text:
\section{Introduction}

X-ray crystallography has been the most popular protein structure
determination technique of both pre- and post-genomic eras. The challenges
of macromolecular crystallography are manifold - after the difficult
steps of expression, purification, crystallization and data collection,
there remains the final and important task of data interpretation
in order to build a model which explains the observed diffractions.
Structural interpretation requires overcoming the phase problem and
often starts with partial and incorrect phases. Typically, semi-automatic
iterative refinement is carried out, gradually improving the model's
quality as indicated by the $R$ and $R_{free}$ factors as well as
decrease in covalent geometry and excluded volume violations. Although
excellent softwares like CCP4 (\cite{ccp4}), Phenix (\cite{phenix})
and {\small \textsc{cns}\xspace} (\cite{cns}) make this task possible, the structure
refinement procedure remains manually-driven hence laborious and subjective.
Due to this, the heterogeneity in structural interpretation of diffraction
data is often ignored in favour of a single-conformer isotropic B-factor
model.

Protein structure is important for its function. But very stable,
rigid proteins cannot exhibit enzymatic activity. This suggests that
proteins have to be stable enough to retain their fold yet dynamic
enough to be functional. Both experimental and computational studies
indicate that single-conformer interpretation of crystallographic
data is not adequate to capture the native state dynamics which is
largely conserved even in a crystal owing to its high solvent content
(\cite{xrayLigReverseBinding}, \cite{crystalfreedom}). Reporting
a multiconformer interpretation of data will make use of the structure
less misleading, especially in the analyses that depend on geometry
such as shapes of binding sites, orientations of sidechains, detection
of non-covalent interactions and so on. While multiple interpretations
are necessary, they should be free from any bias such as that introduced
when different crystallographers solve the same diffraction data.
Multiconformer interpretation will be greatly facilitated by automated
methods.

Thus multiple persuasive justifications emerge for automating the
protein crystallographic refinement task: (a) capturing the dynamics
of protein in the crystalline state (b) removing subjective bias from
the refinement process and (c) reducing the need for precious human
resource. But this goal is hard to achieve in practice. The under-determined
nature of the problem (number of independent observation < number
of parameters) prevents a straightforward solution by minimization.
Even when sufficient restraints exist, minimization methods like conjugate
gradient, steepest descent etc. suffer from the problem of local minima.
Hence use of well-known features of proteins is unavoidable. Automatic
pattern recognition in electron density is very successful in presence
of high resolution data and good phases because it looks for such
features (\cite{arpwarp}). But at medium resolution or given poor
phases, this strategy can get misled.

Our recent efforts with automated crystallographic refinement started
with {\small \textsc{rapper}\xspace}, which is a conformation sampling program for
proteins and uses a genetic algorithm cum branch-and-bound ({\small \textsc{gabb}\xspace})
algorithm. \cite{xrayHetInaccu} showed that multiple interpretations
similar to the deposited structure are possible given the deposited
data, and the divergence in interpretation is correlated to resolution.
With {\small \textsc{rapper}\xspace}, it was demonstrated (\cite{rapperKnowledgeXray})
that when a protein structure is approximately known, it can be refined
to native-like quality, unlike {\small \textsc{mdsa}\xspace} in {\small \textsc{cns}\xspace} which
may get stuck in local minima. Fundamental features of {\small \textsc{rapper}\xspace}
responsible for avoidance of local minima traps were (a) use of fine-grained,
propensity-weighted $\phi-\psi$ maps for backbone sampling (b) use
of backbone-dependent rotameric libraries (c) use of ideal Engh and
Huber covalent geometry (d) mild use of electron density and positional
restraints to guide the sampling process. Later \cite{rapperLowResolution}
demonstrated that a low-resolution dataset can be rescued and interpreted
semi-automatically to obtain structure of a system with great biological
significance.

\cite{rapperKnowledgeXray} observed that automatic refinement becomes
less satisfactory as positional restraints become weaker: the structures
could not be refined if the initial $C_{\alpha}$ perturbation was
of order of \Ang{3} or more. This is not unexpected because larger
positional restraints dilute the information and would make the search
harder. But often a practical problem encountered in crystallography
is that of missing loops, i.e. knowing loop regions with far less
$C_{\alpha}$ positional certainty than the regions with regular secondary
structure. By definition, loops exhibit rich variability in backbone
torsion angles. They are thought to be more dynamic than the protein
secondary structural framework and also functionally more interesting.
Thus it is important to use the available restraints as efficiently
as possible to build loops despite weaker electron density and greater
positional uncertainty while tolerating small positional errors in
the framework.

After determining a single-conformer loop structure, the second important
challenge is to estimate the structural variability of the loops.
It is easy to see by generating artificial data that existence of
structural heterogeneity for a loop results in confusing electron
density. In general, partial occupancies result in weaker density
than full occupancy. Sidechains of the same residue may occupy different
density contours. Overlaps in conformations may lead to significant
loss of shape information. These challenges can be expected to make
the task only harder for minimization-based programs when refining
a multiconformer structure.

Following the reformulation of {\small \textsc{rapper}\xspace} as a versatile modular
software called \textit{Rapper}tk\xspace (\cite{rappertk}), it was essential to
benchmark its performance for all-atom protein crystallographic refinement.
Hence the first result in this work is the all-atom knowledge-based
crystallographic refinement given the positional restraints for the
entire protein, establishing that a similar result as {\small \textsc{rapper}\xspace}
(\cite{rapperKnowledgeXray}) can be achieved. We then demonstrate
that single loops in proteins can be reconstructed to a high quality
with \textit{Rapper}tk\xspace using little positional information. This case is
then extended to include all loop regions and a small error in the
framework to show that the composite {\small \textsc{cns}\xspace}/\textit{Rapper}tk\xspace refinement
approach is suitable in a realistic scenario. Finally, we ask whether
single-loop heterogeneity can be modelled with collections of independently
generated models or ensembles of conformers.

\section{Methods}

\subsection{Overview of iterative refinement}

Each step in refinement procedure consists of: (a) identification
of residues which do not fit well into density, (b) finding contiguous
bands of such residues, (c) rebuilding the bands with knowledge-based
conformational sampling within restraints, (d) optimal sidechain placement
of rebuilt sidechains and (e) refining the resulting model with {\small \textsc{cns}\xspace}.

The fit of a set of atoms to electron density is calculated as the
correlation coefficient between the $\sigma_{A}$-weighted omit map
and $F_{c}$ map for a region around \Ang{1} of the atoms. The maps
used are both generated by {\small \textsc{cns}\xspace} refinement script, hence
they are described on the same grid. Following \cite{RrealJones1991}
and \cite{rapperKnowledgeXray}, the correlation coefficient between
the maps is calculated on the grid neighbourhood around atoms of interest:

\begin{equation}
CorrCoef=\frac{\sum\sigma_{omit}\sigma_{c}}{\sqrt{\sum\sigma_{omit}^{2}\sum\sigma_{c}^{2}}}\label{eqChisqCorrCoeff}\end{equation}

If the correlation is below $0.9$, the atoms are flagged for rebuilding.
Correlation is calculated on all atoms in residues and then on mainchains
only, sidechains only and peptide atoms only. Ill-fitting sidechains
are marked for sidechain reassignment whereas residues with ill-fitting
peptide or mainchain or all-atoms are marked for all-atom reconstruction.

Once the residues are flagged for all-atom rebuilding, contiguous
bands are identified and marked either as N-terminal, C-terminal or
intermediate. Bands are then sampled in random order using the PopulationSearch
algorithm. Each band is attempted 5 times and left as it was if it
cannot be sampled within given restraints. Previously sampled bands
are considered while sampling later bands. N and C terminal bands
are built using forward and reverse techniques and weighted sampling
of $\phi-\psi$ propensities. Building of intermediate regions is
described in a later section.

Once all bands are sampled, all the resampled sidechains are reassigned
using the optimal sidechain placement procedure described elsewhere
(\cite{opsax}).

\subsection{Electron Density Ranker}

Generally a single conformer model would refine better if its occupation
of the $2F_{o}-F_{c}$ map is better - so a model within $1\sigma$
contour is more reliable than $0.5\sigma$. Thus, on output of each
builder, a binary electron density restraint can be applied with a
$\sigma$-level cutoff. But the quality of map is not uniform over
all residues and hence such binary restraint is useful only for ensuring
that model remains within positive electron density. Hence, in addition
to that restraint, an analog ranker is used for electron density that
ranks the possibilities and chooses the better ones. In the population
search algorithm, the ranker asks more children to be generated at
each conformation extension step than the population size (typically
5 times more), ranks them and chooses top-ranking ones to fill the
conformation pool. The ratio of number of children generated to the
population size is termed as the enrichment ratio. The electron density
ranking scheme calculates score of a set of atoms by summing up the
$\sigma$ values in a \Ang{1} region around their coordinates. The
effective $\sigma$ value is calculated by penalising the negative
$\sigma$ and flattening the peaks by upper cutoff, the latter for
better recognition of shape of density rather than spikes, say due
to waters or ions. In addition to filtering children at each step,
the ranker also chooses the best member from the conformational pool
generated, which is returned as the sampled model for the band.

\subsection{Symmetry-related clash cheking}

As described in \cite{rappertk}, \textit{Rapper}tk\xspace uses geometric caching
implemented as Clashchecking grid for efficiently deciding whether
atomic van der Waals spheres are overlapping. This excluded-volume
restraint rules out many unproductive sampling trajectories. When
loop positions are largely uncertain, the existence of symmetry-related
images of the molecule around it acts as an excluded volume restraint
to loop sampling. \textit{Rapper}tk\xspace uses the Clipper (\cite{clipper}) libraries
for crystallographic computing for symmetry-related calculation. Clashchecking
grid uses Clipper's Spacegroup class and symmetry operators therein
to calculate the images of atoms to be added into the grid. Images
within \Ang{20} of the bounding box of given protein coordinates
are considered. First the grid looks for any clashes between sampled
coordinates and their images. Then it is verified that they do not
clash against the rest of the coordinates or their images. In case
of no clashes, the new coordinates and their images are added to the
grid. Removing coordinates from the grid removes their images too.

\subsection{Loop closure}

The typical incremental sampling step in \textit{Rapper}tk\xspace builds $C_{\alpha}^{i}$
and $(i-1)^{th}$ sidechain in the forward mode or $C_{\alpha}^{i}$
and $(i+1)^{th}$ sidechain in the reverse mode. In this context,
loop closure can be formulated as finding the locations of mainchain
atoms $\{ C^{i-1}$, $O^{i-1}$, $N^{i}$, $C_{\alpha}^{i}$, $C^{i}$,
$O^{i}$, $N^{i+1}\}$ and sidechains of $(i-1)^{th}$,$i^{th}$ and
$(i+1)^{th}$ residues. Seamless loop closure of this kind is challenging
because many conditions need to be met: (a) the covalent angles and
lengths should be correct (b) $\phi,\psi$ states of 3 residues should
be in the allowed regions (c) two $\omega$ angles should be adopt
cis or trans conformation, but not be restricted to one or the other
(d) 3 sidechains should be rotameric and (e) van der Waals restraints
should be obeyed.

A sampling procedure was devised for meeting conditions (a), (b),
(c) and (d), while (e) is met using clash-checking restraints. The
sampling procedure is similar to the one described in \cite{rappertk},
but modified to meet the cis $\omega$ state too. First, the two $\omega$
angles are sampled, leading to the center, plane and radius of the
circle on which the middle $C_{\alpha}$ is sampled. The circle is
uniformly sampled. For each sample, the $\{ r,\alpha,\theta,\}-\{\phi,\psi,\omega\}$
mapping is used to build the mainchain atoms. Then the three sidechains
are sampled from a rotamer library. Sampling is continued until a
conformation satisfying all restraints is found.

The problem with this sampling is that the restraint density abruptly
increases at loop closure because it is not clear how to back-propagate
the geometric requirements (a) and (c). Due to this, the sampling
procedure fails often and takes a long time to find a valid sample.
Often an incorrect conformation is built in case of imperfect density
because sampling of $\phi,\psi,\omega$ is not propensity-weighted.
Hence after significant experimentation with this approach, it was
abandoned in favour of a simpler approximate approach.

In the simpler approach, the loop closure is formulated as finding
the coordinates of mainchain atoms $\{ C^{i}$, $O^{i}$, $N^{i+1}\}$
and sidechains of residues $i$ and $i+1$. A $\phi$ sampler is used
to build the $C^{i}$ atom which is required to lie between $0.5$
to \Ang{2} from the $N^{i+1}$ atom. Covalent angles $N^{i}-C_{\alpha}^{i}-C^{i}$
and $C^{i}-N^{i+1}-C_{\alpha}^{i+1}$ are restrained to lie between
$90^{o}$ and $150^{o}$. $\omega$ dihedral angle $C_{\alpha}^{i}-C^{i}-N^{i+1}-C_{\alpha}^{i+1}$
is allowed a maximum deviation of $30^{o}$ from cis or trans conformation.
Two sidechains are sampled for each $C^{i}$ sampled. It is observed
that it is more efficient to close a loop with this method than the
previous.

\subsection{Both-sided sampling}

Bands to be rebuilt can be of three types: the N terminal band, the
C terminal or intermediate. For the C and N terminal bands, forward
and reverse sampling are used respectively. For the intermediate regions,
the most efficient way is to use a both-sided sampling approach as
opposed to only forward sampling. As explained previously (see \cite{rappertk}
in the context of $\beta$-sheet sampling), in both-sided sampling,
residues are sampled in the order $i,k,i+1,k-1,i+2,k-2,...$. In case
of forward or reverse sampling, only a weak loop-closure distance
restraint informs the sampling process of the other end of the loop,
but with both-sided sampling, information at both N and C termini
is actively used. A distance restraint is used between $C_{\alpha}$
atoms at the same sequence distance from both termini, so that the
chance of loop closure remains high despite both sided sampling. Initial
experiments with crystallographic loop building clearly showed that
refinement was better with both-sided sampling than forward-only sampling,
especially with larger loops and weaker positional restraints. Thus,
in this work, we have used forward-only, backward-only and both-sided
sampling for C terminal, N-terminal and intermediate bands respectively.

\subsection{Multiconformer sampling}

This type of sampling constructs many conformations of the same band
simultaneously. Instead of incrementally sampling one band, multiple
models of the band are extended simultaneously. This is achieved by
re-implementing the PopulationSearch algorithm in its plural form
in which each builder is replaced by a set of builders that have same
input and output atoms in different models. Clashchecking is not performed
across models. Electron density ranker uses the combined output of
a set of corresponding builders to calculate the score of a child
conformation. Due to this, the possibility of getting attracted into
higher density is reduced and the chance of occupying the density
generated due to genuine heterogeneity increases. The disadvantage
of this kind of sampling is the obvious increase in conformational
freedom and execution time.

\section{Results}

\subsection{Reproducing {\small \textsc{rapper}\xspace}/{\small \textsc{cns}\xspace} refinement}

The utility of knowledge-based refinement has been demonstrated by
\cite{rapperKnowledgeXray} with automatic refinement of perturbed
starting structures of 9ILB and 1KX8 to an $R_{free}$ almost same
as the deposited structure. When that refinement protocol was closely
reproduced in \textit{Rapper}tk\xspace, very similar results were obtained. Five
proteins were selected in the \Ang{2}-\Ang{3} resolution range from
the {\small \textsc{pdb}\xspace}: 9ILB (\cite{ref9ILB}), 1KX8 (\cite{ref1KX8}),
1MB1 (\cite{ref1MB1}), 1BYW (\cite{ref1BYW}) and 1RN7 (\cite{ref1RN7}).
Five perturbed structures were generated for each of them within \Ang{2}
$C_{\alpha}$ and \Ang{3} sidechain centroid restraints respectively.
20 rounds of both {\small \textsc{cns}\xspace}-only and {\small \textsc{cns}\xspace}/\textit{Rapper}tk\xspace
refinement protocols were performed on these starting models to obtain
the $R_{free}$ statistics summarized in Table \ref{fullprotTable}.
$R_{free}$ figures reported for 9ILB and 1KX8 by \cite{rapperKnowledgeXray}
for same restraints were $0.27(0.01)$ and $0.32(0.01)$ respectively
- the corresponding statistics observed here are comparable.

\begin{table}

\caption{Full-protein testset and refinement statistics for 5 starting models
generated within \Ang{2} $C_{\alpha}$ and \Ang{3} sidechain restraints.}

\begin{center}\begin{sideways}
\begin{tabular}{|c|c|c|c|c|c|c|c|c|}
\hline
PDB Id&
Resolution&
\#AA&
Baseline$^{a}$&
CNS-only&
\multicolumn{4}{c|}{CNS/\textit{Rapper}tk\xspace}\tabularnewline
&
 (\Ang{})&
&
$R_{free}$&
$R_{free}$&
$R_{free}$&
AA-RMSD$^{b}$&
$\chi_{1}$$^{c}$&
$\chi_{12}$$^{d}$\tabularnewline
&
&
&
&
&
&
\multicolumn{3}{c|}{(pdb vs. models, among models)}\tabularnewline
\hline
\hline
9ILB&
2.28&
153&
0.225&
0.266 (0.016)&
0.253 (0.011)&
1.29 (0.08)&
75.2 (2.63)&
53.0 (4.64)\tabularnewline
&
&
&
&
&
&
0.585&
79.100&
55.100\tabularnewline
\hline
1KX8&
2.80&
99&
0.294&
0.317 (0.018)&
0.310 (0.010)&
0.60 (0.02)&
76.2 (1.93)&
50.2 (4.06)\tabularnewline
&
&
&
&
&
&
0.551&
77.800&
57.500\tabularnewline
\hline
1MB1&
2.10&
98&
0.292&
0.333 (0.021)&
0.302 (0.003)&
0.60 (0.02)&
75.8 (2.31)&
56.4 (2.05)\tabularnewline
&
&
&
&
&
&
0.598&
78.300&
59.600\tabularnewline
\hline
1BYW&
2.60&
110&
0.292&
0.342 (0.020)&
0.316 (0.013)&
0.58 (0.06)&
78.6 (3.32)&
57.4 (5.60)\tabularnewline
&
&
&
&
&
&
0.595&
79.700&
58.400\tabularnewline
\hline
1RN7&
2.50&
112&
0.270&
0.366 (0.054)&
0.318 (0.009)&
0.61 (0.04)&
81.0 (2.09)&
53.2 (1.93)\tabularnewline
&
&
&
&
&
&
0.604&
80.300&
54.500\tabularnewline
\hline
\hline
\multicolumn{9}{|c|}{}\tabularnewline
\multicolumn{9}{|c|}{\begin{minipage}[c]{200mm}%
$^{a}$Baselines $R_{free}$ values are computed by running a long
CNS-only refinement starting from the deposited structure.

$^{b}$All-atom RMSDs are in \Ang{} units. First two numbers are
the mean and standard deviations of RMSDs between the PDB and 5 models.
The third number is the mean of RMSDs among 5 models based on $^{5}C_{2}=10$
pairwise comparisons.

$^{c}$$\chi_{1}$ accuracy of a protein structure with respect to
another is defined as the percentage of sidechains in the former that
have a $\chi_{1}$ differing by less than $40^{o}$ from the corresponding
sidechain in the latter. First two numbers are the mean and standard
deviation of the $\chi_{1}$ accuracies between 5 models and the deposited
structure. The third number is the mean of $\chi_{1}$ accuracies
from $^{5}C_{2}=10$ pairwise comparisons among the 5 models.

$^{d}$Same as $^{c}$, but with $\chi_{1}$ and $\chi_{2}$ both
instead of only $\chi_{1}$.\end{minipage}%
}\tabularnewline
\multicolumn{9}{|c|}{}\tabularnewline
\hline
\end{tabular}
\end{sideways}\end{center}

\label{fullprotTable}
\end{table}

Table \ref{fullprotTable} shows the variation in all-atom {\small \textsc{rmsd}\xspace}
and $\chi_{1},\chi_{12}$ values as function of resolution. The reported
{\small \textsc{rmsd}\xspace} is the average of pairwise unsuperposed {\small \textsc{rmsd}\xspace}
between the deposited and each of the composite models and thus can
be said to indicate the inaccuracy in retrieving the deposited model
from an approximate starting model. This inaccuracy does not seem
to be sensitive to the resolution, suggesting that at least in the
\Ang{2.1}-\Ang{2.8} resolution range, an approximate model can be
corrected to a similar quality with respect to the deposited one irrespective
of the resolution.

When the models are compared among themselves in a pairwise manner,
the average {\small \textsc{rmsd}\xspace} and $\chi_{1},\chi_{1,2}$ figures can
be said to represent heterogeneity. This heterogeneity is slightly
lower than the inaccuracy, but the difference is insignificant, i.e.
each model is as far from the deposited structure as from any other
model. Recently the heterogeneity defined similarly has been suggested
to be the minimum uncertainty expected in the coordinates of a single-conformer
model of that structure (\cite{minUncertaintyXrefine}).

An ideal refinement method should start with approximate models and
yield a set of high heterogeneity models each of which agrees at least
as well with the data as the deposited structure, i.e. a combination
of results in \cite{xrayHetInaccu} and \cite{rapperKnowledgeXray}.
Clearly, the protocol used is similar to \cite{rapperKnowledgeXray}
and perhaps expectedly, does not yield greater heterogeneity than
inaccuracy. But when the models are assigned partial occupancies and
combined to create a multiconformer model (Fig.\ref{LoopCollectionRfree},
Section \ref{collectionRfreeSection}), the collection $R_{free}$
values for 1MB1, 1BYW and 1KX8 drop significantly by $1.5-2\%$ than
the deposited structure. This drop suggests that perhaps structural
heterogeneity is captured to some extent.

\subsection{Rebuilding missing loops}

Five structures of various resolutions and no obvious homology were
selected from the {\small \textsc{pdb}\xspace}: 1MB1 (\cite{ref1MB1}), 1BYW (\cite{ref1BYW}),
1KXB (\cite{ref1KXB}), 1RN7 (\cite{ref1RN7}) and 2DBO (\cite{ref2DBO}).
All structures have a single continuous peptide chain between 100-200
residues and no ligands. For each structure, a loop at least 10 residues
long was chosen for rebuilding (Table \ref{loopdataset}). Unlike
the previous exercise, there are no positional restraints on loop
sidechains. $C_{\alpha}$ atoms are positionally restrained, in the
first case with \Ang{5} restraints and later with \Ang{10} restraints.
The loops were rebuilt using the both-sided loop sampling and loop
closure techniques within the iterative refinement protocol. For 4
of 5 loops considered, the \Ang{5} perturbation of the loop can be
corrected to within one point of the baseline $R_{free}$. In the
\Ang{10} case, this performance drops marginally to two points from
the baseline $R_{free}$ for the same cases.

Fig.\ref{loopfig1MB1} shows the large difference in the quality of
{\small \textsc{cns}\xspace}-only and {\small \textsc{cns}\xspace}/\textit{Rapper}tk\xspace refinement protocol.
Every starting model refines to a structure very similar to the deposited
using the composite protocol whereas it gets trapped in local minima
during {\small \textsc{cns}\xspace}-only refinement. The 1RN7 case (Fig.\ref{loopfig1RN7})
is unsatisfactory due to a difficult 5-residue segment in the loop
(Pro-80, Asn-81, Leu-82, Asp-83, Asn-84). As noted by \cite{ref1RN7},
the density for this segment is confusing, perhaps due to underlying
heterogeneity, and consistently misleads the band sampling into a
conformation different from the deposited.

\begin{table}

\caption{Dataset for loop building and refinement statistics.}

\begin{center}\begin{sideways}
\begin{tabular}{|c|c|c|c|c|c|c|c|c|}
\hline
PDB Id&
Resolution&
\#AA&
Loop range&
Baseline &
\multicolumn{2}{c|}{$R_{free}$(CNS-only)}&
\multicolumn{2}{c|}{$R_{free}$(CNS/\textit{Rapper}tk\xspace)}\tabularnewline
&
 (\Ang{})&
&
and size&
$R_{free}$&
\Ang{5}&
\Ang{10}&
\Ang{5}&
\Ang{10}\tabularnewline
\hline
\hline
1MB1&
2.1&
98&
66-75 (12)&
0.292&
0.331 (0.024)&
0.368 (0.022)&
0.304 (0.013)&
0.307 (0.005)\tabularnewline
\hline
1BYW&
2.6&
110&
115-124 (12)&
0.292&
0.329 (0.006)&
0.335 (0.008)&
0.299 (0.012)&
0.314 (0.008)\tabularnewline
\hline
1KXB&
2.9&
158&
206-218 (15)&
0.283&
0.326 (0.007)&
0.337 (0.010)&
0.290 (0.008)&
0.293 (0.017)\tabularnewline
\hline
1RN7&
2.5&
112&
76-95 (22)&
0.270&
0.347 (0.018)&
0.366 (0.022)&
0.311 (0.009)&
0.316 (0.013)\tabularnewline
\hline
2DBO&
2.76&
148&
83-98 (18)&
0.289&
0.332 (0.015)&
0.354 (0.006)&
0.291 (0.010)&
0.291 (0.008)\tabularnewline
\hline
\end{tabular}
\end{sideways}\end{center}

\label{loopdataset}
\end{table}

\begin{figure}

\caption{Loop building exercise for the 1MB1 loop with \Ang{10} $C_{\alpha}$
restraints. Top panel shows the loop in the deposited structure (green)
and starting models generated for it. Middle panel shows the best
$R_{free}$ models (slate) obtained during the CNS-only refinement.
Bottom panel shows the CNS/\textit{Rapper}tk\xspace models (magenta) and the loop
in the deposited structure (green) in all-atom representation.}

\begin{center}\includegraphics[%
  width=100mm]{1MB1_loop10_start.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1MB1_loop10_cns.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1MB1_loop10_rtk.jpg}\end{center}

\label{loopfig1MB1}
\end{figure}

\begin{figure}

\caption{Loop building exercise for the 1RN7 loop with \Ang{5} $C_{\alpha}$
restraints. Panels arranged in a similar way to Fig.\ref{loopfig1MB1}.}

\begin{center}\includegraphics[%
  width=100mm]{1RN7_loop5_start.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1RN7_loop5_cns.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1RN7_loop5_rtk.jpg}\end{center}

\label{loopfig1RN7}
\end{figure}

\subsection{Framework and loops}

Perhaps a more frequently encountered scenario than the previous two
is the one in which both the secondary structure and loops have positional
uncertainty. In such cases, the loop regions invariably are more unreliable
than the secondary structure framework. In order to simulate this
scenario, the framework was restrained to \Ang{1} $C_{\alpha}$ and
\Ang{3} sidechain centroid restraints, whereas loops were restrained
to \Ang{3} $C_{\alpha}$ restraints and no sidechain restraints.
Five models were built for each protein and then iteratively refined
using both {\small \textsc{cns}\xspace}-only and {\small \textsc{cns}\xspace}/{\small \textit{Rapper}tk\xspace}
protocols. The refinement statistics are summarized in Table \ref{frameloopTable}.
Note that the refinement composite refinement statistics are not as
good as in the previous exercises, but still better than the {\small \textsc{cns}\xspace}-only
refinement. Fig.\ref{loopfig1BYWfr} shows a typical contrast between
the {\small \textsc{cns}\xspace}-only and the composite refinement protocols in
this scenario.

\begin{table}

\caption{Dataset and refinement statistics for the loop-framework refinement}

\begin{center}\begin{tabular}{|c|c|c|c|c|c|c|}
\hline
PDB&
Resolution&
\#AA&
Loops&
\multicolumn{3}{c|}{$R_{free}$ }\tabularnewline
&
 (\Ang{})&
&
\#AA (\%)&
baseline&
CNS-only&
CNS/{\small \textit{Rapper}tk\xspace}\tabularnewline
&
&
&
&
&
Mean (Std.Dev.)&
Mean (Std.Dev.)\tabularnewline
\hline
\hline
1MB1&
2.1&
98&
27 (28)&
0.292&
0.352 (0.012)&
0.325 (0.0l0)\tabularnewline
\hline
1BYW&
2.6&
110&
50 (45)&
0.292&
0.400 (0.046)&
0.321 (0.016)\tabularnewline
\hline
1KXB&
2.9&
158&
54 (36)&
0.283&
0.351 (0.022)&
0.336 (0.006)\tabularnewline
\hline
1RN7&
2.5&
112&
33 (29)&
0.270&
0.342 (0.032)&
0.328 (0.008)\tabularnewline
\hline
2DBO&
2.76&
148&
57 (39)&
0.289&
0.370 (0.020)&
0.335 (0.017)\tabularnewline
\hline
\end{tabular}\end{center}

\label{frameloopTable}
\end{table}

\begin{figure}

\caption{Framework/loop exercise for 1BYW. The deposited structure is shown
as thick green ribbon and starting structures as brown ribbon in the
top panel. Middle panel shows the best $R_{free}$ structures from
CNS-only trajectories (slate) and bottom panel shows those from from
the CNS/{\small \textit{Rapper}tk\xspace} trajectories (magenta).}

\begin{center}\includegraphics[%
  width=100mm]{1BYW_frloop_start.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1BYW_frloop_cns.jpg}\end{center}

\begin{center}\includegraphics[%
  width=100mm]{1BYW_frloop_rtk.jpg}\end{center}

\label{loopfig1BYWfr}
\end{figure}

\subsection{Variation of $R_{free}$ with collection size}

\label{collectionRfreeSection}

We define a collection as a set of independently refined structures
which when taken together may capture some aspects of structural heterogeneity.
This term is introduced to distinguish the collection from an ensemble
(see Section \ref{loopCollEnsSection}) which is also a set of structures,
but refined in an interdependent manner.

For the previous exercises of refinement (whole chain, \Ang{5} loop,
\Ang{10} loop and loops with framework), single best $R_{free}$
models from five {\small \textsc{cns}\xspace}/{\small \textit{Rapper}tk\xspace} trajectories are
chosen and combined to create collections, e.g. a three model collection
is created by choosing three lowest $R_{free}$ models from the five
and assigning occupancy of $0.33$ to each of them. A collection is
subjected to a short {\small \textsc{cns}\xspace} refinement and $R_{free}$ at
its end is noted as collection $R_{free}$. Fig.\ref{LoopCollectionRfree}
shows the variation of such $R_{free}$ values as a function of collection
size. The drop in $R_{free}$ is modest and the highest when going
from collection size of 1 to 2 or 3. The collection $R_{free}$ generally
rises for sizes 4 and 5. This indicates the danger of overfitting
due to increase in number of parameters. Thus a straightforward combination
of models does not seem to be the correct way of describing heterogeneity.
Intelligent schemes for parameter reduction need to be investigated
in this regard, such as upper-bounding $B$-factors, enforcing the
same $B$-factor on corresponding atoms across all models and positionally
constraining them together when large variability is not expected.

\begin{figure}

\caption{Variation of $R_{free}$ with collection size in whole chain, loop
and framework-loop exercises. For collection size of 0, baseline $R_{free}$
is shown. For collection size 1, the mean and standard deviation of
best $R_{free}$ models in CNS/{\small \textit{Rapper}tk\xspace} trajectories are
shown. The rest are calculated by combining the best $R_{free}$ individual
models with partial occupancies.}

\begin{center}\begin{tabular}{cc}
\includegraphics[%
  width=90mm,
  angle=90]{collrfreewholechain.pdf}&
\includegraphics[%
  width=90mm,
  angle=90]{collrfreeLoop5.pdf}\tabularnewline
\includegraphics[%
  width=90mm,
  angle=90]{collrfreeLoop10.pdf}&
\includegraphics[%
  width=90mm,
  angle=90]{collrfreeLoopFr.pdf}\tabularnewline
\end{tabular}\end{center}

\label{LoopCollectionRfree}
\end{figure}

\subsection{Mistakes in the composite protocol}

Although the {\small \textsc{cns}\xspace}/{\small \textit{Rapper}tk\xspace} refinement produces
a well-refined structure very close to the baseline $R_{free}$, it
never betters the latter. This is due to imperfections in various
components of the protocol. Identification of residues to rebuild
relies on the $\chi^{2}$ correlation coefficient which can sometimes
be an unsatisfactory substitute for human judgement. This can lead
to unnecessary resampling of satisfactory bands and sometimes incorrect
conformers are not detected. The copying of non-protein atoms from
one round to next may sometimes result in their permanent misplacement.
The sampling problem may result in unsatisfactory bands because the
right conformation must be generated in order to be picked by the
electron density ranker. On the other hand, the ranker may not score
a correct conformation as the best one. If the density for a band
is weak, band sampling may sometimes lead into the density of waters
or ligands. In spite of these difficulties, the refinement statistics
presented in previous sections are satisfactory.

But when restraint radii are increased beyond those used here, the
serious problem of spatial overlap between restraint spheres of two
different bands starts affecting the band sampling. The correct density
for a band then may be occupied by a band sampled before it. In such
case, the correct density is always occupied by the wrong band. For
the wrong band, subsequent {\small \textsc{cns}\xspace} refinement may take it so
far from its correct location that the restraint radii may be too
small to let the band be built correctly again. Further work is required
to get rid of the problem of band overlaps, either by restraint adjustments
or change in the sampling strategy.

\subsection{Modelling loop heterogeneity with collections and ensembles}

\label{loopCollEnsSection}

Conformational diversity is most pronounced in loop regions due to
the relatively smaller number of non-covalent interactions to maintain
order. Absence of good density for a loop when rest of the structure
has good density is a sure indication of the loop's flexibility. Modelling
heterogeneity is challenging because density is generally more confusing
for such regions owing to conformer overlaps and subsequent dilution
of shape information.

Heterogeneity can be modelled with collections or ensembles. Members
of a collection are single-conformer isotropic B-factor models determined
independently of one another. Members of an ensemble are determined
in a highly interdependent manner and have partial occupancies.

Derivation of a collection is a simple way to estimate the unavoidable
uncertainty in structure determination, but it cannot be said to represent
any structural correlations. A major advantage of collections is their
simplicity. A procedure that produces a single model can be executed
multiple times with different random seeds or starting models to generate
a collection. Thus the time taken increases only linearly as the collection
size.

An implicit assumption in the ensemble representation is that the
members are in dynamic equilibrium, making ensemble a much stronger
statement than the collection. Determining ensembles is very challenging
because it is unclear how to determine the number and occupancies
of the ensemble members prior to or during the refinement process.
Another major challenge is the linear increase in the number of parameters
which results in an exponential increase in search space and execution
time.

In order that its output be credible, any procedure that aims to model
the structural heterogeneity must be first validated using artificial
data where the \emph{real} heterogeneity is accurately known. To that
end, we have chosen a significantly simplified kind of heterogeneity
by generating artificial diffraction data in which the underlying
heterogeneity is restricted to a single loop and consists of two equally-occupied
loop conformations. For a loop each from 1MB1, 1BYW and 2DBO, two
conformers were generated by perturbing the loop to within \Ang{3}
$C_{\alpha}$ restraints and no sidechain restraints. All non-protein
atoms (ions, waters etc.) have been removed so as to reduce the density
dilution. Artificial diffraction data were created with the same cell,
spacegroup and resolution as the deposited structure. For self-consistency
in the {\small \textsc{cns}\xspace} forcefield, data was generated iteratively.
The average of the two conformations was considered as the starting
conformation for further heterogeneity modelling.

A collection of 4 members was generated for each loop with {\small \textsc{cns}\xspace}/{\small \textit{Rapper}tk\xspace}
protocol used previously. An ensemble consisting of 2 members was
generated for each loop using multiconformer sampling described previously.
As with the single-conformer protocol, multiconformers were sampled
iteratively and alternatingly with {\small \textsc{cns}\xspace}. Enrichment was
increased to 10 and population size to 200 to be able to build reasonable
models. Positive electron density restraint was enforced on mainchain.

The performance of collections and ensembles can be visually inspected
in Fig.\ref{EC1mb1}, Fig.\ref{EC1byw} and Fig.\ref{EC2dbo} but
it is essential to quantify quality of heterogeneity modelling objectively.
The two important quantities to measure are: the extent to which both
conformers are captured and the extent to which at least one conformer
is captured. The former (multiconformer quality index, MQI) should
quantify how much of the underlying diversity is represented and the
latter (single-conformer quality index, SQI) should quantify how well
at least one of the heterogenous states is modelled.

If conformers $H_{i}$ constitute the true underlying heterogeneity
and $M_{i}$ are the ensemble or collection members which model it,
then MQI and SQI can be calculated as:

\begin{eqnarray}
MQI & = & \sum_{i}min_{j}(Rmsd(H_{i},M_{j}))\label{eqMqiSqi}\\
SQI & = & min_{i,j}(Rmsd(H_{i},M_{j}))\nonumber \end{eqnarray}

where {\small \textsc{rmsd}\xspace} is calculated over the atoms of interest, e.g.
a sidechain or all $C_{\alpha}$ atoms in the loop. Note that these
expressions do not consider the occupancies. Table \ref{ECtable}
quantifies the performance of ensembles and collections using $R_{free}$,
MQI over loop $C_{\alpha}$ atoms, and MQI, SQI over each sidechain.
The ensemble $R_{free}$ values are smaller than those for collections
as expected due to greater number of parameters. $C_{\alpha}$ MQI
suggests that mainchain heterogeneity is modelled better in the ensemble
method. Sidechain SQIs do not show any systematic difference between
the two methods, which suggests that both methods capture the rotameric
heterogeneity to a similar extent. But sidechain MQIs tend to be slightly
better for ensemble than collection. This is not surprising because
in principle, the only limitations on the ensemble method are sampling
and ranking of conformational extensions. Generally the higher density
option is chosen in single-conformer modelling but a lower density
can also be chosen in multiconformer modelling due to a greater number
of atoms to place in the density. This is evident from residues Lys-72
and Tyr-73 in 1MB1, Glu-118, Asp-119 in 1BYW and Lys-86, Lys-87 in
2DBO where collection models are biased towards one of the loop conformations
due to weak density.

\begin{figure}

\caption{Heterogeneity modelling with collection and ensemble for a loop in
PDB 1MB1. The top panel shows the artificially generated loop heterogeneity
with corresponding electron density contoured at $1\sigma$. The middle
and bottom panels respectively show a 4-member collection and 2-member
ensemble model of that heterogeneity.}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1mb1ENS1.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1mb1ENS2.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1mb1ENS3.jpg}\end{center}

\label{EC1mb1}
\end{figure}

\begin{figure}

\caption{Heterogeneity modelling with collection and ensemble for a loop in
PDB 1BYW. Panels arranged as in Fig.\ref{EC1mb1}.}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1bywENS1.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1bywENS2.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{1bywENS3.jpg}\end{center}

\label{EC1byw}
\end{figure}

\begin{figure}

\caption{Heterogeneity modelling with collection and ensemble for a loop in
PDB 2DBO. Panels arranged as in Fig.\ref{EC1mb1}. In the top panel,
the red contours correspond to $0.5\sigma$.}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{2dboENS1.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{2dboENS2.jpg}\end{center}

\begin{center}\includegraphics[%
  width=60mm,
  angle=90]{2dboENS3.jpg}\end{center}

\label{EC2dbo}
\end{figure}

\begin{table}

\caption{Comparison of collection and ensemble heterogeneity modelling styles
with artificially generated 2-conformer heterogeneity for 3 loops.
MQI and SQI are in \Ang{} units.}

\begin{center}\begin{sideways}
\begin{tabular}{|c|c|c|c|c|c|c|}
\hline
PDB&
Baseline&
Collection $R_{free}$&
Ensemble&
MQI-$C_{\alpha}$&
MQI-$C_{\alpha}$&
Residue-wise MQI, SQI for collection;\tabularnewline
&
$R_{free}$&
Mean (Std.Dev.)&
$R_{free}$&
Collection&
Ensemble&
MQI, SQI for ensemble\tabularnewline
\hline
\hline
1MB1&
0.055&
0.127 (0.003)&
0.094&
2.11&
1.42&
\begin{tabular}{ccccc}
&
&
&
&
\tabularnewline
\hline
Gln-67&
4.269&
0.354&
4.285&
0.516\tabularnewline
\hline
Phe-70&
3.575&
0.262&
3.271&
0.670\tabularnewline
\hline
Lys-72&
5.624&
0.889&
2.539&
0.235\tabularnewline
\hline
Tyr-73&
3.316&
0.151&
0.233&
0.115\tabularnewline
\hline
Gln-74&
1.606&
0.349&
1.614&
0.189\tabularnewline
\hline
&
&
&
&
\tabularnewline
\end{tabular}\tabularnewline
\hline
1BYW&
0.159&
0.242 (0.004)&
0.192&
0.79&
0.58&
\begin{tabular}{ccccc}
&
&
&
&
\tabularnewline
\hline
Lys-116&
0.994&
0.325&
1.004&
0.275\tabularnewline
\hline
Asn-117&
1.849&
0.529&
1.861&
0.275\tabularnewline
\hline
Glu-118&
2.171&
0.169&
0.178&
0.049\tabularnewline
\hline
Asp-119&
4.580&
1.833&
3.703&
1.281\tabularnewline
\hline
Val-122&
1.402&
0.279&
1.000&
0.262\tabularnewline
\hline
Ile-123&
0.829&
0.273&
2.559&
0.677\tabularnewline
\hline
&
&
&
&
\tabularnewline
\end{tabular}\tabularnewline
\hline
2DBO&
0.292&
0.312 (0.005)&
0.271&
2.01&
1.10&
\begin{tabular}{ccccc}
&
&
&
&
\tabularnewline
\hline
Asn-84&
0.696&
0.286&
1.710&
0.147\tabularnewline
\hline
Val-85&
0.598&
0.210&
3.202&
1.073\tabularnewline
\hline
Lys-86&
2.160&
0.901&
0.622&
0.153\tabularnewline
\hline
Lys-87&
6.821&
0.456&
3.830&
0.451\tabularnewline
\hline
Arg-89&
0.814&
0.136&
7.495&
1.480\tabularnewline
\hline
Arg-90&
1.739&
0.394&
2.174&
0.442\tabularnewline
\hline
Pro-91&
0.850&
0.220&
1.273&
0.522\tabularnewline
\hline
Ser-92&
0.396&
0.173&
0.576&
0.189\tabularnewline
\hline
&
&
&
&
\tabularnewline
\end{tabular}\tabularnewline
\hline
\end{tabular}
\end{sideways}\end{center}

\label{ECtable}
\end{table}

\section{Concluding Discussion}

The problem of automated crystallographic refinement is interesting,
challenging and has significant immediate practical relevance. An
automated solution for building single conformer models from approximate
spatial restraints, combined with an automated method to explore heterogeneity,
will allow the crystallographic community to revisit and annotate
the entire {\small \textsc{pdb}\xspace} with structural variability information.
Such information will have an impact on all analyses that rely on
accurate coordinate information, like in-silico ligand design and
binding, non-covalent interactions and sequence-structure conservation,
etc. It will also significantly change the understanding of crystalline
state and protein flexibility, benefitting the refinement process.
The main components for successful heterogeneity annotation are reliable
construction of single-conformer models and reliable estimation of
heterogeneity that is predominantly seen in loops. This work has attempted
to develop methods to that end.

Application of the {\small \textsc{rapper}\xspace} approach to crystallographic
refinement (\cite{rapperKnowledgeXray}, \cite{rapperLowResolution})
has the primary benefit of crossing the energy barriers in a non-random
manner, based on knowledge-based sampling instead of kinetic sampling.
As described in \cite{rappertk}, {\small \textsc{rapper}\xspace} has been reformulated
as {\small \textit{Rapper}tk\xspace} recently, creating possibilities of applying
knowledge-based sampling in many different ways. In this work, we
showed that {\small \textit{Rapper}tk\xspace} can be used to automatically refine
the whole protein structure starting from reliable positional restraints
on mainchain and sidechain. This benchmarked its performance vis-a-vis
{\small \textsc{rapper}\xspace} as reported by \cite{rapperKnowledgeXray} for a
similar task. Then we showed that by efficient use of available restraints,
single loops can be modelled in protein structures to native-like
quality with few positional restraints. The efficient use of available
restraints was a result of symmetry-related clashchecking, restraint
propagation using loop anchors and sampling from both anchors simultaneously.
The same strategy could be extended to a more realistic problem of
a large uncertainty in loop regions and an imperfect secondary structure
framework. The {\small \textsc{cns}\xspace}-only refinements, run as controls, showed
the value added by {\small \textit{Rapper}tk\xspace} to the refinement task.

In addition to determination of single-conformer models under differing
restraint qualities, we started addressing the challenge of heterogeneity
assessment in loops. This is indeed a very difficult problem with
fundamental unknowns like number of conformers, correlations within
heterogeneity and relative occupancies. Conformational heterogeneity
can be divided into two types: the simpler sidechain-only heterogeneity
where mainchain is nearly the same and the all-atom heterogeneity
where mainchain also takes distinct conformations. The latter can
be further divided based on the extent of spatial overlap between
the conformers. Sidechain-only heterogeneity is relatively easy than
the all-atom heterogeneity because the density is likely to contain
good cues about diversity. But for overlapping conformers, a visual
inspection of density is less likely to be helpful. There are two
distinct ways to model heterogeneity, which we have termed collections
and ensembles, depending upon interdependence of member conformations.
For single-loop 2-conformer overlapping heterogeneity, we generated
both the collections and ensembles and assessed how well they modelled
the heterogeneity. The main observation was that the collection was
generally biased towards the higher electron density. The ensemble
method, due to more freedom and parameters available to it, manages
to avoid this trap and fit two distinct conformers, leading to better
modelling of heterogeneity than the collection.

Various pitfalls of the composite refinement protocol were recognized
and they need to be addressed in future. Addressing the problem of
overlapping bands will significantly increase the reliability of the
method given very approximate positional restraints, and make the
method more useful in low resolution, large uncertainty cases. Perhaps
many models can be generated for such bands and the best combination
of those models can be used. At lower resolution, use of coarse-grained
sampling (fragment sampling) may also be useful, followed by fine-grained
$\phi-\psi-\chi$ sampling.

From the heterogeneity perspective, a fundamental question to address
would be the estimation of the nature of underlying conformations
before attempting to model it because ensemble sampling must have
prior knowledge of the number and occupancies of its members. Generation
of collections seems the only way for such estimation, for which collection
modelling method will have to be modified suitably to sample within
electron density yet avoid the bias towards higher density. The main
challenge of ensemble sampling is the explosion in conformational
freedom and the work ahead will have to focus on efficiently scaling
this sampling method for larger ensemble size.

\bibliographystyle{marko}
\addcontentsline{toc}{section}{\refname}

redpajama_set_name: RedPajamaArXiv


text:
est une actrice et seiyū japonaise née le  à Ōsaka.

Biographie

Filmographie

Cinéma
 1999 : Gravitation: Lyrics of Love : Suguru Fujisaki

Télévision
 1983 : Olive et Tom (Captain Tsubasa) : Sanae Nakasawa (voix)
 1994 : Mahôjin guru guru : Nike
 1999 : Gravitation : Suguru Fujisaki
 2001 : Mahoromatic : Suguru Misato
 2004 : Kono minikuku mo utsukushii sekai : Ryou Ninomiya
 2005 : Noein : Yū Gotō

Doublage

Jeux vidéo
 Série The Legend of Zelda : Link enfant
 Grandia (Justin)

Notes et références

Liens externes

Actrice japonaise
Naissance à Osaka
Naissance en novembre 1967
Seiyū

redpajama_set_name: RedPajamaWikipedia


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text:
Utah's highest rated restaurant launches fundraiser to keep doors open
November 28, 2022 November 28, 2022 by Stuart Melling
As one known for hyperbole, let me assure you that the headline above is anything but. One of Utah's genuine best are up against the wall. News just sent my way suggests that the multi-award winning Hell's Backbone Grill are facing a very real, and very terminal existential threat.
Earlier today the restaurant took to crowd funding platform Go Fund Me seeking investment to ensure the Boulder gem stays afloat and sails for many more years to come. Owners Blake Spalding and Jen Castle are looking for an initial $324K investment – with an aim of settling up Covid-induced loans as well as securing a permanent home in the remote Southern Utah town. You can check out the full fundraiser there.
The seasonal restaurant – open March through November – is renowned for its commitment to sustainability featuring a menu built around locally produced, and seasonally appropriate cuisine. Dishes are often loaded with veg grown on one of the restaurant's two gardens and six-acre farm; eggs come from an on site pampered flock. Fruit comes from Boulder's heirloom orchards and locally ranched meat stars prominently too. The business is a true farm to table pioneer in the state. You'll find many a local business preaching the local gospel, but few deliver any further than throwing some micro greens on top of a plate and calling it good. HBG are the diametrical opposite.
Back to that headline though, and let me follow up with some cold hard facts. A little over a year ago the restaurant was recognized with an historic James Beard nod, garnering a semifinalist nomination in the Outstanding Restaurant category; that's the best of the very best for the national restaurant awards, and a first and only nomination for a Utah restaurant. No small beans.
Hells Backbone Grill (Peter Horan)
That 2021 achievement comes after a litany of previous accolades from the JBF awards (ostensibly America's answer to Michelin if you will) not least every last local publication over their multi-decade tenure. Speaking of which, Hell's Backbone has been at it for an improbable 23 years at this point, and has won the lot.
Improbable you ask? Boulder has a population of 226 and is effectively a world unto itself once the winter snow sets in. In their fundraiser the duo reckon that the town is the most remote in any of the lower 48 – I'll wager several of you are hitting up Google maps right now.
If you want to contribute to helping keep the lights on, you can do so via: https://www.gofundme.com/f/save-hells-backbone-grill-and-farm – the pot has already increased some $15,000 while I wrote this quick post.
The full note from Hell's Backbone founder's Blake Spalding and Jen Castle reads as follows:
We write to you today with reverence, humility, and immeasurable gratitude. This past week, as we've taken stock of all we're thankful for, you've been at the top of the list. You've always been at the top, ever since we opened a restaurant in a tiny town in the extreme middle of nowhere, and wondered if anyone would show up—and you showed up.
We are sorrowful to say that after 23 years, Hell's Backbone Grill and Farm is at risk of closing our doors. Throughout our 23 years as restaurateurs, we've always identified as deeply self-reliant. Even during three difficult and stressful pandemic years — and even when we received threats for advocating for the monuments — we declined offers from friends to help us fundraise for our survival. We were determined to work things out on our own. We had, after all, opened a restaurant in the most remote town in the lower 48 states–we had confidence in our ability to overcome obstacles.
But we've finally accepted that we need help. Spiraling costs and a shockingly reduced summer visitation to the whole southern Utah region have turned things upside down, and we now recognize that we can't move forward alone. So, with the support and guidance of our community, and with the continued aspirations to make people happy with our food and to be of benefit … we have started a GoFundMe.
Our restaurant's unlikely (ok: damned near impossible) success has had as much to do with your abiding support as with our commitment to the work. Nothing — not one thing — that we've accomplished over the decades could have happened without you. And so, as we try to see a path forward, we see that there is no path forward without you.
We remain optimistic for a new chapter that's brighter than we could have imagined. And we're hopeful that you'll walk through those doors with us, hand in hand. But for us to all get there, we need a whole lot of help.
Areas of Critical Need
We've set a goal that addresses three specific areas of need:
Critical need one: Debt payment
We'll be direct: we need to address the pandemic-related debt load of more than $500K that the restaurant has taken on in the last three years to weather the pandemic. As with countless other restaurants, we received loans and various relief from the federal government, which enabled us to keep our doors open and pay our staff and vendors—but the hard truth is that the loan money wasn't enough, and now those loans have come due. And after a shockingly slow summer visitation year, we're using borrowed money to make payments on borrowed money—which every grown-up knows is a bad idea.
Critical need two: A forever home for Helles
We need to move beyond renting. A down payment on the purchase of a permanent space for Helles here in Boulder would give us a much stronger positioning for a pathway forward. Having rented for 23 years, we need to find a more stable circumstance to feel good about growing older as we do this work. We're exploring several options, our favorite being the possibility of buying the building that we currently rent on the grounds of the Boulder Mountain Lodge—the same beautiful space you know and love—the building that's grown along with us.
Critical need three: Infrastructure tune-ups
If you know us, you know we've always been scrappy and tenacious. We're pros at making do and getting by on little (after all, we started HBG with $3,000 and a year's free rent), but it's not easy on our staff to work with marginal and broken equipment, and there's a whole lot that needs fixing—we're dealing with a hefty amount of deferred maintenance. We've outgrown our thrift-shop origins (and the fridges and pots and pans we've been using since we opened). Any additional revenue could go to much-needed updates to farm and kitchen infrastructure, so we can continue delivering the quality of hospitality that you've come to expect—and maybe even level up a bit.
An Intentional Goal
We've set a GoFundMe goal of $324,000, a symbolic starting point. Really, we need a lot more than that. The number isn't arbitrary—it's 3 X $108,000. In Buddhism, 108 is an auspicious and sacred number, the completion of a cycle of mantras on a mala of beads. (And as many of you know, Blake practices Buddhism, and we have always run restaurant decisions through a Buddhist filter.) This is also an amount we believe will secure our short-term survival. But to be transparent, we hold tightly in our hearts to a bigger dream than surviving. We have so many new things we want to introduce—new visions and events and experiences we're eager to create for you.
Immeasurable Gratitude
Finally, we want to say it again before we sign off: thank you. No matter what happens, thank you for showing up all of these years, and believing in us, and turning our impossible dream into a reality. Thank you for being the backbone of Hell's Backbone. For being our family. We can't express how much you mean to us.
We love you, and we love our restaurant, our monument, the town, and our amazing team. And we love the work we do, growing and preparing and serving beautiful food in a beautiful location to you beautiful people. It's an honor and a privilege, and we want to continue doing it, for as long as we possibly can.
We hope you'll share our fundraiser with your community. And if you'd prefer to make a contribution by check to our fundraising efforts, you can do so to Hell's Backbone Grill, and send it to us at PO BOX 1428, Boulder, Utah, 84716.
Again, you can find the fundraiser at – https://www.gofundme.com/f/save-hells-backbone-grill-and-farm
Featured image courtesy of Chris Michel.
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Stuart Melling
Hi, I'm Stuart, nice to meet you! I'm the founder, writer and wrangler at Gastronomic SLC; I'm also a former restaurant critic of more than five years, working for the Salt Lake Tribune. I've worked extensively with multiple local publications from Visit Salt Lake to Salt Lake Magazine, not least helped to consult on national TV shows.
I'm a multiple-award winning journalist and have covered the Utah dining scene for the better part of fifteen years. I'm largely fueled by a critical obsession with rice, alliteration and the use of big words I don't understand. What they're saying about me: "Not inaccurate", "I thought he was older", "I don't share his feelings". Want to know more? This is why I am the way I am.
This article may contain content provided by one of our paid partners. These are some of the best businesses in Utah. For a list of all our current and past relationships see our partnership history page.
Categories News Tags Blake Spalding, Hell's Backbone Grill, Jennifer Castle
Downtown Salt Lake bar bringing the Christmas fun
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2 thoughts on "Utah's highest rated restaurant launches fundraiser to keep doors open"
Probably should have stayed out of the political topics; especially the monuments. This is the age old federal governmental tactic of procuring state land with no compensation. Its a bit more, but you get the point.
I as a southern Utah resident, I would not only not give money,I would not frequent the establishment. And if you knew what you were doing you would not be paying back covid loans. Also, gofundme takes 15%, at least that is what I saw on a previous, albeit smaller request.
Oh, and the name doesn't sound lime something a Buddhist would name their restaurant.
C Olsen
As sad as this story is on Hell's backbone, they took on way too much debt for starters fully knowing where they are located and the long term results of Covid. If the restaurant cannot sustain itself, and more important, predict it's future to some degree, then it should close it's doors. True of all restaurants. Last time I looked, it is a business not a charity.

redpajama_set_name: RedPajamaCommonCrawl


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N.Y. / Region|Mosque Plan Clears Hurdle in New York
Mosque Plan Clears Hurdle in New York
By MICHAEL BARBARO and JAVIER C. HERNÁNDEZ AUG. 3, 2010
Sharif El-Gamal, chief executive of SoHo Properties, the developer of the project, center, praised the commission's decision. Credit Ozier Muhammad/The New York Times
As New York City removed the final hurdle for a controversial mosque near ground zero, Mayor Michael R. Bloomberg forcefully defended the project on Tuesday as a symbol of America's religious tolerance and sought to reframe a fiery national debate over the project.
With the Statue of Liberty as his backdrop, the mayor pleaded with New Yorkers to reject suspicions about the planned 13-story complex, to be located two blocks north of the World Trade Center site, saying that "we would betray our values if we were to treat Muslims differently than anyone else."
"To cave to popular sentiment would be to hand a victory to the terrorists — and we should not stand for that," the mayor said.
Grappling with one of the more delicate aspects of the debate, Mr. Bloomberg said that the families of Sept. 11 victims — some of whom have vocally opposed the project — should welcome it.
"The attack was an act of war — and our first responders defended not only our city but also our country and our Constitution," he said, becoming slightly choked up at one point in his speech, which he delivered on Governors Island. "We do not honor their lives by denying the very constitutional rights they died protecting. We honor their lives by defending those rights — and the freedoms the terrorists attacked."
But even as the mayor called for the mosque to be embraced, those opposed to the project pledged to aggressively fight it, using both litigation and public pressure. A prominent Republican and foreign policy analyst said he was working with business, civic and political leaders to organize a campaign to persuade architects, contractors and donors to steer clear of the project. He said they would also aggressively scrutinize any donors who supported it.
The Republican, Daniel Senor, a former high-ranking official with the coalition government in Iraq, said that anybody who works with the center "needs to know there is going to be a real stigma associated with this project."
"Do they really want to be involved with something so detrimental, that might set New York back?" he asked.
Lawyers representing a firefighter who survived 9/11 also said they would file a lawsuit on Wednesday to block the city's approval.
The plan for the complex, which began as a local zoning dispute, has quickly snowballed into an intense national debate about the nature of Islam and the meaning of the Sept. 11 attacks.
National Republican leaders, like the former House speaker, Newt Gringrich, and Sarah Palin, the 2008 vice presidential nominee, assailed the proposal, calling it offensive. On Friday, the Anti-Defamation League, an influential Jewish civil rights group, declared its opposition, distressing many in the interfaith community.
The disagreement has underscored how differently the World Trade Center site is viewed by those in New York and those outside of it.
In the city, the space has returned, haltingly, to the urban grid, sprouting new office towers and train stops. But beyond New York's borders, it looms as a powerful symbol of the war on terror and the lives lost on that day.
Those opposed to the project have argued that building a Muslim community center so close to the site where radical Muslims killed about 2,750 people is as much a political statement as a religious gesture, and have demanded that developers find a different location.
Bill Doyle, whose 25-year-old son, Joseph, was killed in the attacks, said many families who lost loved ones at the site were "incensed" about the mosque, viewing it as a tribute to the terrorists behind the hijackings. Part of their anger stems from their broader frustration over redevelopment of the site, and plans for a memorial that would be located beneath street level.
"High up in the air you have a 13-story mosque, outshining the memorial itself," Mr. Doyle said. "It's almost a slap in the face."
Proponents, eager to address the raw feelings the issue has aroused, emphasize that the center would focus on interfaith dialogue, include members of other religions on its board, and feature what its developer called "a Sept. 11 memorial and contemplation space."
Sharif El-Gamal, the developer, said he was reaching out to elected officials, community groups and opponents to directly explain the center's mission and allay any fears about it.
But it has been challenging. On Tuesday, at the final Landmarks Preservation Commission hearing on the project, Mr. El-Gamal said he tried to introduce himself to a Republican candidate for governor, Rick A. Lazio, a vocal opponent of the project.
"He just looked at me and walked by," Mr. El-Gamal said.
On Tuesday morning, the project received its final city approval when the landmarks commission voted 9 to 0 to deny granting historic protection to the building in Lower Manhattan where the $100 million center would be constructed.
Opponents, who have turned out in large numbers to speak out against the plan at public hearings this summer, seemed resigned to losing the vote, and few showed up to protest. After the commission voted, scattered members of the audience shouted "Shame on you!" and "Disgrace!" One woman carried a sign reading, "Don't Glorify Murders of 3,000; No 9/11 Victory Mosque."
There were signs that the intense backlash had left moderate American Muslims uneasy about the plan for such a large center near ground zero.
"There is some ambivalence within the community," said Hussein Rashid, a visiting professor of religious studies at Hofstra University who specializes in Islam in America. "We still want to know who is going to be involved in this. So far, we have heard from just a few Muslim voices. If this is meant to be a community center, who in the community will be involved?"
The mosque presented a potentially challenging political situation for Mr. Bloomberg. Some of his most fervent supporters — religious Jews, working-class whites in the boroughs outside Manhattan and conservative Republicans — opposed the idea.
But Mr. Bloomberg loathes what he sees as old-school ethnic politics. And from the start, he viewed it as a simple question of law and American values, privately expressing consternation at those who sought to block the project. As the fight over the proposal intensified, he encouraged aides to find an occasion for him to deliver a major speech on the topic.
Even some of his friends disagreed with him. But behind the scenes, Mr. Bloomberg was heartened to hear that some of the families of Sept. 11 victims supported his position, and told him so at a fund-raiser for the memorial at the site two weeks ago, aides to the mayor said.
Last Friday, he became all the more determined to speak out after he learned that the Anti-Defamation League, which for weeks has denounced what it saw as bigoted attacks on the planned Muslim center, abruptly announced its opposition.
Mr. Bloomberg was surprised and disappointed, these people said. On Tuesday, when asked about the group's position, the mayor called it "totally out of character with its stated mission."
Mr. Bloomberg generally leaves most speechwriting to his aides, but he became heavily involved in writing his defense of the mosque, revising it until just moments before it was delivered. And he enthusiastically agreed that the speech should be given on Governors Island, in sight of the Statue of Liberty.
"We have come here to see the inspiring symbol of liberty that, 250 years later, would greet millions of immigrants in the harbor," he said, "and we come here to state as strongly as ever — this is the freest city in the world. That is what makes New York special and different and strong."
A version of this article appears in print on August 4, 2010, on Page A1 of the New York edition with the headline: Mosque Plan Clears Hurdle In New York. Order Reprints| Today's Paper|Subscribe


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namespace Aspose.Email.Examples.CSharp.Email.POP3
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redpajama_set_name: RedPajamaGithub


text:
The son of Neptune
Did You Check eBay? Fill Your Cart With Color Today Written by Rick Riordan, Narrated by Joshua Swanson. Download Now! Plus Listen All You Want To Exclusive Podcasts, Audiobooks, Audible Originals & More The Son of Neptune is a 2011 fantasy - adventure novel written by American author Rick Riordan, based on Greek and Roman mythology The Son of Neptune In The Lost Hero, three demigods named Jason, Piper, and Leo made their first visit to Camp Half-Blood, where they inherited a blood-chilling quest: Seven half-bloods shall answer the call, To storm or fire the world must fall
Son Of Neptune Book - Amazon Official Sit
The Son of Neptune was a thrilling, exciting, crazy, and absolutely bizarre adventure, and I loved every second of it
The Son of Neptune is the second book in The Heroes of Olympus series written by author Rick Riordan. It was released on October 4 th, 2011, a week earlier than originally planned
The Son of Neptune (The Heroes of Olympus, #2) by Rick Riordan Goodreads helps you keep track of books you want to read. Start by marking The Son of Neptune (The Heroes of Olympus, #2) as Want to Read
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Having been protected by Percy during the attack, Juno announces Percy's arrival with approval, identifying him as a son of Neptune
THE SON OF NEPTUNE The Kane Chronicles series: THE RED PYRAMID THE THRONE OF FIRE www.rickriordanmythmaster.co.uk. To Becky, who shares my sanctuary in New Rome. Even Hera could never make me forget you. Map 1 Camp Jupiter. I. Percy THE SNAKE-HAIRED LADIES WERE starting to annoy Percy
The Son of Neptune (graphic novel) is the comic book adaptation of the The Son of Neptune. It was published on February 22, 2017 by Hyperion Books Percy is back! And on a quest with Roman demigods. Read Common Sense Media's The Son of Neptune: The Heroes of Olympus, Book 2 review, age rating, and parents guide The Son of Neptune (2011), a young adult fantasy novel by Rick Riordan, is the second entry in the five-book Heroes of Olympus series, which succeeds Riordan's Percy Jackson and the Olympians series ― Rick Riordan, The Son of Neptune tags: customs , declare , mental-pat-down , no-arms , percy-jackson , terminus 1713 like The Son of Neptune by Rick Riordan. Publication date 2011 Topics Hera (Greek deity), Gaia (Greek deity), Greek Mythology, Juvenile fiction, Roman Mythology, Fiction, Prophecies, Camps, Monsters Publisher Disney * Hyperion Books Collectio
Heroes of Olympus Complete Collection 5 Books Box Set -The Lost Hero/The Son of Neptune/The Mark of Athena/The Blood of Olympus by Rick Riordan (2015-06-07) by Riordan Rick | Jan 1, 2015. 4.9 out of 5 stars 62. Paperback A Widow's Guide to Scandal (The Sons of Neptune Book 1) by. Finally my son of Neptune trailer is finished so happy!!! sorry it took so long but it was necessary. i'll be doing sea of monsters trailer now so i can have.. A vocabulary list featuring The Son of Neptune by Rick Riordan, Chapters 1-12. Despite suffering from amnesia, Percy Jackson manages to make his way to Camp Jupiter, where he enlists the help of two new friends to help him on a quest. Here are links to our lists for the book: Chapters 1-12, Chapters 13-24, Chapters 25-36,.. the Son of Neptune. 3 The snake-haired ladies were starting to annoy Percy. They should have died three days ago when he dropped a crate of bowling balls on them at the Napa Bargain Mart. They should have died two days ago when he ran over them with a police car in Martinez
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The Son of Neptune (The Heroes of Olympus #2) Seven half-bloods shall answer the call, To storm or fire the world must fall. An oath to keep with a final breath, And foes bear arms to the Doors of Death. Percy is confused. When he awoke from his long sleep, he didn't know much more than his name. His brain fuzz is lingering, even after the wolf. The official fan made trailer for Percy Jackson The Son Of Neptune The Son of Neptune Audiobook Online. Percy manages to range from them as well as comes across an old woman. The lady presents herself as June as well as tells him that he is a demigod. June offers him a selection: Percy can escape to the sea, where he will certainly live a satisfied life, or he can bring her to Camp Jupiter The Son of Neptune; Page 1. The Son of Neptune. Page 1. I Percy THE SNAKE-HAIRED LADIES WERE starting to annoy Percy. They should have died three days ago when he dropped a crate of bowling balls on them at the Napa Bargain Mart. They should have died two days ago when he ran over them with a police car in Martinez The Heroes of Olympus 2 - The Son of Neptune Audiobook Free download and listen The plot of The Son of Neptune Audiobook Free. Percy is confused. When he awoke from his long sleep, he didn't know much more than his name. His brain fuzz is lingering, even after the wolf Lupa told him he is a demigod and trained him to fight
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The Son of Neptune Page 2 read online free - Novels80 Maybe if he just fell down the mountainwould he survive? He didn't want to risk it—not without something to slow the fall, or a sled, or He looked at Stheno's large silver platter of free samples
What listeners say about The Son of Neptune. Average Customer Ratings. Overall. 5 out of 5 stars 4.8 out of 5.0 5 Stars 46 4 Stars 7 3 Stars 1 2 Stars 0 1 Stars 0 Performance. 4.5 out of 5 stars 4.6 out of 5.0 5 Stars 30 4 Stars 7 3 Stars 2 2 Stars 0.
The Son of Neptune Summary. Percy Jackson is a Greek demigod who has lost his memory and wandered into a Roman demigod training camp. His father is Neptune, the Roman sea god. Also known as Poseidon, the Greek sea god. Percy has been in the wilderness for some time under the protection of Luna, the goddess protector of Rome
The Son of Neptune. The Heroes of Olympus. by Rick Riordan Store Select Amazon Apple Books Barnes & Noble Books-A-Million Bookshop Google Play Indigo Kobo Available Formats ebook & print Buy Now Percy has no memory. Hazel is supposed to be dead. Frank is a klutz. With heroes like these, what could possibly go wrong?.
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The Son of Neptune (The Heroes of Olympus Series #2
The Son of Neptune: The Graphic Novel (Heroes of Olympus Series #2) by Rick Riordan, Robert Venditti, Antoine Dode (Illustrator), Orpheus Collar (Illustrator About The Heroes of Olympus, Book Two: The Son of Neptune. Percy is confused. When he awoke from his long sleep, he didn't know much more than his name. His brain fuzz is lingering, even after the wolf Lupa told him he is a demigod and trained him to fight. Somehow Percy manages to make it to a camp for half-bloods If that wasn't spectacular enough, the old lady Percy is carrying transforms into the goddess Juno and disappears, leaving him with a bunch of astonished Roman warriors. Their leader, a fierce-looking girl named Reyna, steps forward. She calls him a son of Neptune, god of the sea, and orders him to come with her Read The Son of Neptune The Son of Neptune (The Heroes of Olympus #2) Seven half-bloods shall answer the call, To storm or fire the world must fall He is the son of Ares, the god of war. Frank is a little pessimistic because of his low self-esteem. But Frank is a great battle commander and courageous. Franks's weapon is a bow and arrow and occasionly a spear. His last name means Master of Bows so it is evident that he has amazing accuracy using a bow and arrow. Frank also has the power to.
The Son of Neptune - read free eBook by Rick Riordan in online reader directly on the web page. Select files or add your book in reader Reading the Son of Neptune by inspiremyworld95. Books » Percy Jackson and the Olympians Rated: T, English, Family & Friendship, Annabeth C., Jason G., Words: 43k+, Favs: 963, Follows: 1k+, Published: 1/31/2012 Updated: 7/31/2012} 223 Chapter 1: Prolouge. A/N-Hi guys! I wanted to try this out! Tell me how it is The Son of Neptune Audiobook - Rick Riordan (Heroes of Olympus, Book 2). The quest needs to succeed by the Banquet of Fortuna in four days' time.[/expand The Son of Neptune Summary & Study Guide Rick Riordan This Study Guide consists of approximately 49 pages of chapter summaries, quotes, character analysis, themes, and more - everything you need to sharpen your knowledge of The Son of Neptune The Son of Neptune by Rick Riordan (2011, Hardcover) $5.25 + shipping. The Son of Neptune By Rick Riordan Hardcover- Brand New. $11.00. Free shipping. The Son of Neptune [Heroes of Olympus, Book 2] $5.99 + $3.45 shipping. The Son of Neptune by Rick Riordan - Paperback - LIKE NEW. $4.0
The answer may lie in another camp miles away, where a new camper has shown up and appears to be the son of Neptune, god of the sea. . . . If the item details above aren't accurate or complete, we want to know about it. Report incorrect product info. Show more. Shipping options Apr 21, 2014 - Explore Winnie the Weird Wallflower's board The Son of Neptune, followed by 908 people on Pinterest. See more ideas about son of neptune, the heroes of olympus, percy jackson Apr 12, 2015 - Explore Percy Jackson's board The Son Of Neptune on Pinterest. See more ideas about son of neptune, percy jackson, percy jackson fandom Read The Son of Neptune from the story Different War by MsPenguingirl1234 (Mary Jane) with 2,904 reads. past, percyjackson, annabethchase. Last time on Differe.. Read novel online: The Son of Neptune - Page 61. Amazons, forward! On the largest forklift stood a girl who looked like an older version of Reyna, in black combat armor with a glittering gold belt around her waist
The Son of Neptune (eBook) : Riordan, Rick : Percy is confused. When he awoke after his long sleep, he didn't know much more than his name. His brain-fuzz is lingering, even after the wolf Lupa told him he is a demigod and trained him to fight. Somehow Percy managed to make it to the camp for half-bloods, despite the fact that he had to continually kill monsters that, annoyingly, would not. The Son of Neptune Rick Riordan No preview available - 2019. About the author (2011) Rick Riordan is an award-winning mystery writer. He lives in San Antonio, Texas, with his wife and two sons. Percy Jackson and the Lightning Thief, Rick's first novel featuring the heroic young demigod, was the overall winner of the Red House Children's Book.
Follow/Fav The Son of Neptune. By: CombatTombat. Born to Neptune, and raised by Lupa, Perseus Jackson is fated to have a long, difficult life, but also one full of victory, glory, and success. Witness the rise of the man who shaped the future of the godly world, both Roman and Greek. AU, Roman!Percy, and Preyna The son of Neptune wants a boat. Sea travel has never been the Roman way, but he isn't much of a Roman! Octavian, Reyna said sternly, a boat is little enough to ask To betray the Rome we love so much, for a son of Neptune, a god who we do not trust! Before anyone could act, Percy had sprinted across the room and driven his fist so hard into Octavian that a loud ' Crack!' was heard. The augur's head snapped to the side, and he fell, motionless. All looked at Percy, whose face was contorted in rage The Son of Neptune (eBook) : Riordan, Rick : Demigod Percy Jackson, still with no memory, and his new friends from Camp Jupiter, Hazel and Frank, go on a quest to free Death, but their bigger task is to unite the Greek and Roman camps so that the Prophecy of Seven can be fulfilled The Son of Neptune is the second book in The Heroes of Olympus series written by author Rick Riordan.It was released on October 4th, 2011, a week earlier than first planned. The story takes place around six months after the previous book, The Lost Hero
The Son of Neptune (Book) : Riordan, Rick : Demigod Percy Jackson, still with no memory, and his new friends from Camp Jupiter, Hazel and Frank, go on a quest to free Death, but their bigger task is to unite the Greek and Roman camps so that the Prophecy of Seven can be fulfilled The answer may lie in another camp miles away, where a new camper has shown up and appears to be the son of Neptune, god of the sea. Also listen to the first book, The Lost Hero. ©2011 Rick Riordan (P)2011 Listening Library More from the sam The gods—and a few demigods—read Percy Jackson and the Olympians and Heroes of Olympus! Rick Riordan owns the characters. Lucky guy. (Picture will change based on what book I am on) : Step - By Step To Download/Read online [PDF] The Son of Neptune: The Graphic Novel (The Heroes of Olympus: The Graphic Novels, #2) : 1. Click Download or Read Online Button 2. Sign Up to Access [PDF] The Son of Neptune: The Graphic Novel (The Heroes of Olympus: The Graphic Novels, #2) 3. Download/Read online as Many Book as You Like 4
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The Son of Neptune: The Graphic Novel PDF book (The Heroes of Olympus: The Graphic Novels) (The Heroes of Olympus: The Graphic Novels Series) Read Online or Free Download in ePUB, PDF or MOBI eBooks. Published in February 21st 2017 the book become immediate popular and critical acclaim in sequential art, graphic novels books The son of neptune Answers Epictortilla said: Technically no, he is about to as the book ends with the cliff hanger of him introducing his Roman friends to his other family Books : The Son of Neptune (Heroes of Olympus Book 2) (Paperback) This is the second title in this number one bestselling spin-off series from 'Percy Jackson' creator Rick Riordan. This crazy messed up world of gods and monsters is 'Percy Jackson's' reality which pretty much sucks for him
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The Son of Neptune by Rick Riordan The Red Pyramid Characters Hazel Frank The Last Olympian by: Kirsten Cowan The Son of Neptune The Mark of Athena coming Fall of 2012 The Percy Jackson series The Lightning Thief The Kane Chronicles: The Sea of Monsters The Titans Curse Th The Son of Neptune. Page 41 Circe's Island, Percy said. I just remembered. The gorgon's blood—maybe it's starting to heal my mind. TheSea of Monsters. Hyllashe welcomed us at the docks, took us to see her boss
Books related to Heroes of Olympus: The Son of Neptune. Skip this list. More by Rick Riordan Skip this list. Ratings and Book Reviews (60 614 star ratings 60 reviews ) Overall rating. 4.6 out of 5. 4.55. 614. 5 Stars 438 reviews have 5 stars 4 Stars 121 reviews. Heroes of Olympus: The Son of Neptune by Rick Riordan. The easy way to get free eBooks every day. Discover the latest and greatest in eBooks and Audiobooks. Heroes of Olympus: The Son of Neptune by Rick Riordan. Popular Free eBooks! South Ernest Shackleton 55 4; The Keto Diet For Weight Los
The Son of Neptune Graphic Novel is the adaptation of The Son of Neptune into a graphic novel. Camp Half-Blood Chronicles - Wikipedia It is preceded by The Last Olympian of Percy Jackson & the Olympians and followed by The Son of Neptune Before the Son of Neptune transpired, Hazel was forced to live in Alaska by Gaea, or mother nature. When she escpaed, Frank, Hazel, and Percy traveled to Alaska to free the god of death, Thanatos. Canada. Northern Vancouver. This is were Frank, Percy, and Hazel fleeded by to when they were attacked by the Giant. Frank is Candian and his. the son of neptune 3.5M views Discover short videos related to the son of neptune on TikTok. Watch popular content from the following creators: Jamie(@jamietmarie), Libby Wilbur(@punziespalace), Abbie S(@abbie.notabby), Ria ��(@alldaladiesluvria), sinéad(@sineadrobbins) The Son of Neptune (Book) : Riordan, Rick : Demigod Percy Jackson, still with no memory, and his new friends from Camp Jupiter, Hazel and Frank, go on a quest to free Death, but their bigger task is to unite the Greek and Roman camps so that the Prophecy of Seven can be fulfille
The Son of Neptune - Wikipedi
The Son of Neptune. Page 17 Frank laughed. Yeah, wellit's harder than it sounds. We have to get past those scorpions and water cannons on the walls, fight through the inside of the fortress, find the banners, and defeat the guards, all while protecting our own banners and troops from capture. And our cohort is in competition with the. High quality The Son Of Neptune gifts and merchandise. Inspired designs on t-shirts, posters, stickers, home decor, and more by independent artists and designers from around the world. All orders are custom made and most ship worldwide within 24 hours The Son of Neptune is by Rick Riordan. It's the 2nd book in the Heroes of Olympus series. Overall, I liked the way Rick Riordan wrote The Son of Neptune. It was intense at points, but he always.
Video: The Son of Neptune Rick Riorda
Video: The Son of Neptune (Heroes of Olympus, Book 2): Riordan
Book 2: The Son of Neptune (Heroes of Olympus) (31) Book 1: The Lost Hero (Heroes of Olympus) (7) Alternate Universe - Canon Divergence (5) Book 3: The Mark of Athena (Heroes of Olympus) (5) Book 4: The House of Hades (Heroes of Olympus) (5) Book 5: The Blood of Olympus (Heroes of Olympus) (5) Angst (4 Buy The Son of Neptune by Rick Riordan online at Alibris. We have new and used copies available, in 4 editions - starting at $1.19. Shop now The Son Of Neptune In The Lost Hero, three demigods named Jason, Piper, and Leo made their first visit to Camp Half-Blood, where they inherited a blood-chilling quest: Seven half-bloods shall answer the call, To storm or fire the world must fall
The Son of Neptune Riordan Wiki Fando
Title: The son of neptune Rating:Umm something like K Type:Suspence, Adventure, friendship a bit romance,mystery Characters: Percy Jackson, Reyna, Bobby, Dakota, Hazel, Gwen, annabeth Chase, Jason Grace, Piper, Leo some other OC and maybe a bit chars from PJO and of course Mrs.o'lear The Son of Neptune book by Rick Riordan Teen & Young Adult Books > Teen Fantasy Books The Heroes of Olympus, Book Two the Son of Neptune (Heroes of Olympus, the, Book Two) (Part of the The Heroes of Olympus (#2) Series and Camp Half-Blood Chronicles (#7) Series The Son of Neptune (The Heroes of Olympus Book 2) Author: Rick Riordan Publisher: Hyperion Publishing date: 2011 Read the review for The Lost Hero (The Heroes of Olympus Book 1) The Idea. Percy has no memory of his life when he comes across Camp Jupiter The Son of Neptune opens with Percy Jackson being chased by two gorgons. While making his escape, he runs into an old woman named June. The old woman reveals herself to be Juno (Hera) and gives him a choice: provide her with a favor or run away. As a favor, Percy carries her across the Little Tiber, which is a powerful river In The son of Neptune we know that there are two camps one is Greek and one is Roman. Strange things conspire when one tries to cheat fate The Gods keep them separated to avoid any clashes, but a huge threat is prevailing the world and the Gods are too stubborn to realize that
The Son of Neptune (The Heroes of Olympus, #2) by Rick Riorda
The Son of Neptune is the second book of the series The Heroes of Olympus. The first book was The Lost Hero. It was published on October 4th, 2011. These books were written by RIck Riordan The Son of Neptune. The Son of Neptune. Author: Rick Riordan Category: Fantasy Series: The Heroes of Olympus #2 Pages: 129 Status: Update Views: 36
The Son of Neptune (The Heroes of Olympus #2) - Rick
THIS IS AMAZING AND CUTE!!! (I say that about LITERALLY everything on this website cuz' everything is so cute and cool!) also, no joke, this is my profile pic for my phone number XD!! The son of neptune, p.1. The Son of Neptune, page 1 part #2 of The Heroes of Olympus Series . Select Voice: Brian (uk) Emma (uk) Amy (uk) Eric (us) Ivy (us) Joey (us) Salli (us) Justin (us) Jennifer (us) Kimberly (us) Kendra (us) Russell (au) Nicole (au) Try our free service - convert any of your text to speech!.
RR - The Son of Neptune
Neptune (Latin: Neptūnus [nɛpˈtuːnʊs]) is the god of freshwater and the sea in Roman religion.He is the counterpart of the Greek god Poseidon. In the Greek-influenced tradition, Neptune is the brother of Jupiter and Pluto; the brothers preside over the realms of Heaven, the earthly world, and the Underworld. Salacia is his wife.. Depictions of Neptune in Roman mosaics, especially those of. The Son of Neptune (Heroes of Olympus Book 2): Amazon.co.uk: Riordan, Rick: Books Select Your Cookie Preferences We use cookies and similar tools to enhance your shopping experience, to provide our services, understand how customers use our services so we can make improvements, and display ads, including interest-based ads The Son of Neptune is the second book in Rick Riordan's Heroes of Olympus series, released here in the Philippines with relatively little fanfare last October 4, 2011.. Camp Jupiter finds itself reluctantly welcoming in Percy Jackson, a 16-year-old amnesiac demigod under Juno's protection. He's trained though, in a style that no one's ever seen before The Son of Neptune Riordan, Rick AR Quiz No. 146841 EN Percy and his new friends from Camp Jupiter go on a quest to free Death, but their bigger task is to unite the Greek and Roman camps so the Prophecy of Seven can be fulfilled
The Heroes of Olympus: The Son of Neptune - IMD
Read The Son of Neptune Page 57 novel online free. The Son of Neptune (The Heroes of Olympus #2) is a Fantasy novel by Rick Riordan The Son of Neptune 1st ed. This edition was published in 2011 by Disney * Hyperion Books in New York The Son of Neptune (New Cover): 2 (The Heroes of Olympus, 2) by Riordan, Rick. $14.94. Free shipping. Seller 97.4% positive. The Son of Neptune [Heroes of Olympus, Book 2] , Hardcover , Riordan, Rick. $4.50. Free shipping. Seller 99.3% positive. The Cat in the Hat by Dr. Seuss Hardcover Book NEW Frank's gift is a spear made of dragon-tooth. When he plants it in the ground a skeleton warrior appears. As Percy is Son of Neptune, his power is that he can control water. This is my favourite of..
The son of Neptune Item Preview > remove-circle Share or Embed This Item. Share to Twitter. Share to Facebook. Share to Reddit. Share to Tumblr. Share to Pinterest. Share via email. EMBED EMBED (for wordpress.com hosted blogs and archive.org item <description> tags) Want more? Advanced embedding details, examples, and help. This eBook set brings together the first three volumes in the wildly popular Heroes of Olympus series: The Lost Hero, The Son of Neptune, and The Mark of Athena. Longtime fans will want this collection and so will new readers just diving into the series. The Son Of Neptune Heroes Of Olympus Book 2 by Rick Riorda ePub: The Heroes of Olympus Book Two The Son of Neptune The Graphic Novel Will miss this series after the last one. The second book is called A Moment in Time and the third is A Matter of Heart. Plus, the craft s Son, easy, and inexpensive: all you need is wool, soap, water, you The Son of Neptune (Book) : Riordan, Rick : After three demigods named Jason, Piper, and Leo visit Camp Half-Blood, they inherit a quest: to free Hera, a prisoner of Gaia. But who are the other four demigods mentioned in prophesy who will join them in their quest? The answer may lie in another camp miles away, where a new camper has shown up and appears to be the son of Neptune, god of the sea Try this amazing The Son Of Neptune quiz which has been attempted 26 times by avid quiz takers. Also explore over 3 similar quizzes in this category
The Son of Neptune. In The Lost Hero, three demigods named Jason, Piper, and Leo made their first visit to Camp Half-Blood, where they inherited a blood-chilling quest: Seven half-bloods shall answer the call, To storm or fire the world must fall. An oath to keep with a final breath, And foes bear arms to the Doors of Death —Rick Riordan, The Son of Neptune Part of their problem was Percy. He fought like a demon, whirling through the defender's ranks in a completely unorthodox style, rolling under their feet, slashing with his sword instead of stabbing like a Roman would, whacking campers with the flat of his blade, and generally causing mass panic Percy is confused. When he awoke after his long sleep, he didn't know much more than his name. His brain-fuzz is lingering, even after the wolf Lupa told him he is a demigod and trained him to fight. Somehow Percy managed to make it to the camp for half-bloods, despite the fact that he had to continually kill monsters that, annoyingly, would not stay dead The u/_Son_of_Neptune_ community on Reddit. Reddit gives you the best of the internet in one place
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Hilarious moment when black girl wakes up after surgery and thinks she is white goes viral
By Maria Varela
https://news.amomama.com/109769-hilarious-moment-black-girl-wakes-surger.html
A 16-year-old African-American girl who wakes up from surgery is cracking up the internet with her reaction when thinks she's no longer black.
Faith Martin had just undergone surgery and was clearly still high on her medication when she was caught on video waking to a hilarious realization - she thought she'd become white.
In the clip that's immediately gone viral thanks to her brother who shot the video and could be heard cracking up in the background, Faith appears to be in a daze as she tells her mother "I want to be black."
She begins to compare her skin to her relatives then starts dancing while still lying on her hospital bed. Her brother is amused and can't help but chuckle while filming her.
Faith then looks at her mother, lovingly holds her face and tells her,
"'We're not the same color anymore."
The room suddenly breaks into laughter watching the teen in her bizarre, groggy state. They tell her to look at her mother's hand to compare with hers but she stubbornly insists they're not the same by pursing her lips and shaking her head despite her mother telling her, "We the same color."
Her brother described the hilarious incident in his caption.
"My sister got her surgery and she woke up higher than the Himalayas. I guess the medication had her still intoxicated so she started hallucinating claiming her skin to be white instead of black."
It would be safe to assume Faith would later realize her color never changed once her system is clear of medication. This was also the case with an Atlanta teen who woke up after a coma speaking fluent Spanish instead of his native English. 16-year-old sophomore Reuben Nsemoh surprised his parents when he started speaking like a Spanish native and lost his ability to speak English after suffering from a concussion that caused him to go into a coma. According to CNN, Reuben acquired what is known as a foreign accent syndrome which is a rare condition brought about by brain injuries affecting a person's speech patterns.
Story of teen who sang during brain surgery to save her musical abilities still melts hearts
Photos of white influencer went viral in 2018 after criticism for 'pretending to be Black'
Teen goes to bed with headache & falls into coma, wakes up 4 days later with 'surprise' baby
A few dozen cases of foreign accent syndrome have been reported through the years after its first case recorded in 1941. Three years ago, a Navy vet woke up in a Southern California hotel with no memory of his life and speaking only Swedish. In Australia, a bus driver came out of a serious crash with a French accent. In 2016, a Texas woman started speaking with a British accent after having surgery on her jaw.
Experts say these cases are an impairment of the motor control and can be quite complicated since speech involves a coordination of a lot of brain centers to control a lot of moving parts. If one or more of these parts are damaged, timing, melody, and tension of speech may be affected.
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