text:
Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s10858-012-9642-5
- Cite this article as:
- Henriksen, N.M., Davis, D.R. & Cheatham III, T.E. J Biomol NMR (2012) 53: 321. doi:10.1007/s10858-012-9642-5
Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility—using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions—by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5γ and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to "update" older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.
KeywordsRNA structure Molecular dynamics Residual dipolar coupling restraints Bulge structure Force fields Ion binding
Molecular dynamics (MD) simulations are often used with restraints derived from crystallography or nuclear magnetic resonance (NMR) experiments for the end-stage atomistic refinement of biological macromolecular structures (Clore et al. 1985; Brunger et al. 1987a, b; Nilges 1996; Brunger and Adams 2002). Quite commonly, rather quick and standard MD refinement protocols are employed using codes such as X-PLOR (Brunger 1992), XPLOR-NIH (Schweiters et al. 2003, 2006) or CNS (Brunger et al. 1998; Brunger 2007). A refinement protocol might initiate a search for restraint-compatible structures via simulated annealing or distance geometry methods followed by very short (20–200 picosecond) gas-phase MD simulations with applied restraints to further relax and refine the structures. In these refinement protocols, often the force field—specifically the molecular mechanical parameters and force constants that define the covalent connectivity and atomic pair interactions—is rather simplified or crude and the MD simulations are performed in vacuo in the absence of solvent and mobile counter-ions. Despite the limitations of these simplified force fields, excellent results are generally obtained given a sufficiently robust set of experimentally derived data. This latter point is somewhat obvious noting that, if sufficient data from experiment has been collected to define the structure, the force field should not strongly influence or bias the results as the structure should be largely determined by the experimental data. However, when the experimental data is sparse, the structure is dynamic, or when solvent and mobile ions may be critically important elements of the structure, the arguably simple force fields and/or absences of experimental restraint data may lead to "loose" structure ensembles. These loose ensembles may suggest larger ranges of motion where data was absent and/or populate anomalous structures leading to an incorrect interpretation of the structure. A logical step to make up for missing data is to apply optimized biomolecular force fields in MD simulations with explicit solvent and modern simulation protocols. A relevant example involves the refinement of nucleic acid structures from NMR data, particularly in the absence of residual dipolar coupling (RDC) information, where long-range restraint information is absent (Konerding et al. 1999). If the force field and simulation protocols are reasonably robust and ideally experimentally validated, they together with the experimentally derived restraint information should provide a better representation of the structure. Although NMR refinement using modern MD simulation protocols with experimentally derived restraints, optimized force fields for proteins and nucleic acids, and explicit solvent suggests this to be true (Prompers et al. 1995; Kordel et al. 1997; Linge and Nilges 1999; Gouda et al. 2001; Spronk et al. 2002; Xia et al. 2002; Linge et al. 2003), such methods are not widely or routinely applied. For example, in recent years only a handful of NMR structures have been refined in explicit solvent using optimized force fields and modern simulation protocols, and these include the structure elucidation of a peptide (Dolenc et al. 2010), an RNA hairpin (Nozinovic et al. 2010), a DNA naphthalimide adduct (Rettig et al. 2010), a designed metalloprotein (Calhoun et al. 2008), and an RNA receptor/ligand complex (Paulsen et al. 2010a).
Although refinement of NMR structures using modern force fields and simulation protocols, including explicit solvent, appears to produce structures that provide excellent fits to the experimental data, these approaches are not without limitation. Complications relate to dynamics, the time scales of the dynamics, and whether these motions are even accessible on the timescale sampled during the molecular dynamics refinement. Moreover, RNA may populate multiple conformations under the given set of experimental conditions (Al-Hashimi and Walter 2008; Hall 2008; Baird and Ferre-D'Amare 2010; Solomatin et al. 2010; Stelzer et al. 2011). With traditional refinement, high restraint weights may lead to structural representations that are too tight, that hide dynamics, and that limit potential transformations between multiple conformations (James 2001). Essentially, an average structure will be found that may not entirely satisfy all of the experimental data. Procedures such as time-averaged restraints (Torda et al. 1990; Pearlman and Kollman 1991; Schmitz et al. 1993), selective enforcement of restraints over time (Gorler et al. 2000), or ensemble based refinement methods (Bonvin and Brunger 1995; Schwieters and Clore 2007) may help mitigate these issues. However, these methods will further depend on the reliability of the force field representation to correctly sample the accessible conformational space. Ultimately, given a reliable and validated force field, the molecular dynamics simulations without experimental restraints starting from the refined NMR structure should provide an accurate representation of the structure and dynamics over the simulation time scale. However, the force fields are not yet fully reliable, especially in the treatment of RNA (McDowell et al. 2007; Besseova et al. 2009; Hashem and Auffinger 2009; Banas et al. 2010; Deng and Cieplak 2010). Therefore, at present, there needs to be a careful balance between the relative weights of the force field compared to the experimental or structural restraints, with further care levied to understand the limitations of the force fields and implications of specific restraint choices.
In this work we further assess the reliability of more detailed MD structure refinement protocols through the re-refinement of two similar RNA molecules. As part of our larger force field assessment efforts, we have been investigating a variety of RNA structures in free, unrestrained MD simulation to better understand the reliability and flaws of the AMBER nucleic acid force fields (Cornell et al. 1995; Cheatham et al. 1999; Wang et al. 2000; Perez et al. 2007b; Banas et al. 2010; Yildirim et al. 2010; Zgarbova et al. 2011) as compared to available experimental data. Ideal RNA structures for our investigation include those which display some non-canonical structure (i.e., non-helical structure since the AMBER force fields appear to do a reasonable job of modeling nucleic acid helices (Csaszar et al. 2001; Reblova et al. 2003; Beveridge et al. 2004; Dixit et al. 2005; Perez et al. 2007a; Ditzler et al. 2010; Lavery et al. 2010)) and importantly, structures where detailed NMR restraint information is available including NOE derived distance, J-coupling and RDC restraints. Our explorations led us to two published RNA structures that have nearly identical primary sequence, yet the published 3D structures differ significantly.
Together, these two structures and their previously accumulated and published data (chemical shifts, restraints, and chemical shift perturbations) provide an intriguing opportunity to validate the MD simulations, to assess simulation refinement protocols which use explicit solvent and modern force fields, and to ultimately determine whether the significant differences in these structures reported are real or artifacts from the refinement process. We show that re-refinement leads to two very similar structures that appear to better satisfy the NMR data. In addition, beyond the bulge region (which is strongly influenced by packing effects and tertiary interactions), the stem and loop regions around the bulge better match the ai5γ-D5 (PDB: 1KXK) crystal structure than the previously published NMR structures. Yet, the results also suggest that a careful balance between relative weights of the force field compared to the experimental data is required, that the experimental data has to be carefully screened, that there are clear sampling limitations, and that there are still known and emerging force field limitations. Taken together, these observations preclude the use of automation to automatically refine structures. The previous and current success of these methods suggest that some published structures might be improved using explicit solvent MD refinements and that such techniques should be more routinely applied in future structure refinement projects.
Materials and methods
Coordinates and restraint data
The coordinates and restraint data for the ai5γ_NMR and PL_NMR structures were retrieved from the RCSB Protein Data Bank website using the PDB codes 1R2P and 2F88, respectively. Of the ten conformers contained in each PDB file, only the first five were used in simulations. The distance restraint data was thoroughly checked for atom naming mismatches between the restraint file and the PDB files. Mismatched restraints were corrected based on visual inspection of the structures (common mismatches included H62 → H61 or H5′1 → H5′2 transpositions). Distance restraints were weighted at 20 kcal/mol-Å within 0.5 Å of the bounds of the flatwell restraint and at 20 kcal/mol-Å2 outside this range, unless otherwise noted. The applied dihedral restraint data from the two earlier refinements were consolidated and reconfigured to produce a more consistent and liberal set of dihedral restraints and also to eliminate minor differences in the conventions used by the ai5γ_NMR and PL_NMR authors. Backbone torsions of the helical regions (torsions 1:γ–8:γ, 10:ε–14:γ, 19:ε–23:γ, 27:ε–34:ε) were restrained to A-form values (±15°) while those in the remaining non-canonical regions were left unrestrained. The χ torsion angle was restrained to syn (70 ± 30°) for G26 and anti (−160 ± 15°) for all others. Sugar puckers were restrained (i.e., torsions δ, ν1, and ν2) in a similar manner as described in the original publications: for ai5γ-D5, A16 and C25 were restrained to C2′-endo, whereas G15, A17, and A18 were unrestrained, and the remaining were restrained to C3′-endo; for PL-D5, A16, A24, and A25 were restrained to C2′-endo, and the remaining were restrained to C3′-endo. Torsion restraints were weighted at 500 kcal/mol-rad within 1° of the bounds of the flatwell restraint and at 500 kcal/mol-rad2 outside this range, unless otherwise noted. Relative RDC restraint weighting, set with the "dwt" keyword in AMBER, was chosen to be the highest value that did not cause SHAKE errors during simulation (dwt = 0.02 for ai5γ-D5, dwt = 0.01 for PL-D5). Base pair planarity restraints were not applied. All restraints were converted to the AMBER formats using in-house scripts and scripts available in AmberTools. The restraint files used during the refinement are supplied in the Supporting Information.
Building, heating, and equilibrating solvated structures
All MD simulations were performed using the AMBER and AmberTools suites of software (Pearlman et al. 1995; Case et al. 2005). The PDB structures were parameterized using the AMBER ff99bsc0 (Perez et al. 2007b) force field, and were solvated in an icosahedral TIP3P (Jorgensen et al. 1983) water box out to at least 10 Å in each direction from the solute followed by net-neutralization with Na+ ions and addition of ~200 mM NaCl using the Joung and Cheatham ion parameters (Joung and Cheatham 2008, 2009). The Na+ cation was chosen for initial investigations due to the salt crystallization artifacts seen with the earlier K+ parameters (Auffinger et al. 2007), despite the fact that K+ was used in the NMR buffer. The Na+ cation was used throughout this work, albeit with the improved parameters, in order to maintain consistency with our older data. In total, the solvated systems contained between 9,000 and 12,000 residues, corresponding to approximately 29,000–37,000 atoms. After building the coordinate and parameter/topology (prmtop) files, the positions of all the ions in the coordinate files were randomized using ptraj, ensuring that ions were at least 6 Å from an RNA atom and 4 Å from each other. The particle mesh Ewald method (Essmann et al. 1995) was used to handle electrostatic interactions using a 9 Å cutoff with default parameters (including an ~1 Å grid spacing, cubic spline interpolation, and a direct space cutoff tolerance of 0.000001). Lennard-Jones interactions were also treated with a 9 Å cutoff and the pairlist built to 10 Å was automatically rebuilt if any atom moved more than 0.5 Å since the previous update. Each system was first relaxed with 1,000 steps each of steepest descent and conjugate gradient minimization while RNA atom positions were restrained with 25 kcal/mol-Å2 positional restraints. Continuing with the same positional restraints, the system was slowly heated from 100 to 300 K over the course of 100 ps at constant volume. After heating, the system was repeatedly minimized (1,000 steps each, steepest descent and conjugate gradient) and equilibrated at constant pressure (for 50 ps each round) using gradually weaker positional restraints (5.0, 4.0, 3.0, 2.0, 1.0, and 0.5 kcal/mol-Å2). For restrained simulations, distance and torsion restraints as previously described were enforced during each step of the equilibration process. A final equilibration step for the restrained simulations was included following the 0.5 kcal/mol-Å2 position restrained equilibration. This step consisted of a relaxation period of 2 ns at constant pressure without positional restraints and with distance and torsion restraints at 10 % of normal strength.
Restrained production simulations
Simulation details and nomenclature for the various refinements
No. of simulations
Simulation notes
Unrestrained simulation
Distance and angle restraints enforced
Distance, angle, and RDC restraints enforced
Modified distance and angle restraints enforced
Modified distance, angle and RDC restraints enforced
Unrestrained simulation
Modified distance and angle restraints enforced
Distance, angle, and RDC restraints enforced
Modified distance and angle restraints enforced; additional heating step
Modified distance, angle, and RDC restraints enforced
The structure refinement protocol presented here significantly extends the procedure used to generate the ai5γ_NMR (Sigel et al. 2004) and PL_NMR (Seetharaman et al. 2006) structures. Specifically, significantly longer MD simulations were performed including explicit solvent and mobile counterions, modern force fields, and proper treatment of the long range electrostatic interactions. Longer simulation, and in some case heating, provided significantly more sampling of potential RNA structure and helped identify where structures may have otherwise been trapped due to previously insurmountable barriers. In contrast, both earlier publications report using CNS to generate an extended structure, followed by the selection of 100 starting structures generated from different random initial velocities. The starting structures were relaxed using high-temperature, torsion-angle dynamics, slow cooling using distance and angle restrained molecular dynamics, and minimization. Total molecular dynamics for each structure in the earlier refinement protocols did not exceed 250 ps. The PL_NMR structures were then further refined based on the RDC data using a more extensive protocol.
All PDB and trajectory structures were visualized using UCSF Chimera (Pettersen et al. 2004). Structure snapshots were also generated using Chimera. RMSD values were generated using AMBER's ptraj module and results were plotted using Grace or Microsoft Excel. Distance and dihedral measurements were calculated and analyzed using ptraj and in-house scripts. Clustering was performed in ptraj (Shao et al. 2007) using the following settings: average-linkage algorithm, cluster count set to 5, rms similarity metric on base heavy atoms only, and sieve set to 5. To generate representative structures for the restrained simulations, the average structure of the dominate cluster for each trajectory was minimized with full restraints. Grid analysis (Cheatham and Kollman 1997) was performed using ptraj and visualized in Chimera. Occupancy analysis of water and Na+ was performed using the hbond command in ptraj.
Initial results with unrestrained simulations
Prior to running the restrained simulations, we performed a set of ~100 ns unrestrained simulations using the ai5γ_NMR and PL_NMR starting structures in order to evaluate the AMBER ff99bsc0 force field; a summary of all of the simulations performed is provided in Table 1 and a figure highlighting the structural and sequence differences is shown in Fig. 1. These simulations, named ai5γ_UR and PL_UR, respectively, were built and equilibrated using the same procedure as for the restrained simulations except without any of the steps related to the distance, torsion, or RDC restraints. The initial results from these simulations led us to begin a more thorough investigation using restrained simulations for two reasons. The first is related to the structural compactness of the ai5γ_NMR and PL_NMR structures. One of the more striking differences between the published ai5γ_NMR and PL_NMR structures is the overall structural length, as measured from the top of the tetraloop to the bottom of the lower helix. The PL_NMR structures display a compacted global conformation that is consistent with and almost as compacted as the x-ray structure of ai5γ-D5 (Zhang and Doudna 2002; Seetharaman et al. 2006), whereas the ai5γ_NMR structures are much more extended (Figure S1). In contrast, when we compared the average structures for the unrestrained ai5γ_UR and PL_UR simulations, both sets of structures adopted the more compact conformations. Visual inspection of the ai5γ_UR trajectories revealed that the end-to-end distance of the structures underwent an approximately 15 Å compaction within the first 10 ns of the simulations. This rapid compaction on the MD simulation time scale suggests that the extended structure is not compatible with the force field, a force field as discussed in the introduction that is known to fairly reasonably model many RNA structures. The PL_UR structures, whose starting structures were already more compact, underwent no appreciable compaction during simulation. This likely leads to the rather significant difference in plateau RMSD values between the ai5γ_UR and PL_UR simulations (Figure S3). Note that although the RMSd values plateau and appear relatively small, at least in the case of the PL_UR structures with RMSd values in the ~3–6 Å range, structural disruption in the bulge and loop regions was evident. However, the similarity of the two sets of unrestrained simulation structures after MD simulation led us to wonder if perhaps the conformation of these two molecules were more similar than the conventionally refined structures suggest or if the minimalist gas-phase refinement protocol employed previously was insufficient to refine the structures.
The second reason these simulations encouraged us to perform a more detailed analysis was related to the localized loop and bulge structural features. We found that during both the ai5γ_UR and PL_UR simulations these regions experienced significant structural degradation. For instance, at various times in the five independent simulations the loop conformation would transition to a pathological, yet stable, geometry that often persisted for the rest of the trajectory. It was soon clear that accurate modeling of these molecules could not be achieved using the MD force field alone, and thus we decided to perform restrained simulations using the original NMR data. While the RNA force field parameters have considerably improved (Cornell et al. 1995; Cheatham et al. 1999; Foloppe and MacKerell 2000; Wang et al. 2000; Perez et al. 2007b; Banas et al. 2010; Yildirim et al. 2010; Denning et al. 2011), our results suggest the geometries of the refined structures presented in the current study are primarily determined by the experimental restraints. The explicit solvent environment and updated force field play a secondary yet critical role, as the resulting structures appear to be improved compared to those obtained using conventional methods.
Restrained simulations produce conformational rearrangements in ai5γ-D5 and PL-D5
Structural statistics for ai5γ-D5 and PL-D5 representative structures
No. of structures
No. of distance restraints
No. of dihedral restraints
No. of RDC restraints
Avg. RMSd of distance (Å)
Avg. RMSd of dihedral (°)
Avg. RMSd of RDC (Hz)
Avg. RMSd from ideal bonds
Avg. RMSd from ideal angles
No. Distance viol. > 0.2 Å
No. Angle viol > 5.0°
Overall heavy atom RMSD
Avg. RMSd from mean
2.58 ± 0.84
1.41 ± 0.29
0.66 ± 0.32
0.83 ± 0.12
1.69 ± 0.45
1.14 ± 0.28
Avg. RMSd pairwise
4.09 ± 1.20
2.10 ± 0.87
1.04 ± 0.46
1.30 ± 0.24
2.56 ± 1.04
1.70 ± 0.76
Pairwise RMSD measurements
Pairwise RMSD
ai5γ_NMR vs. PL_NMR
6.03 ± 1.00
ai5γ_NMR vs. ai5γ_DAR
7.18 ± 1.52
ai5γ_NMR vs. ai5γ_mDAR
5.61 ± 1.22
ai5γ_NMR vs. PL_DAR
6.55 ± 1.69
ai5γ_NMR vs. PL_mDAR
6.18 ± 1.34
PL_NMR vs. ai5γ_DAR
4.29 ± 0.52
PL_NMR vs. ai5γ_mDAR
2.71 ± 0.15
PL_NMR vs. PL_DAR
2.67 ± 0.63
PL_NMR vs. PL_mDAR
2.21 ± 0.40
ai5γ_DAR vs. PL_DAR
3.33 ± 0.61
ai5γ_DAR vs. PL_mDAR
3.23 ± 0.70
ai5γ_mDAR vs. PL_DAR
2.33 ± 0.82
ai5γ_mDAR vs. PL_mDAR
1.95 ± 0.40
ai5γ_DAR vs. ai5γ_mDAR
3.13 ± 0.66
PL_DAR vs. PL_mDAR
2.00 ± 0.88
The differences between the PL_NMR structures and PL_DAR structures are not as drastic as those for ai5γ. In contrast to the results for ai5γ, the intra-structure pairwise RMSD is higher for PL_DAR (2.56 Å) than for the PL_NMR (1.30 Å). One of the most obvious differences between the PL_DAR and PL_NMR structures is that during equilibration and relaxation, residue A25 of model 4 shifted from a partially extruded position in the minor groove to a stacked position within the helix. Thus the PL_DAR ensemble has three of five structures with A25 stacked, whereas the PL_NMR structures have two of five (ignoring structures 6–10 in the published PDB structure file). All five representative structures for PL_DAR show U9 participating in a hydrogen bond with either A24, A25 or G26. In the two structures with A25 extruded, U9 interacts with A24. Of the three structures with A25 stacked in the helix, two show U9 interacting with A25 and one shows a hydrogen bond between U9 H3 and G26 N7. During simulations of the former case, U9 shifts back and forth between interactions with A24 and A25. In the latter, the U9 H3–G26 N7 hydrogen bond is particularly stable throughout the trajectory, leaving A24 and A25 stacked above U9. In all five simulations, these interactions close the "hole" described by Seetharaman et al. in reference to the PL_NMR structures. Interestingly, for the three structures with A25 in the stacked position, G26 no longer "packs into the major groove against G8" (as in all five PL_NMR structures), but points away from the major groove with the base plane parallel with that of U9. For the two structures with A25 extruded into the minor groove, G26 remains oriented towards the major groove.
Troubleshooting problematic or unusual regions in the refined ai5γ-D5 and PL-D5 structures
On closer inspection of the refined models, some features of both the ai5γ_DAR and PL_DAR structures seemed problematic. For ai5γ_DAR, there were three distance restraints in the bulge region with consistently large upper bound violations during simulation (these restraints connected the following atoms: U7 H1′-A28 H2, G26 H8-A28 H1′, G26 H8-U7 H3). Two of these involved the atom G26 H8. On closer inspection it seemed possible that these two restraints were responsible for the severe kink in the backbone noted by the authors of ai5γ_NMR structures as being a very unusual conformation. Given the high upper and lower bounds of these restraints, one of the corresponding authors (Samuel Butcher, personal communication) suggested to us that these restraints were derived from weak NOEs that may be mediated by spin diffusion. Even though the U7 H1′–A28 H2 restraint violation was probably a side effect of the other two restraints, we decided to investigate the effect of removing all three problematic restraints. Identical simulations to ai5γ_DAR were then run with the three aforementioned restraints removed to generate the simulations that are designated as ai5γ_mDAR.
Additionally, the unrestrained simulations suggested that the positioning of the A25 residue in the PL_DAR structures might also be problematic. Although the unrestrained simulations are imperfect due to force field deficiencies, we never observed A25 extruded into the minor groove during the 100 ns of unrestrained simulations. As mentioned previously, we also found that one fully restrained simulation showed A25 move from the extruded position to the stacked position and we therefore considered whether this conformation might be preferred. To investigate this, we tested several different equilibration and relaxation conditions before choosing a procedure for the modified restrained simulations. Many of the conditions resulted in either one or two of the three extruded structures transitioning to a stacked structure. Critically, in none of these conditions did A25 transition from a stacked conformation to an extruded conformation. In one condition, all three of the extruded structures transitioned to stacked structures. This condition involved three alterations to the PL_DAR simulation procedure: (1) the removal of the Watson–Crick base pair restraints, but not the NOESY restraints, between residues G8 and C27 (which are not present for A8/U27 in ai5γ_DAR), (2) increasing the weight of distance restraints from 20 to 50 kcal/mol as well as increasing the G26 χ dihedral restraint from 500 kcal/rad to 1,000 kcal/rad, and (3) heating to 700 K with restraints at 8 % strength to allow structural relaxation, followed by a smooth increase of restraint weight to 100 % strength prior to the production simulation. The rationale for removing the Watson–Crick base pair restraints was to allow structural transitions in the bulge region that may otherwise be hindered. The changes to the restraint weighting were made to ensure the enforcement of distance restraints and prevent G26 from flipping to the anti conformation (a frequent problem for previous attempts) while the heating period was intended to enhance conformational sampling. These simulations were named PL_mDAR.
Analysis of the bulge and loop regions in optimized, restrained simulations
The removal of the three problematic restraints from the ai5γ restraint list makes a significant difference in the bulge region of the ai5γ_mDAR structures. First, the intra-structure pairwise RMSD of the ai5γ_mDAR structures (1.04 Å) is significantly lower than ai5γ_DAR (2.10 Å) (Table 2). The sharp kink observed in the bulge of the ai5γ_DAR structures is replaced by a smooth, upward trending backbone in the ai5γ_mDAR structures (Fig. 4, green). Rather than being drawn below the major groove face of the A8-U27 base pair, C25 is positioned above A8-U27 and its base plane is parallel with A8′s in the ai5γ_mDAR structures (Fig. 3, bottom). This positioning puts U9 directly in between the A24 and C25 bases and during simulation U9 alternates hydrogen bonding between A24 and C25. Occasionally, C25 N3 or O2 also form a hydrogen bond with the A8 H61, H62 atoms. In contrast to the ai5γ_DAR structures, four of the five ai5γ_mDAR have the base plane of G26 perpendicular to the helical axis, thus pointing away from the major groove.
The other non-canonical structure of interest, the GAAA tetraloop, is reasonably similar between the NMR structures and among each of the restrained simulation structures. These structures are also closer to the earlier crystal structure. Whereas the RMSD values for residues 10–23 are 1.64 and 1.79 Å when the original NMR structures are compared to the crystal, the re-refined structures yield values of 1.37 and 0.92 Å (for ai5γ and PL, respectively). However, some variation in the backbone torsions are observed, such as that of the γ torsion of residue A16, which we attribute to the bsc0 modifications of the AMBER ff99 parameters which disfavor γ in the trans configuration (Perez et al. 2007b). In both the ai5γ_NMR and PL_NMR structures, γ is in the trans configuration while in each of the ai5γ_mDAR and PL_mDAR structures this torsion is in the gauche+ position. In addition to the backbone differences, the base orientations in the tetraloop were compared between the NMR structures and the restrained simulation structures. Previous work by Correll and Swinger (2003) identified aspects of the GNRA tetraloop that commonly vary between one of two conformations. The first of these involves the planarity of the "NRA" portion of the tetraloop with respect to the underlying base pair of the upper helix (i.e., the planarity of residues 16–18 with respect to the base pair formed by residues 14 and 19 in the case of D5). When the NRA bases are planar with the underlying base pair, the conformation is referred to as the "standard orientation". If the NRA bases depart from planarity, typically tilting upward and away from the underlying base pair, the conformation is named the "altered orientation". In both cases, the three NRA bases remain stacked together. Visual inspection of both the NMR structures and representative structures from our restrained simulations reveal that nearly all of the tetraloops adopt the altered orientation. This contrasts with results from unrestrained simulations in which the standard orientation seems to be favored.
Deviations from ideal geometry when using the AMBER force field
One problem which might deter researchers from using the AMBER force field is the slight deviations of covalent bond angles from ideal geometries. Deviations outside of the expected covalent bond angle range were observed for all of our AMBER refined structures as well as other recent structures (Paulsen et al. 2010b; Tolbert et al. 2010) when submitted to the ADIT-NMR (AutoDeposit Input Tool for NMR structures: http://deposit.bmrb.wisc.edu/bmrb-adit/) from the RCSB website (http://www.rcsb.org). The origin of the deviations results from the use of shared, transferable and rather generic atom types for the nucleobases (such as CT for all tetrahedral carbons and OS for O2′, O3′, O4′, and O5′) rather than specific types for each different atom to more accurately represent nucleoside geometry. Although the deviations (on the order of ~5° or less) are outside the range observed in experimental databases (Clowney et al. 1996; Gelbin et al. 1996), these deviations are within the range of thermal fluctuation and likely have a small impact on the overall structural quality due to compensation by the many degrees of freedom in large biomolecules. Addressing these deviations will require an overhaul of the atom type naming system used by the AMBER force field and is the subject of ongoing research. In addition to the deviations observed for covalent bond angles, some deviations were also observed in base planarity (on the order of 0.1 Å rmsd or less), which likely reflect restraint strain on the relatively soft improper torsion parameters used in the AMBER force field to maintain planarity.
Comparision of representative ensembles with and without RDC restraints
A comparison between the simulations run with and without RDC restraints (DAR and DA, respectively), suggests that the RDCs primarily affect the structural compactness of these RNA structures, but not the local conformations. Both the ai5γ_mDAR and PL_mDAR simulations resulted in an increase of overall structural length (measured by the distance between A16 P and C34 P). The average structure length for ai5γ_mDA and ai5γ_mDAR, taken from representative structures, was 47.0 and 54.5 Å, respectively. The increase was less dramatic for PL, for which the average structure length was 52.8 and 53.4 Å for PL_mDA and PL_mDAR, respectively.
These results are consistent with a recent study by Tolbert et al. (2010) of a purely A-form RNA double helix, which suggested that a wide range of A-form structures are accessible when using only distance and torsion restraints. In contrast to what was observed by Tolbert et al., the addition of orientational restraints resulted in structural expansion, not contraction. However, the differences between the simulations (pure helical RNA vs. non-canonical RNA, implicit solvent vs. explicit solvent) preclude further conclusions.
Solvation and Na+ density during simulation
In addition to structural information, the publications describing both the ai5γ_NMR and PL_NMR solution structures also included data describing chemical shift perturbations observed upon titration of D5 RNA with Mg2+. The results for ai5γ_NMR, which only tracked 1D proton shift changes, differ somewhat from those for PL_NMR, which included more detailed 2D 1H–13C and 1H–15N chemical shift data. However, these differences could be attributed to either a real difference in structure, simply a difference in the experimental techniques, or perhaps a combination of these differences. A detailed examination of Na+ position during the simulation provided a plausible explanation for the results observed in the Mg2+ binding experiments. Our simulations were performed with monovalent salt due to the lack of good parameters for divalent cations, the absence of polarization, and conformational sampling limitations. Although the replacement of Mg2+ with monovalent salt can destabilize RNA, for small RNAs high monovalent salt concentrations are generally a good substitute for physiological Mg2+, and typically provide similar structures (Draper 2004; Draper et al. 2005). We studied Na+ and water binding during simulation using two techniques. First we generated density grids to map positions of highest density over the course of an entire set of simulations. Second we probed the occupancy of Na+ and water within a defined radius for atoms of interest.
Another region of interest is the bulge (residues 9, 24, 25, and 26; residues 8 and 27 can be included as well). Chemical shift perturbation analysis showed that H1′ protons in residues 9 and 25 of ai5γ_NMR were greatly affected by Mg2+ titration, whereas only the aromatic carbon and nitrogen atoms in residue 24 of PL_NMR were affected. As was observed for the triad region, we suspect that carbon and nitrogen chemical shifts in residue 24 would have also been perturbed in ai5γ_NMR if they had been monitored. However, the shifts of C1′ in residues 9 and 25 of PL_NMR were not affected by Mg2+ as was seen for the analogous H1′ for ai5γ_NMR. The simulation data clearly accounts for this difference. Visual inspection of the Na + density grid for ai5γ_mDAR reveals a large, high density region straddling the minor groove surface of residues 9, 25 and 27 (Figure S5, top). Na+ ions in this region are likely stabilized by the O2 atom of U9, C25, and U27. The positioning of this high density region is therefore quite close to the H1′ atoms of these same residues and occupancy analysis using a 5 Å cutoff reveals that these H1′ atoms are among the nearest to Na+ during simulation (Fig. 8). In contrast, no such high density region is observed for PL_mDAR (Figure S5, bottom), and the H1′ atoms of U9, A25, and C27 are not near Na+ during the simulation (Fig. 8). It is likely that Na+ binding in this region of PL-D5 is not supported for two reasons: (1) the occurrence of adenine at residue 25, rather than cytosine in ai5γ-D5, places a hydrogen atom in the binding region, rather than an oxygen atom, and therefore does not support metal binding and (2) replacement of the A8-U27 base pair in ai5γ-D5 with the G8-C27 base for PL-D5 produces a stronger base pair and does not allow the twisting conformation that permits ai5γ-D5′s U27 O2 to interact with a Na+ atom.
The large simulated Na+ density near G15 explains the Mg2+ induced chemical shifts for G15 in both ai5γ_NMR (Sigel et al. 2004) and PL_NMR (Seetharaman et al. 2006) and its location in the major groove is consistent with previous NMR work with cobalt(III) hexamine (Rudisser and Tinoco 2000)). It is less clear why A16–G19 C1′ shifts are so heavily affected in PL_NMR, whereas only the H1′ of G15 is affected for ai5γ_NMR. For A16–A19, inspection of the 2D 1H–13C spectra for H1′–C1′ reveals that the majority of the chemical shift occurs in the carbon dimension (Supplementary Figure 2 in Seetharaman et al. (2006)) and thus would not be revealed in the ai5γ_NMR results where only 1H shifts were measured. However, a significant shift does occur in the proton dimension of the PL_NMR G19 H1′–C1′ cross peak, while G19 H1′ of ai5γ_NMR is unaffected by Mg2+. Another puzzle is why so many atoms of the tetraloop region have large chemical shift perturbations (according to the PL_NMR results) when the ion binding appears to be limited to the major groove according to the simulation Na+ density grid results. Inspection of the medium density solvation shell around the tetraloop region reveals that the base atoms are much more exposed to the bulk solvent than base atoms in the rest of the RNA molecule (data not shown). We propose that this lack of solvent shielding may explain the large Mg2+ induced chemical shift perturbations in the tetraloop.
Simulated annealing with the mDAR restraint sets
To compare the results of our explicit solvent refinement with traditional refinement methods, we performed simulated annealing on the completely extended ai5γ and PL RNA, both in vacuo and with Generalized-Born (GB) implicit solvent, using the mDAR restraint sets. One notable feature of in vacuo results is the presence of sharp kinks near the bulge for both the ai5γ and PL ensembles. Given that these kinks are not observed in the GB ensembles or the explicitly solvated ensembles, these results suggest that in regions with sparse distance restraints, such as the bulge, the lack of a solvation environment can lead to anomalous conformations. Other than the kinked bulge conformation in the in vacuo ensemble, the local conformation of both simulated annealing ensembles are very similar to the explicity solvated ensemble. In contrast, comparison of the average structural length of the GB ensembles reveals that they are somewhat more extended than the explicit ensembles: for ai5γ_mDAR the average structural lengths are 56.9 and 54.5 Å for the GB and explicit, respectively; for PL_mDAR the average lengths are 58.2 and 53.4 Å. In contrast to what was observed for the explicit ensembles, the pairwise RMSD was higher for ai5γ_mDAR GB ensemble (2.54 Å) than that of the PL_mDAR GB ensemble (0.74 Å). The reasons for these differences likely lie with the representative structure selection method. Whereas the explicit solvation representative structures were chosen by their proximity to the centroid of the major cluster during a long simulation, the simulated annealing structures are simply the lowest energy structures which satisfy restraints from a few hundred simulated annealing cycles. It is possible that performing 10,000 or 20,000 simulated annealing cycles (a similar quantity to the frame count in the explicitly solvated simulations) would produce representative structures that are in better agreement with the explicit results. However this has yet to be investigated.
The results presented in this study are immediately relevant to research in experimental structure determination and, more specifically, to refinement of RNA structure from NMR data. First, for structure refinement projects, we find that currently available MD tools with modern simulation protocols, force fields, inclusion of water and mobile counterions, and longer molecular dynamics simulations, offer robust environments for probing structural features that may not be adequately modeled by older and more conventional structure refinement techniques. Our restrained simulations of ai5γ-D5 and PL-D5 produced a set of refined structures that differed significantly from the previously published NMR structures and offer new insights into the similarities and differences of these RNA molecules. For instance, the simulation refined ai5γ_mDAR structures are much more compact than the original NMR structures and more closely resemble both the NMR and simulation structures of PL-D5. Moreover, for regions outside the bulge region (which is strongly influenced by sequence, packing and tertiary interactions) the re-refined structures better match the ai5γ-D5 crystal structure (PDB: 1KXK). We also were able to identify and troubleshoot potentially incorrect regional conformations in the conventionally refined structures of both molecules. For instance, we uncovered three problematic long range distance restraints in the ai5γ-D5 bulge, which when removed generated a smoother backbone trajectory in the bulge region, lower RMSD values, and fewer restraint violations. We also found that in three of the five PL-D5 structures, residue 25 was apparently trapped in a partially extruded conformation that upon heat annealing converted to the stacked conformation having lower RMSD values and fewer restraint violations. Noticing the problematic regions required MD simulations orders of magnitude longer than were previously and typically applied in order to highlight trapped or metastable conformations. Finally, the pairwise RMSD values for the simulation ai5γ_mDAR structures are lower than those for PL_mDAR, which is the reverse of what was found using conventional structure refinement. This is likely due to the greater flexibility in the bulge region of PL-D5 observed during the significantly longer and less-tightly restrained simulations used to re-refine the structure. The need to carefully evaluate the choice of NOE and restraint assignments (to not only check for misnaming, incorrect assignments, and/or potentially anomalous spin diffusion)—coupled with the potential for conformational trapping—suggest that automated NMR refinement of RNA structures remains a challenge. Our results suggest that the refinement of RNA structures requires a careful balance between the strength of the experimental restraints and the influence of the force field, and perhaps most importantly, the resulting structures require careful validation and assessment.
The publication of a self-spliced group IIc intron crystal structure (Toor et al. 2008) reveals that the D5 bulge adopts a different conformation in the context of the entire intron than in isolation. Therefore, while our refinement of the original NMR structures provides new insight into the isolated RNA hairpins, the functional relevance of these structures in the context of the intact intron remains unclear. However, the results clearly suggest that the bulge conformation is sensitive to the surroundings and sequence, and that the re-refinement leads to structures that are more consistent with the available crystal structures. Our simulations also suggest that these hairpin structures, which differ at only three positions, are much more similar than the older conventional refinement techniques indicated. These results also improve our understanding of how differences in primary sequence affect 3D structure, and provide insight into conformational flexibility, solvation, and ion binding. The isolated D5 bulge apparently samples a range of conformations, while tertiary interactions in the context of the entire intron select and stabilize a flipped out conformation necessary to assemble a catalytically active intron (Toor et al. 2008). MD simulations with explicit solvent and updated force fields can potentially uncover minor conformers that nevertheless have functional relevance. The flipped out conformation need not be the lowest energy structure observed by NMR, merely accessible with sufficient frequency to be captured by tertiary contacts in the intron. Accurate "ground state" structures determined from NMR data and explicit solvation MD provide a starting point from which to investigate the dynamical behavior of RNA. The methods employed in this paper should also aid researchers who use structural databases to further refine their models.
Remaining unanswered questions relate to potential disorder and/or dynamics in the bulge and loop regions. MD simulations without experimentally derived restraints are unable to maintain the expected structure; given this, movement away from the experimental structure does not represent true dynamics, but suggests deficiencies in the force field. Researchers may be interested in the minimal set of restraints required to maintain experimentally valid structures. We propose that the minimal restraint requirements for maintaining accurate structure using the AMBER force field would include as many distance restraints in non-canonical regions as possible and orientational restraints such as RDCs to maintain proper structural compaction. Although structures consistent with the experimental data can be maintained via the application of restraints, such restraints will tend to inhibit conformational transitions and dynamics. Given this, it is unclear if the representative structures found in re-refinement completely represent the ensemble of frequently accessed conformations or simply represent the lowest energy structures without a proper depiction of the true disorder or dynamics sampled at room temperature. For instance, depending on the choice of experimental restraints, two GAAA tetraloop conformations, each with distinct solvation and ion binding properties were observed. Both are consistent with experiment, however as exchange was not observed between "outward" and "inward" structures during the re-refinement, speculation on the relative populations or conformational dynamics are not possible. On the other hand, given that both are observed experimentally and are likely nearly iso-energetic, it is likely that both are populated and in dynamic equilibrium. To further resolve these questions through simulation will require improvements in the underlying nucleic acid force fields.
The AMBER force field is continually being developed and refined to produce improved simulation results. Generally these improvements are evaluated in the context of unrestrained simulations. However, evaluating force field performance is difficult given the huge diversity in RNA structure. Our unpublished work suggests that canonical A-form RNA is relatively stable in unrestrained simulations for long periods of time. In contrast, non-canonical regions frequently populate conformations which are not observed in experimental structures suggesting a force field problem. One notable RNA motif for which this occurs is the UUCG tetraloop. Progress towards improving RNA torsional parameters is underway, including recent force field modifications that improve the glycosidic χ in RNA (Banas et al. 2010; Yildirimet al. 2010; Zgarbova et al. 2011).
Supplementary data
Supplementary data to this article can be found online. This includes a ZIP file containing all of the restraint data files in AMBER format used in this work along with PDB files of the final structures. In addition, a PDF file is supplied that provides six supplementary figures S1-S6. PDB codes for the coordinates are 2LPS and 2LPT, BMRB accession numbers are 18274 and 18275.
The authors thank Sam Butcher and Kwaku Dayie for helpful discussions. We also directly acknowledge the Pittsburgh Supercomputing Center, the National Institute for Computational Science at the University of Tennessee, and the Center for High Performance Computing at the University of Utah for access to additional computational resources. This work was supported by the NIH (GM-081411) and the National Science Foundation XSEDE award for computer time (TG-MCA01S027).
Open Access
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.


text:
Camshaft Position Sensor Replacement
The camshaft position sensor notifies the powertrain computer (PCM) when the number one cylinder is about to reach top dead center of its compression stroke. This information is used to control ignition timing and the timing of the fuel injectors. When replacing the camshaft position sensor, make sure that there are no obvious oil or coolant leaks that could ruin the new sensor.
How to Replace the Camshaft Position Sensor
Note: There are two common locations for the camshaft position sensor. These are general summaries of how to replace each type.
A. The sensor is located in the timing cover, close to the timing chain
1. Disconnect the negative battery terminal
2. Remove the electrical connector to the camshaft position sensor
3. Remove the holding fastener and the sensor
4. Clean the hole where the old sensor was located
5. Install the new sensor in the same position
6. Tighten the fastener and reconnect the electrical connector and negative battery terminal
7. Clear any PCM codes and perform a Drive Cycle Test Drive to verify the repair
B. The sensor(s) is located on or near the valve cover(s) of the engine
1. Disconnect the negative battery terminal
2. Disconnect the electrical connector, loosen the fastener, and remove the old sensor
3. On some engines, the valve cover and timing cover may have to be removed for access
4. Clean the mounting area of the old sensor
5. Install the new sensor, tighten the fastener, and connect the electrical connector
6. Install the valve cover(s) and timing cover if removed
7. Connect the negative battery terminal
8. Clear any PCM codes and perform a Drive Cycle Test Drive to verify the repair
Note: After replacing the camshaft position sensor, the powertrain control module needs to have the camshaft adaption reset. This is so the PCM can recalibrate itself to the new camshaft position sensor.

subset: commoncrawl


text:
Starz Orders Gritty Ballet Drama Starring 'Black Swan' Dancer
After pirates, ballerinas are heading to Starz next. The network has ordered a new drama series titled "Flesh and Bone" about a young ballet dancer, who has a distinctly troubled past, as she joins a prestigious ballet company in New York.
The "dark and gritty" show will "unflinchingly explore the dysfunction and glamour of the ballet world." It is created and executive produced by Moira Walley-Beckett, who was serving as a writer and producer on AMC's "Breaking Bad".
Starz gave the ballet drama a greenlight after dancer Sarah Hay ("Black Swan") was cast in the lead role of Claire. "From the beginning, we all agreed this project could not go forward unless we found a world class dancer who had the ability to convey the widest range of emotions that Moira has written," says Starz CEO Chris Albrecht. "This is one of the most challenging and dynamic characters I have even seen and am thrilled to say we found our Claire in Ms. Hay."
Walley-Beckett adds, "Finding the perfect Claire has been an almost impossible task. It was truly like looking for a unicorn because the demands of the role are so specific and challenging. I was looking for a beautiful and transcendent ballerina who is not only a brilliant dancer but also a gifted actress, and who also possesses a fierce curiosity and enthusiasm about the brave demands of the work. Sarah Hay embodies the role of Claire just as I dreamt her."
Claire is described as "a beautiful, soulful and deeply emotionally wounded young woman who possesses an innate innocence and fragility while at the same time harboring self-destructive tendencies and a vaulting ambition. She is a transcendent ballerina, capable of reaching the sublime, but her inner torment and aspirations drive her in compelling, unforeseeable ways."
Production on the new series will begin early this year in New York City for a 2015 premiere.
- Super Junior's Leeteuk Gets Robbed in Switzerland
- Kardashian-Jenner Sisters Throw Surprise Birthday Party for Khloe
- Evidence of Song Joong Ki and Song Hye Kyo's Rendezvous in Bali Revealed
- Living Life to the Fullest! Brad Pitt Caught Cozying Up to Sienna Miller and Elle Macpherson
- Louis Tomlinson Was Feeling Left Out of One Direction: 'What Have I Really Done to Contribute Here?'
- Gwyneth Paltrow's Goop Accused by NASA of Scamming Customers With Healing Stickers
- Liev Schreiber Spotted With Another Woman Amid Dating Rumors With Gerard Butler's Ex Morgan Brown
- Future and Chris Brown Are Total Womanizers in Sexy 'Pie' Video


text:
Überlandstrasse 1098600 Dübendorf
Display on the map
Maya Bachmann Brunold
Anja Gieger
Account Manager, Mitglied GL
Christina Kaegi
Mitglied Geschäftsleitung
Ansgar Schäfer
Managing Partner
We believe successful searches are the result of both hard work and in taking all of the steps necessary to ensure success. Our succesful performance over the years has created a reputation for excellence which frequently leads to multiple assignments from established clients, as well as attracting new ones. We attribute our record of success to the diligent application of our proven process for identifying, screening, evaluating, and recruiting quality candidates. For quality results, filling a position must involve establishing a good relationship, not just a hiring transaction.


text:
Seize the Day, no dream is too impossible, no distance too far.
Self descriptions aren't usually very accurate...find out about me yourself!
Reading, travelling, learning new things/languages, singing in the shower and with friends in karaoke, window shopping, watching movies/travel shows/discovery channel, listening to all types of music
The list is possibly endless, since every place has its own charm
kathwindy hasn't logged any travels yet.
Hello! My name is Katherine and I'm a Singaporean who has lived in Singapore all 26 years of my life. If you have any questions about my country, I'll be more than happy to help. Need a tour guide to show you around in a low-budget and time-saving way? I'll do it for free, as long as you share your travelling experiences and information about your home country with me. My e-mail add: firstname.lastname@example.com
kathwindy hasn't been active in the wiki travel guide yet. Have you?


text:
Product Reference: 39630
Why not View a video about this product?
See the world from a unique perspective
Step into the future with SunnyCam HD video recording sunglasses. The SunnyCam glasses allow you to record High Definition 720p video to the included 16GB memory card and shoot amazing photos comfortably between your eyes. This offers you a true of point of view (POV) recording experience with footage that no other product can offer.
Features a HD video recorder
The SunnyCam glasses feature a built in pinhole camera that will let you shoot amazing 720p HD video. With the advanced auto focus sensor the glasses capture beautiful sharp video footage with every use. All while the built in microphone captures conversations, music, or any other audio.
Convenient hands-free and mount-free recording
Perfect for recording on the move or when performing any activity requiring the use of two hands, the discreet and stylish SunnyCam lets you leave heavy helmet mounts or other camera attachments behind. Ideal for sports and outdoor use, the SunnyCam can be used for endless applications including sports coaching / training, security, and construction.
Allows you to take photos, record movies or audio
The SunnyCam glasses now come equipped with three different user modes. Not only can you shoot HD 720p video, the SunnyCam now allows you to shoot JPG photos and record WAV audio files. Record your life in images with beautiful photos at the touch of a button or capture life through sound with the audio only mode.
Protective polarized interchangeable lenses
The SunnyCam glasses have changeable lenses which are 100% UV protected and have 99% polarized efficiency. Each of the lenses offer enhanced visual performance by increasing visual acuity and improved colour contrast. Dark and clear lenses are included and a wide range of colour options is available
Connect and share your experiences with others
The SunnyCam glasses easily connect to any PC/Mac, tablet or mobile with USB access. Simply plug in your SunnyCam glasses and watch your videos instantly and show the world recorded through your eyes. The SunnyCam glasses save in AVI and JPG formats, which allows for the files to be instantly ready to be uploaded and shared online.
Includes essential SunnyCam accessories bundle
The SunnyCam has a great range of accessories included; a 16GB memory card and adaptor, car charger, USB cable, carry case, cleaning cloth and glasses string.
Technical Specifications:


text:
Member Since: November 30, 2012
Country: United States
I bought one of those boards, plugged it to my pc and got the main firmware working. Then i tried to program some new firmware to it and now the green led just stays on and my pc can't recognize it anylonger. Is the board just broken now or is there something I can do?
No public wish lists :(


text:
For Hamilton's concerned citizens and activist groups, the Hamilton City Hall auditorium is a familiar hangout. A month ago, Rocky Mountain Laboratories (RML) used the venue to present its case for adding a new biosafety level (BSL) four facility—which would contain some of the world's most deadly pathogens (see "Containment area," by Jed Gottlieb, May 15, 2003). Last week the auditorium played host to two more meetings centered around the proposed expansion.
The first, on Wednesday, June 25, was put on by Friends of the Bitterroot, the Coalition for a Safe Lab and Women's Voices for the Earth (WVE), and pointed out the shortfalls these groups see in the Environmental Impact Statement (EIS) supporting the expansion. The second, on Thursday, June 26, was hosted by the lab to collect public comment on the EIS.
At the beginning of Wednesday's meeting, Friends of the Bitterroot President Jim Miller told the crowd of about 200 that the EIS is inadequate because it doesn't address the risk of level four pathogens like anthrax or Ebola virus escaping into the community. Miller—who stresses that his organization has not decided whether it is for or against the expansion—says the lab knows that an outbreak is a possibility, but has chosen to ignore that possibility.
After sifting through 1,700 pages of documents obtained through a Freedom of Information Act request, Friends of the Bitterroot found a memo the group considers a bombshell. Friends was told in writing by a FOIA officer that the memo was written by National Institute of Allergy and Infectious Diseases Intramural Research Director Tom Kindt, but hasn't been able to confirm this with Kindt. The memo reads, in part:
"The RML campus is located in rural Western Montana, well removed from major population centers. The location of the laboratory reduces the possibility that an accidental release of a biosafety level 4 organism would lead to a major public health disaster."
As Miller read the memo to the crowd, a chorus of gallows laughter rippled through the room, and one frustrated citizen, deep in the back, called out: "Why are we expendable? Because we're a small community. I say they build it out in the desert." Miller's logic is that while the chance of a containment breach is low, it is a possibility that needs to be considered so local medical workers, law enforcement personnel and citizens know how to handle themselves if remote possibility ever becomes reality.
"It's our community that could suffer the consequences of their failure," Miller told those gathered. "We deserve better, and our country deserves better…We thought that a radiation release from a nuclear power plant was near impossible, but yet we had Three Mile Island."
After Miller and representatives from the Coalition and WVE presented their responses to the EIS—all the groups have serious reservations about the statement—the public took the floor. But only a handful of questions were asked of the presenters. Instead, a sequence of angry rants focused on an EIS that citizens don't think addresses concerns of decreasing property values around the lab, lack of additional training for law enforcement, negative traffic impacts, increased noise pollution, the possibility of leaking lab sewer pipes and others.
A constant theme running through the comments was curiosity as to why lab and city officials weren't at the meeting to allay these concerns. On the presenters' table, name placards sat in front of each chair, but behind the names of Mayor Joe Petrusaitis and lab Director Dr. Marshall Bloom, the chairs were empty. Both Petrusaitis and Bloom were invited, but decided not to come.
Bloom says that Friends requested his attendance at one of the lab's community liaison meetings several weeks before, and Bloom made it clear then that no National Institute of Health representative would attend.
"The reason I declined to attend is very simple," Bloom says. "The questions that were going to be asked are complicated…There's no way I could have sat there and answered any question with any degree of certainty or accuracy unless I brought with me a team of five experts."
Bloom tries to be as accurate as he can at all times with any questions he receives from the public, he says. Under the circumstances of the meeting, he says he couldn't be expected to provide all the information requested.
Asked why he didn't attend, Hamilton Mayor Petrusaitis wrote in an e-mail to the Independent, "The Community Liaison Group never voted to have this meeting. If anyone wants to have a meeting, that's their right. The official EIS meeting was the next day. The same people were there, so was I and other officials. We are not worried about the Lab impacts. All our concerns were addressed in the EIS statement. The Lab is saving lives. People are dying all over the world and bio-terrorism is a real and present danger. Where are your priorities, let's put people first!, not politics!"
The following night at RML's meeting, much the same tone of animosity remained. The meeting began with lab officials giving a presentation and informing the public that this would be a "scoping" meeting, and not a forum in which every question would be answered. Instead, the meeting's purpose was to learn what the public wants to know about the expansion. The majority of verbal comments at the RML meeting centered on concerns about the EIS' lack of comprehensiveness.
"Not everything is fleshed out in [the EIS], that's why you call it a draft," says Bloom. "It's obvious that there will be a number of other points that will be raised in a final EIS. As to whether we'll be doing [a completely new] draft, I don't know."
At this point, it remains unclear whether a new draft will be done, and if certain questions will ever be answered. Hamilton's residents are asked to continue waiting, and hope that answers eventually come.


text:
Now here's a little known fact about me: I have pretty much given up on hair salons. *shock* *awe*
Oh, yes! I generally find salons to be a huge waste of time. . . and money. Why? Well — apparently, my hair is the most complex hair type in the world. It's true! I sit in the chair and instantly, highly-skilled, trained professionals are rendered clueless. Now I have enjoyed a few good experiences here & there (Phyto Universe/NYC is awesome — Ask for Ron…De Ja Vous/NYC does amaze blowouts — ask for David, Eli, or Ricky), but for the most part, I shy away from salons and do my own hair at home.
I am regularly invited to partake in services offered by celeb stylists, colorists, etc. . . I generally, respectfully decline — these things rarely end well for me. My hair proves to be too challenging for most. And frankly, I tire of sitting there having to walk "professional stylists" through the do's and don't's of combing, brushing, blowing, curling, and styling my hair. It really is a bit much!
Enter Dana's Loft, a quaint "look-away-and-you-might-miss-it" hair salon on Manhattan's too-cool-for-school lower east side. Upon my arrival I was greeted by Dana. Yep, "the" Dana of "Dana's Loft". She was kind and welcoming. Before putting product to head, Dana asked lots of questions about my hair, styling products, hair care routine, hair goals, etc. Oh, hey now. . . that's a rarity! Dana's initial intake process immediately put me at ease. I really appreciate that she took the time to ask about the state of my hair and how I maintain it. It's quite rare that stylists do this — Instead they tend to quizzically examine your hair, make you feel even more self conscious about your less than pristine ends, and then recommend a host of products to repair your ol' tired-looking hair. It's enough to make a girl cry. . . ask me how I know :/
Well there were no tears during my visit to Dana's Loft — only cheers! And here's another thing that I really enjoyed during my visit. . . Dana was the only person to handle my hair. Remember the good old days when your stylist handled your hair from start to finish? Well this still exists at Dana's Loft. . . perhaps it was a slow day or perhaps it was my lucky day — not sure, but it made me feel really good to have my hair handled by just one person. I didn't have to worry about two or three different people in the mix.
For my visit, I decided to live a little and try out extensions. I'm generally not that girl but every now and then you have to go for the gusto. Dana added a few layers of hair from Creme de la Creme Tami Roman collection (Caution: the website plays music. . . a small pet peeve of mine). I'm rather easy going when it comes to hair so I stressed wanting a very natural look. And Dana executed just that. I left feeling like my exact hair wishes were met. Dana was so sweet, so thoughtful, and above all – extremely knowledgeable. I left knowing that I'll definitely return. If you're in search of a super good hair salon experience, try Dana's Loft.


text:
Sign in or Create a profile and we'll remember where you stopped.
Don't show me this message again.
When a rap producer is found shot to death in his studio, detectives focus on his protege, whose demos link him and a friend to a second murder. As the investigation unfolds, Southerlyn's opinions put her sharply at odds with both McCoy and Branch.


text:
Now GameWorks, the company he helped create, has folded its Austrian operations - its first foray into Europe - after just six months. The venture had been backed by multi-millionaire Egon Putzi, who has confirmed it is now insolvent and owes several million pounds.
Worldwide, GameWorks boasts 15 outlets - 13 in America, one in Brazil and one on the Pacific island of Guam after the demise of the Vienna and Klagenfurt outlets. The Vienna centre, on Mariahilferstrasse, was on the same site as the ill-fated Marks & Spencer flagship store that folded last year, and also offered a restaurant, two bars and a café.
Putzi had intended to expand the Klagenfurt location into a vast entertainment complex, including a stadium, cinema and shopping centre, but the project was stopped due to the large debts that investors refused to honour.
He said most debts had been run up by the rebuilding and furnishing of the hi-tech GameWorks site in the capital and that he had no choice but to register his company as insolvent. 'It means all investments in Carinthian Arena GameWorks and GameWorks Vienna have to be regarded as losses,' he said.
The failure shocked some of its Austrian management, who had predicted expansion. Harald Rohrer, Putzi's partner in the venture, said: 'The creation of an outlet in Villach had been fixed and plans were being examined for further branches in Linz, Graz and Salzburg as well as a second one in Vienna.'
Gerhard Fandl, GameWorks business director, predicted the Vienna centre would receive a million visitors in its first year when it opened last October. Even a fortnight ago, he denied rumours it was about to shut. 'The Vienna branch is not closing. We've had an excellent response so far,' he said.
- More about:
- Austria
- Bankruptcy
- Brazil
- The Super-Rich
- Vienna


text: Students will ordinarily apply to the program the second semester of their sophomore year. Students must have, at the time of application, a cumulative GPA of 2.5 or higher to qualify. Students will submit to the advisory committee an essay explaining why they wish to participate in the program, and show they will be able to meet the program requirements over the next two years. They will also be expected to provide two letters of reference (one of which should be a faculty reference). Please note: Students who plan to take part in the College's International Studies Program during their junior year must consult with a member of the Committee to determine whether they will be able to meet the Program's requirements while abroad.


text: Posted November 4, 2012 Within the past year, the multiyear contract extension the Saints announced for Sean Payton in September 2011 was voided by the NFL, making the suspended head coach a free agent after this season and casting questions on how long he will remain in New Orleans, according to league sources. http://espn.go.com/nfl/story/_/id/8592214/sean-payton-deal-new-orleans-saints-voided-nfl-sources-say What this could mean? Could Sean leave the saints? Could the Panthers try and pick him up if Rivera is fired at the end of the season? I will say safe money is that he stays with the Saints, just because the NFL voided his contract does not mean that he has any intention of going anywhere, I do believe he has a better chance going to Carolina then Dallas(he made a more then subtle slight at Jerry Jones after the Saints won the super bowl but the media doesn't remember that), which I believe is a Dallas fan and national media pipe dream. thoughts I think the best we can hope for is he goes somewhere out of the NFCS, he's not going to leave the Saints for the Panthers so forgetaboutit! Share this post Link to post Share on other sites


text:
Who we are
We are a team of psychiatrists and psychologists, all with psychotherapeutic background and experience. We have at least one specialist psychiatrist and psychologist for every area who can thus cooperate and augment each other in solving the particular problem. Such cooperation is especially essential in many types of suffering such as eating disorders, addictions, or sexual dysfunctions.
Head of the team: MUDr. Tomáš Rektor is a psychiatrist and psychotherapist, a candidate in the Czech Psychoanalytic Society, also a graduate of deep SUR therapy courses; individual self-experience in "gestalt therapy", trained in body-work and non-verbal techniques.
MUDr. Nataša Škárková is a psychiatrist, psychoterapist and supervisor, trained in psychoanalytic psychotherapy and cognitive behavioral therapy.
Mgr. MgA. Helena Bezděková is a psychologist and psychotherapist. She completed trainings in gestalt psychoterapy, coaching and courses in body-work therapy and non-verbal techniques.
Mgr. Kateřina Baumruková is a psychologist and psychotherapist. She has experience in human resources and currently undergoes complex psychotherapeutic training in systemic psychotherapy.
Mgr. Livia Didičová is a psychologist and psychoterapist trained in gestalt psychotherapy. She also underwent courses in body-work therapy, traumatherapy, psychomotoric psychotherapy.
Mgr. Veronika Buchler is a psychologist and psychotherapist. She is a graduate of system psychotherapy training. Her main focus is on psychotherapeutic work with children and families.
Mgr. Jakub Kuchař is a psychologist, psychotherapist. He is a candidate of psychoanalysis, he underwent group self-experience in gestalt psychotherapy and courses in existential psychotherapy, drama therapy.


text:
sku DSETP0061BLGOPIL1
Black / Gold Square P0061 PillowBlacks And Grays Blacks And Grays Pillows Pillows Synthetic Synthetic Under $100 Under $100 Under $500 Under $500 Square Square Medium Medium Casual Casual Outdoor Indoor Outdoor Indoor Pillows Pillows
Free Shipping
Delivery within 2 weeks on average.
Product Information
100% Poly Propylene Square Black Pillow.
Rug & Home is proud to offer free shipping with every rug, art, mirror, lamp, and accessory purchase on our website.


text:
Quebec's Environment Minister says the province could block a project to transport Alberta oil through Quebec if it is found to pose a serious environmental hazard.
Enbridge Inc. proposes to use a pipeline that currently runs through Quebec to transport Western Canadian crude from the oil sands to a Montreal refinery to gain access to the eastern market.
In an unexpected outburst on Wednesday, Environment Minister Daniel Breton responded to the project by saying this was the 50th anniversary of the election on the nationalization of hydro-electricity in Quebec and the province will never accept a project imposed on it by Ottawa and Alberta that it hasn't approved itself.
"What I see is Alberta wanting to transport its oil on our territory without our consent. … Are we masters of our own territory or not? " Mr. Breton said before heading into a Parti Québécois caucus meeting, without elaborating further.
Premier Pauline Marois's advisers, perhaps fearing the minister's comments would be interepreted as a declaration of war on Alberta and its oil industry, talked to Mr. Breton before holding a news conference.
Mr. Breton was an environmental activist opposed to the development of the Alberta oil sands before entering politics. When he spoke to the media later, his tone was considerably softer, saying the Quebec government will first demand to be kept informed before ruling on the project.
"They cannot go over our heads because it's on our territory and we have environmental reasons to make sure everything goes well," Mr. Breton said. "If it doesn't go well, we can't go ahead. If it goes well, we'll go ahead. But we need to have all the information."
Mr. Breton said that environmental concerns in Nebraska over the construction of a pipeline have stalled efforts by Alberta and Ottawa to export crude from the oil sands to the United States. And there's similar opposition to the construction of a pipeline through British Columbia to export Alberta oil to Asian markets. "Before they come east, we want a say over the matter," Mr. Breton said.
He said he is concerned an oil spill from an Enbridge pipeline in Kalamazoo, Mich., in 2010 could repeat itself in the province. Alberta crude, he said, was found to be more corrosive on older pipelines and could result in spills.
The PQ government is juggling environmental concerns with the economic benefits Alberta crude would have in creating jobs in Montreal's depressed oil-refinery industry.
In an interview in the first week of November, Enbridge CEO Al Monaco said he has been "trying" to arrange a meeting with the PQ. But he expressed confidence that the company would be able to proceed there with little opposition, in part because of the jobs tied to refineries in the province that stand to benefit from using cheaper Western Canadian crude.
"So far Quebec's actually been pretty receptive … and obviously there's a lot of economic benefits to keeping the refineries in Quebec running," he said.
"Remember those refineries have high-priced feedstock right now," he said. Montreal refineries now import crude from places like the North Sea and offshore Africa, where oil sells for nearly $25 (U.S.) a barrel more than North American crude.
"Having cheaper feedstock and having Canadian crude to that market makes a lot of sense," he said. But, he acknowledged, "it's going to be like everything else today: We have to work through it and explain what we're doing. But I'm hopeful that we'll get support."
Mr. Breton said he will be meeting Mr. Monaco "pretty soon." Natural Resources Minister Martine Ouellet said she wants to be on hand to weigh the full impact of the project, arguing in favour of securing other supply sources for the province.
"There are economic advantages with respect to costs and it also represents an alternate source of supply. But we also have to examine the environmental impact. What kind of oil will be transported in the pipeline? Is it light crude? Will it be conventional oil or not? We are still waiting for answers," she said.
While the federal National Energy Board will hold public hearings on the project, Quebec said it will also undertake public consultations of its own but has yet to define the type of environmental review process it will initiate.Report Typo/Error
Follow @nvanderklippeon Twitter:


text:
|Members List
|Today's Posts
|Mark Forums Read
|Photo Gallery
greatlakes4x4.com is the premier Great Lakes 4x4 Forum on the internet. Registered Users do not see the above ads.
|Thread Tools
|Search this Thread
|May 24th, 2012, 09:22 AM
Direct Drive FTW
Bump Steer?
I want to run a double triangualted 4 link up front. The engine is in the rear and there is very little limiting link placment in the front. If I use a rack and pinion mounted on the axle from PSC that will control any bump steer correct? I don't see how it would not. But, maybe I missed somethimg I could catch now avoiiding a costly mistake.
|May 24th, 2012, 09:33 AM
Senior Member
Join Date: 08-24-06
Location: K-zoo
Posts: 1,148
iTrader: (7)
Mentioned: 0 Post(s)
i'm curious, but how were you going to run the steering link from the wheel to the rack and pinion and still let it cycle with the suspension? Normally isn't the rack and pinion fixed to the frame? I don't quite follow this.
|May 24th, 2012, 05:03 PM
Direct Drive FTW
Enlighten me as to why it is a brain dead idea?
I don't think you need a crappy picture. Think of a rack and pinion mounted in the same manner as a double hydro ram. Now think of a slip shaft drive shaft going from the rack to the firewall. There is a smaller shaft parallel to the floor behind the firewall connecting to the steering wheel. As the suspension cycles the steering shaft extends and contracts. Because the rack and pinion is hydraulic it should resist bump steer. This should give me as much flexibility as with a full hydro running a 4 link while still providing a manual interaction in the event of a loss of hydraulic pressure. I would think at extreme droop of over 15" the steering shaft may move slightly as the u joints align. But, for daily driving with a travel of 10"(max) I do not see it causing any issues. Although feedback is helpful and perhaps I am missing something.
If I can not do it I will need to run a standard cross over steering system with assist. I will also need to run a 3 link with a pan-hard bar because using a triangulated 4 link or 3 link with a watts linkage will bump steer like crazy.
I am not doubting you at all. I simply just want to know why it is a brain dead idea?
|May 24th, 2012, 05:08 PM
Web Wheeler :(
Join Date: 10-08-08
Location: Grand Rapids
Posts: 1,705
iTrader: (10)
Mentioned: 2 Post(s)
I have nothing helpful to add. I would like to see pictures though just because im curious.
"Insert Comedic and or ego boosting quote questioning another members sexual orientation here"
|May 24th, 2012, 05:20 PM
Direct Drive FTW
This is what I am pretty much copying with more travel, heavier duty components, and mounts.
After reading this a few times I am pretty sure it will work with no issue. Any one have any reason why it will not or any freaky quirks I can expect?
|May 24th, 2012, 06:55 PM
Senior Member
Join Date: 11-05-05
Location: Freeland, mi
Posts: 5,515
iTrader: (31)
Mentioned: 52 Post(s)
There is a buggy in the latest issue of crawl right with the rack and pinion set up you are talking about. I believe the price of the correct rack that can withstand the abuse an offload rig can put out will prevent you from doing it though. It will in no way be cheap. Why not just run a three link front with a track bar and conventional steering maybe with a hydro assist? Or is that too simple and proven?
|May 24th, 2012, 07:59 PM
welfare wheeler
Join Date: 03-03-06
Location: hazel park, mi
Posts: 6,857
iTrader: (41)
Mentioned: 7 Post(s)
with limited travel, and not so extreme use i think it could work.
with extreme travel and serious abuse it would be tricky to make it live very long i think.
i have never seen a slip joint in a steering shaft that could handle serious articulation/flex, not that it could not be designed though.
i wonder about the steering strength(can it turn big tires off road effectively)?
can it handle the abuse of say 36" tires(lateral forces applied by big tires hitting trail obstacles at speed)?
seems like alot of work and money. a 3 link and hydro ram work fine.
i dont think bump steer would be an issue though.
|May 24th, 2012, 09:09 PM
Senior Member
Imagine one side of the axle stays still while the other droops, the axle will effectively move towards the side, at best it will pivot at a point somewhere near the link point on the side that stays stationary. With the steering wheel stationary, that will mean the the steering shaft going into the rack will turn, steering the tires. There's the bump steer you think your avoiding. If it doesn't steer, it will turn the wheel in your hands. Better keep your fingers clear.
Then there's how much strength you would need in the rack itself (good luck finding one that will hold up to a heavy tire/wheel combo, not to mention being slammed sideways in an off road situation), and the amount of slip you would need in the steering shaft, plus the rag joints or u-joints in that shaft will take alot of abuse.
I don't see any way this could be done and made more functional then a basic crossover, or full hydraulic.
|May 24th, 2012, 09:17 PM
Senior Member
Build what you want, if you think it will work. Good luck.
|May 24th, 2012, 11:16 PM
Direct Drive FTW
Thanks for talking some sense into me. I knew about the cost to do this right and was ok with it considering what I wanted in the lines of travel. But, a 3 link leaves considerable less room for error, gives me a lot more freedom in mounting the bags, and is more reliable. A 3 link is ultimately more stout and that is overall what is most important in this build. I will just set the panhard for the 8" street height, when the clearance drops to 4" at higher speeds or raises to 15" for trail use the panhard will pull the axle.
With somewhere around 200" of hydraulic hose I really don't feel comfortable with full hydro at highway speeds. So the 3 link with assist is the best option.
Thanks again.
|Thread Tools
|Search this Thread


text:
|Thread Tools
pinion nut
i have a '72 C10 while checking my u-joints in search of a vibration problem, I noticed the nut that retains my pinion yoke was loose. Can someone help me with this one? What would cause this to happen? Can I just torque it up a little or Am I going to find myself rebuilding the whole axle? thank for any input....B/w72
I have seen them come loose. If it were mine I would take the nut off, clean it and the pinion threads with brake cleaner, apply some red loctite and put it back on. It doesn't need to be real tight, you don't want to change the crush sleeve any so maybe 30 foot pounds.
And go from there. See how it drives and check for noises.
72, that is supposed to be a locker nut and I beleive it has a specific torque. Locktite if you want, but put the proper torque on it.
This nut does not have a specific torque. When the rear is set up it has to be tightened until a specified amount of torque is required to turn the pinion. I would locktight it and tighten it to about 30 ft. Lbs. Then take a punch and peen the end of the nut to the threads. Basically you are making a bad place in the threads so the nut will not be able to back off. The rear should have made a whining noise when you decelerated since the loose nut would have allowed the pinion to move away from the ring gear. I have seen this condition on several GM products. Hope this helps, Toni
[ March 29, 2003: Message edited by: hrracecar ]

Concidering it takes over 100 lbs of torque to crush a sleeve, I cant see it staying tight with a 30 lb retighten. Dont have a book in front of me, but I bet there is a spec.
The reason why your pinion nut was loose is because something collapsed the crush sleeve more than the original preload did. What causes that? Dropping the clutch at high rpm, or any other heavy overload of the driveline. This is the main reason why drag racers toss the collapseable sleeve and go with a solid machined spacer.
Retorqueing the nut will not bring the pinion back into spec. If you do a backlash check on the ring gear you will find it to be excessive and this will eventually destroy both the pinion and the ring gear. Pull the cover and check the backlash with a dial indicator, if it is still in spec consider yourself lucky. Likely you'll find it is too much and the axle needs to be rebuilt with a new crush sleeve and torqueing procedure to ensure proper bearing preload. You might be able to pull the yoke reinstall a new crush sleeve and retorque but I don't recommend it, it's loose for a reason.
The other possibility is so much wear has taken place in the bearings on the pinion that the crush sleeve reached the end of it's "spring" and is no longer doing it's job of maintaining the proper preload on the pinion bearings, in either case a rebuild is in order.
The reason why mfg's don't use a machined spacer is because it does not expand to accomodate wear in the pinion bearings and is much more expensive to set up.
If you do nothing but retorque the nut then you are inviting disaster in the future. There is no retorque spec on a crushed sleeve because torquing the pinion is a one time deal. I imagine your loose pinion is causing a portion of your vibration problem.
There is a guy here on the forum that rebuilds axles for a living and he can back this up. Where are you Axle Bastard?
[ March 30, 2003: Message edited by: 4 Jaw Chuck ]

She may not look like much, but she's got it where it counts, kid. I've made a lot of special modifications myself.
Han Solo
[quote]Originally posted by TurboS10:
Concidering it takes over 100 lbs of torque to crush a sleeve, I cant see it staying tight with a 30 lb retighten. Dont have a book in front of me, but I bet there is a spec.
Yes it takes sometimes 300 foot pounds to get the sleeve to crush but this would be a new sleeve. The idea in this one situation is to snug the nut down onto the sleeve but not crush it any more.
No one can predict what happened to cause the nut to back off, for all we know someone may have changed the seal and not tightened it enough.
I don't care how many professionals have "done it right" and swear it's the only way, they have to protect their ***** to avoid a call back. I have tightened several pinion nuts as I described
on vehicles I keep track of like my own, my family and friends. You know when one of them fails. The pros only know of a failure when the customer brings it back which doesn't always happen.
If you want an answer that will ensure your rear end will never fail here it is, have it totally rebuilt ,
if you want to take a chance and do something several people have done with success just tighten the nut. Thats about a $600-$1200 difference---you decide. Anyway if tightening the nut guarantees a failure (which depends on personal opinion) your still stuck to rebuilt the whole rear.
I would tighten the nut and drive off.
You know 'technicaly' 4-Jaw is right as always but I will say there are also many times that we do things in the field that you're not supposed to with limited side effects. Since I know that every time I replace a leaky pinion seal I remove that nut only to retighten it just enough to keep it on without crushing the collar any more I would probably try Jasons idea as well. If the rear goes south then you're rebuilding anyway. I have also had the oportunity to see cars with front end hub bearings making noise (FWD bearing assemblies pressed into steering knuckle) only to find out some hack didn't tighten down the axle nut and it came loose causing noise and some nice play in the front end. Now ideally you should remove the knuckle, replace the bearing, and then reinstall. On more than one occasion I have simply retightened the axle nut and that's all, a definate 'by the book' no-no. In the long run it's up to the owner, fix it right immediately for no future worries or take a 50/50 chance to save some out-of-pocket cash right now.
Makes sense to me now. I see what you are saying, Jason. 30 ft/lbs just sounds like so little concidering the original pressure used to set the sleeve. I guess if all the spring is gone, it is all you can do.
[quote]Since I know that every time I replace a leaky pinion seal I remove that nut only to retighten it just enough to keep it on without crushing the collar any more...

How about measuring break-a-way torque? Wouldn't that take some of the guess work out of it?
[quote]Now ideally you should remove the knuckle, replace the bearing, and then reinstall.

Remove the steering knuckle?
-Hub Tamer-
[quote]Originally posted by KULTULZ:
Remove the steering knuckle?

Yes, remove the steering knuckle. Unless you have a Hub Shark or equivalent it's the only way you are going to press in a new bearing assembly on a FWD car. I have tried cheaper units and they suck not to mention they beat the tar out of your impact gun since that is what drives out the bearing assembly with those devises. Our shop, as do most, has a hydaulic press and on most cars I just assume take out the knuckle and use the shops press.
[ March 30, 2003: Message edited by: dmorris1200 ]

One more note, one of the guys I work with had a HUBSTER (think that was what it was called). It looked exactly like the Hub Tamer in the video clip. It sucked. The only thing I could say about that tool is things aren't always as good as they're advertised to be. The Hub Shark is much sturdier but with all adapters runs about $600 and still beats the tar out of your impact gun.
And yes, these are just my opinions. There are probably guys who don't mind hammering their impact gun for ten minutes driving out a bearing assembly (some really fight ya) but I prefer a press if available. I suppose if your a do it yourselfer at your house renting one of those from your local auto parts stores might not be a bad idea but I would still probably rather take the knuckle out and take it to a shop with a press and have it done that way. JUST MY OPINION!
[ March 30, 2003: Message edited by: dmorris1200 ]

Uh...Did you click on the link I gave for the Hub Tamer? And it is nowheres near six hundred dollars.
Hub Shark-
[quote]Originally posted by KULTULZ:
Uh...Did you click on the link I gave for the Hub Tamer? And it is nowheres near six hundred dollars.
Hub Shark-

Uh... yes I did. Take a second to figure it out. The Hub Shark is $400 online, now tack on the mark-up from the tool truck driver and that's just about the $600 the guy I work with paid for it (give or take a few dollars-it was an estimate you know).
Uh... I will repeat again... in my opinion the Hub Tamer is a cheap knock-off of the Hub Shark and I wouldn't buy it.
Uh...also again why would I want to beat a couple hundred dollar impact gun to death with either of those tools when I have a hydraulic press at my disposal???? You do realize I can remove most steering knuckles in a matter of minutes to use the press.?.
[ March 30, 2003: Message edited by: dmorris1200 ]

|Recent Transmission - Rearend posts with photos
|Currently Active Users Viewing This Thread: 1 (0 members and 1 guests)
|Thread Tools


text:
Peanut Butter Blondies RecipeRead Reviews Be the first to add a review
5 7 9
The kids I baby-sit for love these moist chewy bars. There's plenty of peanut butter flavor plus a yummy chocolate frosting and a sprinkling of peanut butter chips.
Recommended: 15 Recipes Using Leftover Halloween Candy
- 3/4 cup creamy peanut butter
- 2/3 cup butter, softened
- 1 cup packed brown sugar
- 1/2 cup sugar
- 2 eggs
- 1 teaspoon vanilla extract
- 1-3/4 cups all-purpose flour
- 1 teaspoon baking powder
- 1/3 cup milk
- 1 cup peanut butter chips
- 1/4 cup butter, softened
- 1/4 cup baking cocoa
- 2 tablespoons milk
- 1 tablespoon light corn syrup
- 1 teaspoon vanilla extract
- 1-1/2 cups confectioners' sugar
- 1/3 cup peanut butter chips
- In a large bowl, cream the peanut butter, butter and sugars until light and fluffy. Beat in eggs and vanilla. Combine the flour and baking powder; add to creamed mixture alternately with milk, beating well after each addition. Stir in chips.
- Spread into a greased 13-in. x 9-in. baking pan. Bake at 325° for 35-40 minutes or until a toothpick inserted near the center comes out clean (do not overbake). Cool on a wire rack.
- For frosting, in a small bowl, combine the butter, cocoa, milk, corn syrup and vanilla. Gradually add confectioners' sugar; beat until smooth. Frost brownies. Sprinkle with chips. Cut into bars. Yield: 2 dozen.
Originally published as Peanut Butter Blondies in Taste of Home April/May 2005, p31
Reviews for Peanut Butter Blondies
Please Log In or Join to add a rating and review.
Click stars to rate
Any changes to your rating or review will appear where you originally posted your review
Reviewed Jul. 23, 2011
"So yummy! You won't be sorry you tried these. Very rich- a little bit goes a long way. It's best to let the icing sit for a while to thicken up before icing blondies. Enjoy!"
Reviewed Jul. 2, 2011
"I made these for a 4th of July picnic and they were a big hit. Highly recommended!"
Reviewed Nov. 17, 2010
"These Peanut Butter Blondies are great. easy to make. Give them a try, you will be glad you did. Yummy! Yummy!"
Reviewed Sep. 9, 2010 Edited Feb. 22, 2015
"These bars are delicious! I don't know what makes them so perfect, but every time I take them somewhere I get compliments galore! I do bake them for 30 minutes or less though, instead of the 35-40 minutes recommended."
Reviewed Sep. 16, 2009
"Those do look good, I bet my SS class would love them."
Reviewed Aug. 20, 2009
"These do sound good. Can't wait to make them."


text:
TOM PETTY: He's Got His Hypnotic Eye on You
Photo by Frederick M. Brown/Getty Images
Tom Petty and The Heartbreakers are back today with their first album in four years, Hypnotic Eye.
Petty had a specific goal in mind for the band's 13th studio effort. He tells us, "I was trying to prove to myself and a younger generation that you can have an album that won't start to get bad three songs in."
The words "hypnotic eye" do not appear anywhere on the album except for the title. Petty tells us, "The album is kind of a loose observational take on maybe American culture these days..."
Petty and The Heartbreakers start a tour in support of the album on Sunday in San Diego with Steve Winwood in the opening slot.