low ticket price and then add various fees at checkout. When you do a thorough end to end price comparison, you'll discover why so many people look to Headline Tickets for all of their discount Broadway theater ticket needs. At Headline Tickets, you pay only the cost of the ticket and the delivery method you choose, and we never add extra fees at the end, so you always know up front exactly how much you will pay when you click to make a purchase.
If you purchase tickets through us and your plans change, in many cases you may have the option to resell the tickets through us. You might just make someone's day by making available tickets to a show that they wanted to see that was previously sold out.
You'll save time and money purchasing your theater tickets from Headline Tickets, and we've got tickets to all of the current Broadway shows.
Tips For Getting the Broadway Musical Tickets
Shop early to get the best access to good seats at great prices. It can also be helpful to start clicking on the ticket pool early instead of waiting for the cheap tickets to go on sale, because this helps the site recognize what you are looking for to alert you when the tickets are released for sale online.
Open multiple browser windows or tabs to compare ticket prices and seating locations on various sites or on ours. This can speed up your ability to find the right deal, as clicking back and forth between options in the same window takes a lot of extra time and you may miss out on a deal that comes up because you were looking elsewhere and couldn't get back in time.
Familiarize yourself with the venue and the seating charts to determine which seating areas are preferable for you.
Mobile Apps
To save you time and offer convenience, we offer mobile apps for iPhone and Android devices to ensure that you will have instant access to the hottest Broadway shows on the go so you don't have to sit in front of your computer at home or at work waiting to purchase discounted Broadway tickets. Download the app today so that you never miss out on great tickets at low prices simply because you were on the move and couldn't get to your computer.
Save Even More
Be sure to always check out our Discount Code page to save even more money! We have multiple codes available on any given day, and our codes do change, so you should always check the discount codes page before ordering to ensure that you get the lowest prices available.
Purchasing Tickets
Time is of the essence in purchasing discount tickets for popular Broadway shows. To ensure you don't miss great ticket deals because of delays in the process of entering your information, be prepared with credit card, address and and any other relevant information before you sit down to purchase tickets. Decide beforehand if you will want to purchase ticket insurance to avoid delays. The better prepared you are with all of the necessary information, the better your chances to ensure that you can get the best seats available at the best prices.
Check Back Often
If we sell out of tickets for the show you want to see, make sure to check back often, because often people purchase tickets but their plans change last minute. Because we offer the option for customers to resell their tickets, you may find last minute that the sold out show you want to see suddenly has tickets available.
What Are the Best Current Broadway Shows in New York?
Cirque du Soleil Paramour
Shuffle Along
The Lion King
Les Miserables
A Long Day's Journey into Night
What Was the First Broadway Theatre?
The history of Broadway theater dates back to the 1700s, when when actor-managers Walter Murray and Thomas Kean established the first resident theatre company at The Theatre, downtown on Nassau Street. The Theater held just under 300 people and presented Shakespeare plays and ballad operas. More theaters were built during the 1800s as demand for entertainment increased in Manhattan. Beginning in the 1900s, more theaters were built in what is now known as the Theater District area of Broadway, which is located between 42nd Street and 53rd Street in Midtown, all around Times Square. The Broadway theaters in the Theater District used electric signs with bright white lights to advertise shows, so the Broadway Theater district became known as "The Great White Way".
What Should I Wear to a Broadway Theatre?
Many people love to dress up to go to the theater and make an elegant evening of it. However, there is no dress code in Broadway theaters, so if you have a ticket you will be admitted. New York City is a pedestrian city and tends to be very casual with regard to dress, with people coming from all walks of life and who may be going out for the evening straight from work or from some other destination. Broadway theater venues are no exception. Many people prefer to wear whatever is most comfortable for them when they go out to the theater or other entertainment venues. Broadway theaters tend to be highly air conditioned, so jeans are common attire, and it might not be a bad idea to bring a sweater if you tend to get cold easily.
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