text:
We introduce a statistical method for evaluating atomic level 3D interaction patterns of protein-ligand contacts. Such patterns can be used for fast separation of likely ligand and ligand binding site combinations out of all those that are geometrically possible. The practical purpose of this probabilistic method is for molecular docking and scoring, as an essential part of a scoring function. Probabilities of interaction patterns are calculated conditional on structural x-ray data and predefined chemical classification of molecular fragment types. Spatial coordinates of atoms are modeled using a Bayesian statistical framework with parametric 3D probability densities. The parameters are given distributions a priori, which provides the possibility to update the densities of model parameters with new structural data and use the parameter estimates to create a contact hierarchy. The contact preferences can be defined for any spatial area around a specified type of fragment. We compared calculated contact point hierarchies with the number of contact atoms found near the contact point in a reference set of x-ray data, and found that these were in general in a close agreement. Additionally, using substrate binding site in cathechol-O-methyltransferase and 27 small potential binder molecules, it was demonstrated that these probabilities together with auxiliary parameters separate well ligands from decoys (true positive rate 0.75, false positive rate 0). A particularly useful feature of the proposed Bayesian framework is that it also characterizes predictive uncertainty in terms of probabilities, which have an intuitive interpretation from the applied perspective.
Citation: Hakulinen R, Puranen S, Lehtonen JV, Johnson MS, Corander J (2012) Probabilistic Prediction of Contacts in Protein-Ligand Complexes. PLoS ONE 7(11): e49216. https://doi.org/10.1371/journal.pone.0049216
Editor: Peter Csermely, Semmelweis University, Hungary
Received: August 16, 2012; Accepted: October 5, 2012; Published: November 14, 2012
Copyright: © 2012 Hakulinen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Academy of Finland grants no. 121301, 251170 and ERC grant no. 239784. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Atomic level structures are an important source of information for inferring functional aspects about macromolecules and ligands binding to them. For instance, this is illustrated by the substantial amount of existing algorithms and structural data modeling software created for molecular docking and scoring purposes [1], [2], [3], [5], [6]. The Protein Data Bank (PDB) [7] offers the central public access to macromolecular structure files.
Although there is already a large amount of structural data available, it is by no means straightforward to model it reliably. There are several reasons for this, such as the inevitable errors present in experimental results and the "averaging" nature of the measurement process used in the construction of x-ray diffraction data. Moreover, along the conversion from a measurement to a structural coordinate file, several computational approximations and the subjective choices of experimentalists will influence the final outcome. Among the latter sources of variability, two major issues are flexibility of the molecules and computational constraints implemented in the refinement process. The first one is related to thermal motion and static disorder, and the second to biochemical a priori information that is always used in the refinement of a structure to create a coordinate file [6], [8]. These are accompanied by crystal packing effects, which also originate from the flexibility of the molecules, uncertainty in orientation and location of small molecules, including water.
It can be argued that for addressing the above-mentioned issues, statistical modeling provides the most promising approach, given its ability to capture uncertainties and errors in data. To meet these goals we introduce a Bayesian statistical method for evaluating atomic level 3D interaction patterns of protein-ligand contacts. Our work is motivated by the previous findings in Rantanen et al., [1], [2], [3] which showcased the usefulness of this kind of a multidisciplinary approach. However, given computational speed related constraints, it has not been possible to pursue these previous Bayesian methods further in contact preference exploration. Therefore, the method discussed here focuses on providing rapid means of computing, together with adjustability and robustness of the statistical model. The latter aspect refers in this context firstly to the constraint that two points in close proximity to each other (with respect to the system size) should not get very different contact preference hierarchies without an easily tractable reason. Secondly, in terms of robustness, the preference prediction model has to be balanced between adhering too closely to the possibly biased overall number of different types of contact atoms in the training data set and using a sole comparison of the probability densities defined for each contact atom type of a molecular fragment. Finally, the adjustability is concerned with both the model structure and the chemistry-based classification of molecular fragments. In our illustrations we consider 24 molecular fragment and 13 contact atom types, exhibiting interactions like hydrogen bonding, dispersion (e.g. aromatic-aromatic) and interactions between charged groups.
The main purpose of this paper is to show how a Bayesian statistical modeling approach can be utilized to make naturally ranked predictions about contact preferences, such that the model itself can be flexibly updated in the presence of novel data and other auxiliary information. Basically, this method is developed to retrieve information to be used in a knowledge-based scoring function. There exist several well performing scoring functions [9], [10] that utilize the experimental knowledge through inverse Boltzmann relation from statistical thermodynamics [11]. These functions depend only on distance between atoms, e.g., a ligand atom and a binding site atom. Our method differs from them in that also directional information is incorporated in the model, which has been shown in case of hydrogen bonding to still significantly improve evaluation of binding energetics from experimental data [12].
Three basic scoring function tasks have been defined [9], of which enrichment of ligands was tested with our method. The test was done through separating catechol-O-methyltransferase (COMT) ligands from decoys using logistic regression on a set of 27 small molecules having similar properties. The receiver operating characteristics (ROC) [13] for the results show that the probabilistic contact preferences give reliable information about the relative affinities in intermolecular contacts. These probabilities can be applied to intramolecular contacts as well. In practice, they are used as part of a molecular docking and scoring routine. The method described in this paper will be integrated as a functionality in the molecular modeling environment BODIL [4].
The structure of the article is as follows. First, data collection and the modeling approach are described. Thereafter, results from several case-studies are presented. Last, implications of the results and some future prospects are discussed.
Materials and Methods
0.1 Data collection and processing
We used PDB as the main source of data in this work. Training data for the model was collected from a set of approximately 28000 structure files published before January 2009. The files were selected using the criteria presented in Table 1. A reference dataset for model validation was selected under the same criteria as the training set and contained 10361 structure files published between February of 2009 and August 2011. X-ray structures form the biggest group of data present in PDB. The bulk of a structure file is the coordinate section, but there is also chemical and biological information, interpreted as metadata, which is necessary for constructing a sensible predictive model. The type of metadata that is most directly deducible from the experimental observations is the atom type. The atom type classification can be considered sufficiently reliable for the higher resolution (<2.5 Å ) structures, however, with at least one exception, which corresponds to the nitrogen (N) and oxygen (O) atoms in a carbamoyl group (-CO-NH2). In this group, O and N cannot be distinguished solely on the basis of x-ray diffraction data, because of the symmetric structure of the group and very similar electronic densities around both O and N. This is a prime example of a regularly encountered error in the metadata, which, however, can be corrected by reversing the coordinates of O and N. The ligand metadata would impose this error when for instance hydrogen bonding with the ligand would require an acceptor (O) contact, but a donor (-NH2) contact is given a closer coordinate location in the structure file.
A considerably more difficult problem to handle is the influence of the constraints used in the refinement of the protein structure from experimental data. These constraints generate some unreliability in the coordinates, because only conformations with restricted geometries are allowed for the amino acid chain, which together with limited resolution can lead to artificially distorted conformations of ligand structures. In practice this means that the refinement involves fitting an alleged structure to the experimentally determined electron density map, which does not define all structural features uniquely, especially when the resolution is low [8].
Molecular fragments of pre-defined types (see Tables 2 and 3) were searched from coordinate ligand structure files in PDB. The search was based on atom types, chemical connectivity and geometry, and the identified fragments were then labelled for use in the extraction of coordinate data from protein structure files. To obtain unique fragment orientations, atoms from within a functional group were, when possible, chosen for the fragment definitions. In order to build a predictive model, the set of 24 fragment classes in Table 2 was used while collecting a dataset of approximately 70,000 contacts, representing the 13 contact atom types, i.e. target classes in Table 3.
Regarding the contact classes in Table 3, for example, the class C3 represents a pure van der Waals contact [14] and class C4, a hydrogen donor in a possible weak hydrogen bond in addition to a van der Waals contact [15], [16]. Aromatic carbons (C5, f11) can participate in both of the typical C3 and C4 interactions [17]. Halogen bonds have a role in biological processes [18] and therefore Fluorine [19], Chlorine, Bromine and Iodine are considered as so called fragment Main-atoms, as shown in Table 2. The target atoms in proteins, identified with three distance criteria ( Å for alleged H-bonds and charged groups, Å for probable dispersion and Å for halogen bonds), were classified during the search using three criteria: 1) element, 2) amino acid residue and 3) side or main chain atom. The interaction was defined as between a fragment type and target type, or between nuclei, mediated by protons and/or electrons.
A fragment was defined by an atom triple: Main-atom, Atom1 and Atom2, and at least the Main-atom was given the following characteristics: element, covalent bond count, aromaticity and possibly functional group, see Tables 2 and 3. These characteristics were used in collecting data from PDB, resulting in coordinates with metadata. The aromaticity of an atom was decided using PDB Ligand Dictionary through PDBeChem [20].
In addition to classification, target atoms have to be put in one coordinate system, i.e. fragments are superimposed. This was done using an elementary translation-rotation: first the database coordinates of the Main-atoms were translated to origin, which creates a new dataset ( below in eq. 1), and then a rotation operation was defined to connect the fragments reference frame to a common coordinate system. This requires solving the following matrix equation:(1)where refers to a 33 rotation;, and , i.e., is the cross product of with .
Thus, we have the equation,(2)which was solved for each fragment. The resulting was then used in the translation-rotations of the respective target atom position vectors to the common coordinate system. When the data is collected as mentioned above, as well as classified and coordinate systemized in this manner, the process results in a collection of three dimensional distributions of points that present measured relative positions of specified atoms with respect to specified fragment types. These distributions were then modeled with 3D probability densities described below.
0.2 Statistical modeling
To obtain predictive distributions for contact preferences we utilize a Bayesian framework where the observed 3D coordinates in the training data are modeled with interconnected parametric 1D densities, such that the parameters are provided a priori uncertainty characterizations in terms of probability distributions. The prior distributions enable regulation of parameter estimates in order to prevent them from depending solely on the observed data, which is desirable especially under the circumstances where the data generation process is known to harbor intrinsic biases. Also, regularization of model parameter estimates with the prior information is most crucial when certain class pairs have only very sparse training data, in which case unsmoothed estimates can be strongly biased.
The core distribution we utilize for characterizing coordinate variability is the von Mises-Fisher distribution (vMF) which is widely applied for modeling directional data. Separate probability densities for all three coordinates were necessary in order to capture the properties of the target atom clouds in a uniform setting (details provided below). Spatially the most complex (multimodal) observed differences in target atom distributions are found around the main direction of the fragment, and to a somewhat lesser extent with respect to distributions of polar angle, i.e. angular deviation from the main direction, see Figures 1 and 2. The distance distributions are given a priori as many modes as the corresponding polar angle distributions have, though in most cases they practically form a unimodal density, but not always. This is explained more thoroughly later in this section. The variables and parameters of the densities used in our work are specified in Tables 4 and 5.
It is formed by main chain carbonyl oxygens (C14) around fragment type (f29) (amino nitrogen singly bonded to a planar structure). In the reference frame where targets are modeled, polar angle value corresponds to what is in this paper called the main direction of the fragment — the direction of the vector from Atom1 to Main-atom, and corresponds to the plane that includes Main-atom in the origo and to which the main direction is perpendicular. Azimuthal angle measures angular deviation from the plane of the fragment, so that the center of the smaller cluster below the fragment is (in the model frame) approximately in direction . A fragment is defined by determining the characteristics of an atom triplet: Main-atom, Atom1 and Atom2. Main-atom is covalently bonded to Atom1, and Atom1 is covalently bonded to Atom2. Chemical properties of the Main-atom primarily determine the class of a fragment.
It is depicted in the same reference frame with the density. They can be interpreted as overlayed such that elevations in the density correspond to dense areas in the cloud of data points. The main direction of the fragment, as described in the caption of Figure 1, is defined by in the reference frame of the model, but corresponds in this figure to , where is the polar angle and is the azimuthal angle.
Let denote a generic set of parameters specifying a density in a 3D space. Then, the probability density in spherical polar coordinates for fragment class and target class is assumed to be of the piece-wise defined form(3)where , the divide and divide to non-overlapping intervals. The limit is the maximum distance used in collecting the target atom locations for a fragment class and target class pair (). The intervals and are associated with weight . The then, are non-overlapping intervals of associated with the weights (see below) and the different density components and parameters are defined using conventional nomenclature as:(4)Equations 4 show the forms of the densities in the particular coordinate system, or reference frame, that is used for modeling and in which the main direction of the fragment coincides with the positive z-axis. A likelihood function is obtained as a product from the values of the components of the density (eq. 3) for (or ) points representing each of the included regions in the sample space,(5)where(6)(7)
The structure of the density (eq. 3) was chosen based on investigations of the forms of the target atom clouds. For example, use of normal densities for the distance data was supported by large p-values in Kolmogorov-Smirnov normality test and the numbers of components needed in the angle dependent part of the density (i.e. and ) were automatically chosen based on frequency distribution of binned data. This was done for both angles ( and ) by connecting the heights of the adjacent bars of the histogram, creating a sequence of values, in which every change of sign corresponds to a local minimum or a maximum. The number of maxima was restricted to the interval [1], [5], was used to represent the number of modes in the density. The number of maxima was restricted by either reducing or increasing the number of bins in case the result would be outside the given interval.
In the separation of variables, azimuthal angle and distance are conditioned on a polar angle interval, see the equations in (4). The angular part reflects arc-like structures around the main direction of the fragment (for examples of this see last paragraph of section Examples 3 and 4). The angular deviation from the main direction is the leading variable in the sense that a multimodal azimuthal angle distribution (i.e. around the main direction) and a unimodal distance distribution are defined separately inside each polar angle segment. The idea behind this is that the peak of a polar angle density is an indicator of the strength of the interaction between a fragment and a target. The smaller the angle, the stronger the interaction, and if there is any effective multimodality in the distance density, the modes should coincide with the modes of the polar angle density. The azimuthal angle distribution thus completes the directional structure within each polar angle mode.
The uncertainties of the distance and the azimuthal angle variances are difficult to model due to the data generation process, the limitations of which were discussed above, and therefore, we use the standard maximum likelihood estimates calculated marginally from observed coordinates. On the other hand, the means , and are central parameters representing a measure of the strength of the interaction between the fragment and the target.
0.2.1 Parameter prior densities.
A prior distribution in Bayesian statistics can either be used to model uncertainty about a parameter or to include a priori knowledge, or beliefs, in the model [21]. Both of these uses are necessary for our modeling purposes.
Prior densities can be chosen in various ways such that they are either conjugate distributions for a particular likelihood function, or some other probability densities possessing required statistical properties, such as an asymmetry. Priors utilized here for individual parameters in the model densities are summarized in Table 6. In Table 6, is the zeroth order modified Bessel function of the first kind. For the von Mises and Normal distributions, conjugate priors are used (for the mathematical derivations related to these distributions see [22], [23], [24]).
Our prior density for the parameters is a slightly modified version of the distributions considered in [25] and [26]. The density has the form:(8)
In equation (8) hyperparameters and represent measures of modeler's belief in the expected values of the concentrations. The larger the value of the hyperparameter, the more the prior is concentrated around or . A default choice for any of these hyperparameters is the number of observations available for calculating the estimates for and .
0.2.2 The posterior distribution.
In Bayesian statistics, learning from observations takes place through the posterior distribution which is accessible from the joint probability density defined for the data and the parameters. For our piece-wise defined likelihood, the joint density is formally defined as(9)
The posterior density (eq. 9) has the same functional form as the prior density (eq. 8), but with updated parameters. As shown in the equation (9), the prior parameters , , and are updated to , , and , respectively, in the usual manner in Bayesian inference. In contrary, the remaining parameters are determined either directly from the data or given a suitable value based on chemical knowledge.
In order to define contact preferences, every fragment class and target class pair has to be specified with some characteristics. Maximum a posteriori (MAP) estimates are in this respect suitable when the associated densities are (piecewise) unimodal. The MAP estimates of the parameters are defined and updated with new data according to the formulae in Table 7. The prior parameter was given a constant value Å2) and was defined separately for each fragment type.
0.2.3 Updated parameters and the probability mass in a reference volume.
In our method, to evaluate the plausibility of a contact atom type in a given spatial area, the probability mass within in this volume is evaluated. The mass is calculated using the model densities (4) with updated parameters, see Table 7. The spatial area, or volume, is defined by a distance interval and a solid angle (i.e. intervals polar and azimuthal angles), and can be arbitrarily located. The volume that contains all target locations is defined through the intervals , and for the distance, polar angle and azimuthal angle, respectively. The cutoff is the maximum distance used when collecting data for a fragment class and target class pair (). The size of the volume can be chosen to be large, when for example contact preferences on either side of the fragments plane are investigated. Alternatively, the size of the volume can also be small, depending on the situation under investigation.
The spatial information content of the model is coded in the particular functional form of the probability density, but if one would solely rely on probability densities, it could easily happen that a scarce contact atom type could get a hierarchically higher preference position than a relatively often encountered type. This could happen in a spatial area where all the few contacts of the former type are observed. These kinds of problems are avoided and results for different contact types made more directly comparable by supervising the model such that chemically more likely contacts are paralleled, as well as the less likely. The model supervision was here achieved by multiplying the probability masses with target type specific weights that are calculated from three parameters electronegativity, softness and mean distance. Softness of an element , one from the group G = {C, N, O, S, F, CL, BR, I}, was defined as twice the mean value of hardness among the elements in G, minus hardness of the element . Numerical values for absolute hardness were taken from Parr et al. [27]. The electronegativity and softness were used to represent the tendency of an element to obtain partial charge in a compound. The third parameter, mean distance, is a measure of the strength of the interaction between a fragment and a target, and the numerical value given to it was the arithmetic mean of the distances in the training data. These parameters are used to calculate the weights as proportional to Coulomb force between the partial charges at the mean distance, i.e.(10)where and are the obtained partial charges for a fragment Main-atom and a target atom, respectively, and is the mean distance between the Main-atom and the target atom. The motivation for this prior is that the intermolecular interactions are mainly electrostatic, despite of the fact that they occur in many different forms, e.g., between a permanent dipole and an induced dipole, or between two induced dipoles, known as a London dispersion.
In the above formulation, it is assumed that a generic a priori information can be accurately utilized when modeling an interaction between and . It would also be possible to use calculated energies of some simplified fragment-target model as the a priori information, but the described approach is chosen because of its simplicity and independence of molecular details, which follows from utilizing element specific, measurable parameters, i.e. ionization energy and electron affinity [27]. Calculated prior probabilities relevant for this study are given in Tables 8 and 9.
0.3 Hierarchy calculations
In order to calculate the spatially dependent hierarchies around an arbitrary reference point(11)we defined intervals in spherical polar coordinates:(12)which define a volume that includes , e.g. , and . The reference point is defined in the reference frame that is used for modeling the data, and in which the fragment is in the (-z)(-x)-plane. The Main-atom (see section Data collection and processing) is at the origin, Atom1 on the negative z-axis and Atom2 in a point , i.e. in the plane defined by the negative z- and x-axes. On the other hand, in the reference frame used for the graphical representations in this article, (eq. 11) is transformed to(13)which is in a reference frame where the fragment is in (-x)(-y)-plane, see Figures 3 and 1.
Here would be located in the center of the volume, both radially and with respect to the solid angle. The center of the volume of Example 2 (not shown explicitly) is located on the axis defined by the vector connecting Atom1 to Main-atom, i.e. on the positive x-axis in this figure.
The probability masses in the volume defined by (eq. 12) are evaluated using the model densities with the updated parameter values. Technically the calculations are done either with series expansions, see e.g. the equations 7.1.1., 7.1.7., 7.1.22 and 9.6.34 in [28] or directly as Riemann sums.
The -specific probability mass is the factor that gives a contact atom type its rank, in the fragment class related, and around a reference point defined hierarchy. Namely, the bigger the mass, the more probable the contact atom type. The volume can cover a larger portion of the neighborhood of the fragment, for example, the hemisphere on either side of the plane of the fragment.
A hierarchy can also be defined for example among the fragment types (), with respect to a representative of a target class around . The motivation for choosing the probability density and the estimation procedures of the model parameters as described in Methods, is that they provide a rapid and flexible way to capture the relevant features of the target atom distributions, without relying on fine details of the target atom clouds, which are potentially misleading due to the intrinsic uncertainties in the data generation process.
The functionality of the introduced Bayesian method of finding hierarchies is here illustrated by a number of case-studies. The calculated hierarchies are compared with contact atom type counts found in the reference data, in the volume surrounding the reference point , representing a target atom location. The hierarchies and the reference frequencies are not expected to be perfectly congruent, due to the fact that the reference data set is not exhaustively large. Therefore, they are expected to simply show some of the specific features around the reference point . In order to illustrate the reliability of the results, standard errors for all results were determined with a bootstrap type procedure [29], which is described together with the corresponding results.
Example 1: A typical location of a hydrogen bonding partner
First, we consider a reference point equal towhich is located just below the plane of the fragment, with a polar angle deviation of to the left from the main direction, see Figure 3. The intervals (eq. 12) used wereand the results calculated around for fragment classes f2, f5, f8, f11, f18, f22, f23 and f27 (see Table 2) are given in Table 10 below. They are also represented graphically for f2, f5, f26 and f27 in Figure 4. The error bars represent separately standard errors for the model based probabilities and the frequencies in reference data. They were defined by calculating both quantities in a set of 1,000 centered volumes, that were slightly different in shape and size. To investigate how the inclusion of additives as ligands in the training and reference data sets would affect the model-based rankings, we divided the data into two sets using a list of approximately 770 additives that are found in PDB [30]. Contact data for these additives was removed from the original set to create a new additive-free set. Then, based on the model trained with the new dataset, contact hierarchies were calculated and probability masses were compared with reference data that also lacked the additives.
. Comparison of contact atom frequencies with model based probabilities for fragment classes f2, f5, f26 and f27. Hierarchy is given by the calculated probabilities, which are represented as circles. The circles are joined with a line to illustrate tendencies among target classes. The bars represent the fractions of target atoms belonging to a particular class. Both sum to one over target classes. Also shown are standard errors for both the frequencies and the model based probabilities (joined with lines and centered around the mean values). The contact atom counts in reference data for f2, f5, f26 and f27 were 165, 126, 16 and 49, respectively.
We replicated the calculations in Example 1 using the additive-free data and the results are shown in Figure 5 and Table 11. In addition, a comparison of the original and new contact atom counts is presented graphically in Figure 6. Comparisons of the rankings reveal that they are nearly identical in the two situations. Nevertheless, some discrepancy does occur and this example illustrates the need of a careful consideration about what structures can be included as representative information to the training data. If predictions based on multiple different training sets are extensively monitored and discovered to be sufficiently similar, it is possible to consider the use of a universal training data collection where the different sets are merged.
Reference point : comparison of contact atom frequencies with model based probabilities for fragment classes f2, f5, f26 and f27. Hierarchy is given by the calculated probabilities, which are represented as circles. The circles are joined with a line to illustrate tendencies among target classes. The bars represent the fractions of target atoms belonging to a particular class. Both sum to one over target classes. Also shown are standard errors for both the frequencies and the model based probabilities (joined with lines and centered around the mean values). The contact atom counts in reference data for f2, f5, f26 and f27 were 65, 126, 16 and 49, respectively.
Fragment classes f2, f5, f26 and f27, the same as in Example 1 related hierarchy figure.
Example 2: A possible halogen bond geometry
The second reference point isi.e. on the positive x-axis when defined in the reference frame of Figure 3. The intervals (see eq. 12) used wereTable 12 presents probabilities for classes f13, f17, f18, f20, f21, f34, f35 and f36 and the results are represented graphically for f17, f20, f34 and f36 in Figure 7. The error bars represent separately standard errors for the model based probabilities and the frequencies in reference data. As in the previous example, they were defined by calculating both in a set of 1,000 centered volumes, that were slightly different in shape and size.
. Comparison of contact atom frequencies with model based probabilities for fragment classes f17, f20, f34 and f36. Hierarchy is given by the calculated probabilities, which are represented as circles. The circles are joined with a line to illustrate tendencies among target classes. The bars represent the fractions of target atoms belonging to a particular class. Both sum to one over target classes. Also shown are standard errors for both the frequencies and the model based probabilities (joined with lines and centered around the mean values). The contact atom counts in reference data for f17, f20, f34 and f36 are 4, 7, 0 and 1, respectively.
Examples 3 and 4: Two distances in a direction perpendicular to the plane of the fragment
The third and fourth reference points, in the reference frame of Figure 3, are
The intervals (see eq. 12) used wereand the corresponding volumes are centered around the negative z-axis, as shown in Figure 3. The results are represented graphically for fragment classes f2, f5, f11 and f22 in Figure 8. The error bars represent separately standard errors for the model based probabilities and the frequencies in reference data, calculated in the same manner as in the previous examples.
and . Comparison of contact atom frequencies with model based probabilities for fragment classes f2, f5, f11 and f22. Hierarchy is given by the calculated probabilities, which are represented as circles. The circles are joined with a line to illustrate tendencies among target classes. The bars represent the fractions of target atoms belonging to a particular class. Both sum to one over target classes. Also shown are standard errors for both the frequencies and the model based probabilities (joined with lines and centered around the mean values). The contact atom counts in reference data for f2, f5, f11 and f22 are 158, 163, 5 and 7, respectively.
Hierarchies calculated when the additives were excluded, as discussed in Example 1, are presented in Figure 9. It is seen that a larger discrepancy in the model-based rankings compared with the reference data occurs when the reference data are extremely sparse, which makes the unsmoothed relative frequencies highly volatile. To also visualize more generally how our method smooths scatter data about different types of contacts, 3D-plots of estimated densities are shown in Figures 10, 11, 12.
These are relating to example 1. Reference points and : comparison of contact atom frequencies with model based probabilities for fragment classes f2, f5, f11 and f22. Hierarchy is given by the calculated probabilities, which are represented as circles. The circles are joined with a line to illustrate tendencies among target classes. The bars represent the fractions of target atoms belonging to a particular class. Both sum to one over target classes. Also shown are standard errors for both the frequencies and the model based probabilities (joined with lines and centered around the mean values). The contact atom counts in reference data for f2, f5, f11 and f22 are 111, 67, 1 and 6, respectively.
Showing differences in spatial arrangement of contacts among three target classes - C4 (alpha carbon), C10 (amino nitrogen) and C12 (carboxyl oxygen). The scatter plots contain all the target atoms found in the fragment class f6 training data for these target classes.
Showing differences in contact arrangements for two target classes - C8 (carbamoyl nitrogen) and C9 (imidazole, guanido or indole nitrogen), and also distance dependence for class C9. The scatter plots contain all contacts found in the training data for these fragment and target class pairs. Note that the target atom clouds in the middle and on the right are the same.
showing distance dependence for target class C14 (carbonyl oxygen). The scatter plot contains all C14 target atoms in the training data and the densities show which directions are emphasized at distances 2.7, 2.98 and 3.26 Å.
Example 5: Direct contacts of R-norepinephrine
Here we consider the hydrogen bonding and aromatic interaction preferences of norepinephrine (also known as noradrenaline; PDB ligand identifier: LT4). The molecular environment of this example is the norepinephrine binding site in chain B of human phenylethanolamine N-methyltransferase (PNMT) from PDB entry 3HCD. PNMT catalyses adrenaline synthesis with coenzyme S-adenosyl-L-methionine (AdoMet). In the structure of 3HCD AdoMet is replaced by its demethylated form S-adenosyl-L-homocysteine (AdoHcy) to study the binding mode of LT4 [31]. The x-ray resolution of the entry 3HCD is 2.39 Å , which is near the upper limit considered in our study (2.5 Å , see Table 1). As discussed previously, this means that some precaution is necessary while deducing interactions from the structure. Consequently, the statistical nature of our method is helpful, since the probability densities can indicate a certain relative location that is associated with what is experimentally observed in other structures. This enable the quantification of the relation in question as a probability.
LT4 has three hydroxyl groups, a terminal amino group and a six-carbon aromatic ring as its functional groups. The most preferred target class for any of these functional groups is defined as a class for which the product of the probability density peak value (eq. 3) and the class conditional prior probability (see Tables 8 and 9) has the highest numerical value.
Two of the hydroxyl oxygens are bonded to the aromatic ring (cathecol hydroxyl groups), and based on the model, prefer a nitrogen from histidine or arginine side chain as a contact. The aromatic ring carbons then prefer aromatic carbons, i.e. phenylalanine, histidine, tyrosine or tryptophan is a likely contact residue. The aromatic carbons also have strong contacts from the hydrophilic targets, for example carboxyl oxygens. The hydroxyl group of the aliphatic tail prefers lysine and the terminal amino group prefers glutamic acid/glutamate or aspartic acid/aspartate.
These a priori preferences are not related to LT4, instead only the 3D structure of the interactions is. Some directional aspects related to this example are illustrated in Figures 13, 14, 15, 16, 17. Figures 14 and 15 present hydrogen bonding contacts for the R-norepinephrine tail. There are two carboxyl oxygen (C12) contacts for the LT4 hydroxyl group, namely GLU B219 and ASP B267. The former is at a distance less than the maximum length used in this study for a oxygen donor and oxygen acceptor hydrogen bond, i.e. 3.14 Å3.30 Å . According to the model, its direction of approach is not typical for a hydrogen bond, but because the same GLU B219 residue is simultaneously a contact for the adjacent amino group, the actual preferred direction is such that it allows the carboxyl to bond with both of these functional groups in LT4. Therefore this is considered a direct contact. Regarding the amino group, the direction of approach of the GLU B219 carboxyl oxygen is typical for a hydrogen bond (almost optimal), only somewhat shifted to a direction that facilitates the double contact described above, see Figure 15. The latter carboxyl, ASP B267, is in a more typical direction, but even further apart, and it is confirmed from PDB entry 3HCD water locations that this contact is a bridged hydrogen bond, not a direct contact.
Also depicted in the figure is the proximity (closest 3.4 Å ) of the TYR B222 aromatic ring to the amino group of LT4. Though the distance and orientation of the ring fit well to the hydrogen bond donor - aromatic ring interaction scheme, the relative direction is such that TYR B222 corresponds to probability density values smaller than 20% of the peak value. Therefore, this might not be a strong contact for the amino group, but possibly has a guiding task in the binding process. The depicted distance between amino group and the methyl group donating sulfur atom is 5.66 Å.
Three amino acid residues (ASP B267, GLU B219 and TYR B222) were considered as contacts.
The residue GLU B219 carboxyl oxygen is located in a typical direction of a class C12 hydrogen bond acceptor for this functional group (fragment class f26).
The distance to both degenerate maxima is 2.52 Å.
In the figures on the right, the double contacts are created by a degeneracy that follows from the way the fragments are defined. Namely, the third atom (Atom2, see Section Data collection and processing) of a fragment in a molecule can frequently be chosen from more than one possible atom and each choice creates its own probability density, including a maximum. These densities are connected through a rotation around the covalent bond between the Main-atom and Atom1.
The TYR B222 contact for the hydroxyl of LT4 tail, see Figure 14, has an aromatic ring that can serve as a hydrogen bond acceptor. Therefore, in case the LT4 tail hydroxyl group would act as an acceptor in the above described hydrogen bonds (i.e. with water and GLU B219) it could in principle donate its hydrogen to a weak hydrogen bond with the aromatic ring of TYR B222, because the closest atom of the ring is at a distance of about 3.5 Å and the ring is facing toward the hydroxyl group. Consequently, as for the LT4 amino group this is not a strong contact, but perhaps has a guiding task in the binding process.
Example 6: Separating COMT ligands from decoys in a subset of DUD
Here we demonstrate the usefulness of the estimated contact probability masses in discriminating between appropriate and poor binders using logistic regression, see e.g. [32]. In the current application context, logistic regression model connects a binary response variable (here ligand/decoy), with explanatory variables describing the modeled system. The outcome is a probability indicating how likely it is for a system to belong to either of the two response groups. Our testing was done by retrieving a set containing 6 out of the 11 Catechol-O-methyltransferase (COMT) ligands and 19 out of the total 468 decoys from the Directory of useful decoys (DUD) [33]. Two extra ligands were added, namely dopamine and BIA 3-335 (PDB Ligand identifiers LDP and BIA, respectively), by retrieving their structures from ZINC database, see [34]. These 27 small molecules (ZINC codes in Tables 13, 14 and 15) were chosen so that the DUD molecules have high mutual resemblance, especially so that the decoys have an aromatic ring with at least two primary oxygens bonded to neighboring carbons (in hydroxyl groups typically). This is because all except one COMT ligand in DUD have this type of a structure, and the exception is different only in that the ring is non-aromatic. The two extra ligands were included for reference, because they are known good binders, for BIA [35], and should have clearly higher preference to binding than an average decoy.
The search for the binding mode of the small molecule in the binding pocket (from PDB ID 1H1D) was started by orienting the molecule such that two of the primary oxygens would coordinate with the magnesium ion (Mg2+), participating in the COMT function (see [35]), and here taken as part of the binding site. Then, predefined rotamers of the small molecule were rotated around the axis connecting the two coordinating Os, and to a lesser amount around a second axis. Direct contact probabilities between the small molecule and the binding site were calculated. Probabilities for the two coordinating Os were excluded to emphasize contacts for the rest of the molecule. Rotamers and orientations with close intra- or intermolecular contacts were removed using distance criteria, though the plausibility of a rotamer could be evaluated using probability masses for intramolecular contacts.
For each small molecule, the rotamer and orientation with highest probability were found and the probabilities were then used in a logistic regression model that mimics a docking screening task. Two explanatory variables were used in the logistic regression model: the total probability mass of direct contacts, divided by the mass of the molecule (pmPerMass) and the ratio of the number of hydrophobic and hydrophilic fragments (Phob/Phil) in the molecule.
It is well known that in an actual binding affinity calculation for a ligand-protein pair in solution, one needs to consider energetics of direct contacts, water and metal mediated contacts, desolvation and entropy. The variable pmPerMass is here considered to be a measure of the binding energy of direct contacts, whereas the variable Phob/Phil reflects desolvation properties and perhaps tendency for water mediated contacts. Configurational entropy doesn't have in this study any obvious representative, because for the numbers of rotatable bonds (RotBonds) no predictive role was identified. Results of the predictions based on logistic regression are shown graphically in Figure 18. Values for the putative explanatory variables are given in Tables 13, 14 and 15.
The green circles represent ligands and the red circles decoys, as they are classified in DUD, when all 27 considered molecules are included in the model. The two extra ligands (see text) have index values 7 and 8 (PDB identifiers LDP and BIA, respectively). Blue step curve gives the mean probability that was obtained from bootstrapping over the two decoy subgroups to calculate standard errors for the logistic regression (error bars representing these are centered at the mean values).
The molecule that was most highly ranked, BIA 3-335, is a known tight binding inhibitor [35]. It is also heaviest of the 27 molecules included in the example; approximately 360 hydrogen masses compared to the more typical value that is between 150 and 250. The variable pmPerMass is intensive, i.e., the size of the molecule should not directly influence it's value, and it is assumed that success in predicting relative binding affinities for smaller candidate binders depends strongly on the accuracy of this variable.
Probabilities derived from the logistic regression are on average over 0.5 for the molecules in the alleged ligand group and below 0.5 for the alleged decoys, which represents a natural threshold between a ligand and a decoy in a screening process. Two exceptions in the ligand group are the low scoring molecules with index values 1 and 4. The ligand with index value 1 has the third highest pmPerMass, but quite low Phob/Phil value, while the ligand with index 4 has a low value for both (see Table 13). Hence, if both these molecules are considered as good binders, our approach does not contain enough information to reveal this. On the other hand, there are not necessarily experimental data available on the binding affinities for the decoys, which in this study were chosen to resemble the ligands as much as possible, each starting with the two, to Mg2+ anchoring primary oxygens bonded to an aromatic ring. This means that some decoys might be reasonably good binders. Nevertheless, based on the logistic regression model, molecules in the ligand group have on average clearly higher probabilities () to be ligands than the molecules in the decoy group (). In summary, in the set of 27 chemically similar small molecules, containing 8 experimentally defined ligands, 6 highest ranked molecules were from the ligand group. Consequently, the receiver operating characteristics (ROC) [13] in the screening experiment are: true positive rate TPR = 0.75, false positive rate FPR = 0 and accuracy ACC = 0.93. A ROC curve was produced by using discriminating thresholds having either different TPR or FPR. The ROC curve is given in Figure 19.
The 12 threshold probabilities separating ligands from decoys that were used for calculating the characteristics are 0.05, 0.1, …, 0.3, 0.4, 0.5, 0.65, 0.75, 0.8 and 0.9.
One important aspect that has not been considered here, is whether the active form of COMT is a monomer or a multimer. If it is a multimer, it would be interesting to investigate how informative this characteristic is about the properties of suitable ligands. Additional potential molecular characteristics for further study are the flexibility of the binding site and the features of the binding modes having the highest probabilities (pmPerMass).
Predictions about unresolved binding sites, or ligands, can be made by building the preferred contact patterns from the molecules included in a set of functionally classified fragments. In out method, these contact patterns are composed of probability masses calculated for the fragments to have a specific kind of contact in a spatial area. When, for example, the binding affinity of a molecule is studied and the probability masses are defined for an entire molecule, they can be used in a docking and scoring procedure. The absolute binding affinity would be given by the total energetics of the binding process in a thermodynamic setting, including direct and bridged contacts, desolvation and entropy. It is presumed, that using a fragmentation where the fragments have distinct and unique contact patterns, the probability densities described here contain information beyond the chemical complementarity, namely on energetics (for results in this direction, see [36]). This is reasonable by an analogy with quantum mechanics, because it can be argued that the probability masses are proportional to the amount of binding energy, which are needed in evaluating the binding affinities. In our setting this means relative binding affinities, i.e. rankings over a set of ligands and decoys.
The results obtained in Example 6, show a level of reliability that is typical for a successful scoring function, see e.g. reviews [9], [10]. Our experiment revealed that potentially very reliable information could be retrieved when our probabilistic method is combined with an effective search routine. An important aspect is that the ligand and decoy molecules were similar, i.e. the decoys used were 'drug-like' [29]. This is based on that they typically had masses between 150 to 250 hydrogen masses, contained both hydrophobic and hydrophilic fragments throughout the structure and were chosen so that each can be anchored to the magnesium ion in the COMT binding site. This should make separation of ligands from decoys challenging and be ultimately based on finer details of the binding affinity, because no decoy was readily rejectable. In a docking and scoring routine such a method can also be used to find the most favourable orientation for the most favourable rotamer, or conformer, of a small molecule in a binding site, i.e. the pose. When adjusting the method for calculations of the absolute binding affinities, the same difficulties will be faced as for any knowledge-based scoring function, see [9].
In addition to the quality of the fragmentation, the reliability of the data is a central issue in the prediction of contact preferences and some issues related to this were discussed in Sections 1 and 2. When choosing the structure for a prediction model, it is essential to understand the data generation process; in principle from the experimental measurement to the coordinate file. Regarding the special characteristics of the experimental method, x-ray diffraction is sensitive to thermal motion in the crystal. This weakens locally the electron density map, and since electron density maps are precisely the starting point in structure refinement, such an effect should preferably be assessed. In the further refinement, constraints are used in order to keep the protein structure within chemically acceptable boundaries. It thus follows that the ligand atom positions have uncertainty which is not straightforward to quantify. Possible approaches to quantification could be exploration of the effects of the constraints on a theoretical basis or using structures refined with different constraints. On the other hand, PDB files contain substantial amounts of metadata that could potentially also be used in modeling. An example of this are the b-factors, which can be used for incorporating thermal motion in the model. Another example is provided by the occupancies that are needed to take into account the more long lasting local displacements, i.e. alternative conformations in the crystallized protein-ligand complexes [8].
Though the results in the example sections are given with standard errors [29], performance of our model in predicting favourable intermolecular contacts could be more quantitatively verified in the future when more extensive reference sets of sufficiently high quality become available. The approximately 10,000 structure files from PDB used as reference data did only give a preliminary test for certain fragment types. This is mainly because of the 3D nature of the problem, since in order to obtain a good spatial resolution, the frequencies need to be defined in less extensive volumes.
Following a reviewer's suggestion, we added a ROC curve to Example 6, which makes it more informative.
The hierarchies illustrated in our examples show both spatial dependence and reliability. They can also be evaluated quite rapidly from coordinates within a classification, typically in seconds, or in tens of seconds. Thus, tentatively our approach can be used to study structural aspects of biochemical reactions or as a tool in predicting the most favourable binding modes and separating ligands from decoys, as described in the examples. A plausible future test would be to create a hierarchy among a group of ligands and compare their binding probabilities to experimentally measured binding affinities, e.g. those of KiBank database referred to in DUD. Test on each stage of the docking and scoring procedure has to be successfully conducted, before it is shown that the method is applicable for the purpose. Then it can be directly compared, e.g., with the knowledge-based potentials that are only distance dependent.
Reliable evaluation of binding affinities for potential ligands of a binding site, would be a desirable feature of a virtual drug design screen, see for example [9], [37]. As discussed, the distance and direction dependent probability masses obtained with the approach described here, are taken to provide direct information on relative binding affinity, which is supported by the results in Example 6. Regarding further development of our modeling approach, both statistics- and chemistry-based generalizations and improvements are possible, including the obvious expansion to all imaginable molecular fragment types.
Bayesian predictive modeling in the normative sense as defined in [38] provides a potential approach to representing contact preference distributions. Such a predictive model could exploit directly the 3D structures of the probability densities (eq. 3) that model the contact atom positions, instead of considering density parameters as the main characterization of the spatial information. The obvious disadvantage of such an approach is the considerably increased computational effort needed to derive approximations to the sought after predictive distributions.
The reliability of an inferred hierarchy depends, on one hand, on how successfully the error from experimental methods and structure refinement is quantified in terms of the used probability densities. On the other hand it depends on how realistically the chemical likelihood of a contact atom type and the bias in the data set are taken into account. The latter are here incorporated as prior information, see equation (10), which guides the model with chemistry-based knowledge. A third fundamental area for chemistry-based improvements are the classifications (Tables 2 and 3). For example, a classification can be envisioned where the covalent bond count of Atom1 (see Data collection and processing) would be used as one of the characteristics defining the fragment, which would then remove the degeneracy described in section Example 5. This kind of more structural way of defining the fragments would expand the classifications, but should also give fragment definitions that are closer to being unique.
Author Contributions
Analyzed the data: RH. Wrote the paper: RH JC. Conception of the work: RH JC MSJ JVL SP. Designed the model: RH JC. Designed the software used in analysis: RH.
- 1. Rantanen V-V, Denessiouk KA, Gyllenberg M, Koski T, Johnson MS (2001) A Fragment Library Based on Gaussian Mixtures Predicting Favorable Molecular Interactions. J Mol Biol 313: 197–214.
- 2. Rantanen V-V, Gyllenberg M, Koski T, Johnson MS (2003) A Bayesian Molecular Interaction Library. J Comput Aided Mol Des 17: 435–461.
- 3. Rantanen V-V, Gyllenberg M, Koski T, Johnson MS (2005) A Priori Contact Preferences in Mole- cular Recognition. J Comput Biol Bioinform Res 3: 861–890.
- 4. Lehtonen JV, Still D-J, Rantanen V-V, Ekholm J, Björklund D, et al. (2004) BODIL: A Molecular Modeling Environment for Structure-Function Analysis and Drug Design. J Comput Aided Mol Des 18: 401–419.
- 5. Kitchen DB, Decornez H, Furr JR, Bajorath J (2004) Docking and Scoring in Virtual Screening for Drug Discovery: Methods and Applications. Nat Rev Drug Discov 3: 935–949.
- 6. Scapin G (2006) Structural Biology and Drug Discovery. Curr Pharm Des 12: 2087–2097.
- 7. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, et al. (2000) The Protein Data Bank. Nucleic Acids Res 28: 235–242.
- 8. Ravi Acharya K, Lloyd MD (2005) The Advantages and Limitations of Protein Chrystal Structures. Trends Pharmacol Sci 26: 10–14.
- 9. Huang S-Y, Grinter SZ, Zou X (2010) Scoring Functions and Their Evaluation Methods for Protein–Ligand Docking: Recent Advances and Future Directions. Phys Chem Chem Phys 12: 12899–12908.
- 10. Huang S-Y, Zou X (2010) Advances and Challenges in Protein-Ligand Docking. Int J Mol Sci 11: 3016–3034.
- 11.
Mandl F (1988) Statistical Physics, 2nd Ed. Chichester, England: John Wiley & Sons. Chapter 2.
- 12. Kortemme T, Morozov AV, Baker D (2003) An Orientation-Dependent Hydrogen Bonding Poten-tial Improves Prediction of Speci.city and Structure for Proteins and Protein–Protein Complexes. J Mol Biol 326: 1239–1259.
- 13.
Egan JP (1975) Signal Detection Theory and ROC Analysis. New York: Academic Press.
- 14.
Finkelstein AV, Ptitsyn OB (2002) Protein Physics: A Course of Lectures. London, UK: Academic Press.
- 15. Desiraju G (1996) The C-H···O Hydrogen Bond: Structural Implications and Supramolecular De- sign. Acc Chem Res 29: 441–449.
- 16. Scheinert S, Kar T, Gu Y (2001) Strength of the CαH···O Hydrogen Bond of Amino Acid Residues. J Biol Chem 276: 9832–837.
- 17. Grimme S (2008) Do Special Non-covalent π– π Stacking Interactions Really Exist? Angew Chem Int Ed Engl 47: 3430–3434.
- 18. Auffinger P (2001) Halogen Bonds in Biological Molecules. Proc Natl Acad Sci U S A 101: 16789–6794.
- 19. Howard JAK, Hoy VJ, O'Hagan D, Smith GT (1996) How Good is Fluorine as a Hydrogen Bond Acceptor? Tetrahedron 52: 12613–12622.
- 20.
Dimitropoulos D, Ionides J, Henrick K (2006) Using MSDchem to Search the PDB Ligand Dictionary. Current Protocols in Bioinformatics UNIT 14.3.Available: http://onlinelibrary.wiley.com/doi/10.1002/0471250953.bi1403s15/full. Accessed 9 August 2012.
- 21.
Gelman A, Carlin JB, Stern HS, Rubin DB (1995) Bayesian Data Analysis. New York: Chapman and Hall.
- 22.
Bernardo JM, Smith AFM (1994) Bayesian Theory. Chichester, England: Wiley.
- 23.
Mardia KV, Jupp PE (1999) Directional Statistics, 2nd ed. New York: Wiley.
- 24.
Mardia KV, Kent JT, Bibby JM (1979) Multivariate Analysis. London: Academic Press.
- 25. Guttorp P, Lockhart RA (1988) Finding the Location of a Signal: A Bayesian Analysis. J Am Stat Assoc 83: 322–329.
- 26. Nuñez-antonio A, Gutiérrez-peña E (2005) A Bayesian Analysis of Directional Data Using the von Mises-Fisher Distribution. Communications in Statatistics — Simulation and Computation 34: 989–99.
- 27. Parr RG, Pearson RG (1983) Absolute Hardness: Companion Parameter to Absolute Electroneg-ativity. J Am Chem Soc 105: 7512–7516.
- 28.
Gautschi W (1972) Error Function and Fresnel Integrals. In: Abramowitz M, Stegun IA, editors. (1972) Handbook of Mathematical Functions with Formulas, Graphs, and Mathematical Tables. Washington, D.C.: National Bureau of Standards Applied Mathematics Series 55 Tenth Printing. pp. 295–330.
- 29. Nicholls A (2008) What Do We Know and When Do We Know It? J Comput Aided Mol Des 22: 239–255.
- 30. Strömbergsson H, Kleywegt GJ (2009) A Chemogenomics View on Protein Ligand Spaces. BMC Bioinf 10 ((Suppl 6)) S13.
- 31. Drinkwater N, Gee CL, Puri M, Criscione KR, McLeish MJ, et al. (2009) Molecular Recognition of Physiological Substrate Noradrenaline by the Adrenaline-Synthesizing Enzyme PNMT and Factors In.uencing Its Methyltransferase Activity. Biochem J 422: 463–471.
- 32.
Bewick V, Cheek L, Ball J (2005) Logistic regression.Crit. Care 9: Statistics review 14. Available: http://ccforum.com/content/9/1/112. Accessed 4 June 2012.
- 33. Huang N, Shoichet BK, Irwin JJ (2006) Benchmarking Sets for Molecular Docking. J Med Chem 49: 6789–6801.
- 34. Irwin JJ, Shoichet BK (2005) ZINC–a Free Database of Commercially Available Compounds for Virtual Screening. J Chem Inf Model 45: 177–182.
- 35. Bonifácio MJ, Archer M, Rodriques ML, Matias PM, Learmonth DA, et al. (2002) Kinetics and Crystal Structure of Catechol-O-Methyltransferase Complex with Co-Substrate and a Novel In- hibitor with Potential Therapeutic Application. Mol Pharmacol 62: 795–805.
- 36. Morozov AV, Kortemme T, Baker D (2004) Close Agreement Between the Orientation Dependence of Hydrogen Bonds Observed in Protein Structures and Quantum Mechanical Calculations. Proc Natl Acad Sci U S A 101: 6946–6951.
- 37. Gilson MK, Zhou H-X (2007) Calculation of Protein-Ligand Binding Affinities. Annu Rev Biophys Biomol Struct 36: 21–42.
- 38.
Geisser S (1993) Predictive Inference: An Introduction. London: Chapman & Hall.


text:
There are currently no reviews for this product. Please help us and others by writing a review.Review This Product
Vivid Arts Black & White Shih Tzu Ornament
We are committed to Low Prices.
If you find any of our products on the price promise pages advertised, and in stock and immediately available, at any of our competitors we will endeavour to match the price.
Product Code: XRL-SZ16-B EAN: 5055195025401
This stunning sitting Black & White Shih Tzu ornament is hand painted to give it a realistic look. It has an exquisite finish to make this a delightful addition to any home, garden or patio.
Made from resin means that this ornament is durable during harsh weather and frost. This also means it is suitable for both indoor and outdoor use making it the perfect gift for any dog or animal lover.
Key Features:
- Made from resin
- Hand painted
- Frost & Weather proof
- Outstanding durability
Dimensions (Approx):
- Height: 28cm (11ins)
- Width: 15cm (6ins)
- Depth: 27cm (11ins)
Questions and Answers
Have a question for this product? Go ahead and ask us! We try to reply within a few hours but it may take longer on some questions.Ask us a Question

subset: commoncrawl


text:
Forever Living Products - Oman
Khaled Khayati
MUSCAT - Villa No. 240, Azaibah Street, backside of Zubair Automotives, Azaibah
IBRI - Opposite to Al Sulaif Park, Land No. 7338, Muscat-Ibri Highway
SALALAH - Al Wadi Area, Plot No. 31, Block G, Building No. 188, Thamreet Street (13052), Near Salalah Public Park
- Phone: +968 24 618098 (Muscat) +968 2 578 4990 (Ibri) +968 2 336 1330 (Salalah)
- Email: firstname.lastname@example.com
Shop the Forever products here.
Become a Forever Business Owner
- Purchase your favorite products at discount (and sell them for up to a 43% profit)!
- Be your own boss. Be in business for yourself, but not by yourself.
- Help your friends and family look better, feel better and live the life of their dreams!
Please contact the Country Office for more information on how to sign up and join!


text:
Holiday Inn Palm Beach-Airport Conf Ctr
Holiday Inn Palm Beach-Airport Conf Ctr
1301 Belvedere Rd.
1301 Belvedere Rd.
West Palm Beach, Florida
United States
Check In: 4PM
Check Out: 11AM
Check-in Age: 21
Book With Us Advantage
- Best Price Guarantee
We promise you access to the guaranteed best available prices, or your first night is free.
- Online Reservation Guarantee
Your room is guaranteed.
- No Booking Fees!
We do not charge any booking fees for making reservations directly with us.
- Data Privacy and Site Security
IHG takes your privacy seriously and works to protect you. All personal information you provide is encrypted and secure.
Welcome to Holiday Inn Palm Beach-Airport Conf Ctr
Our Hotel's West Palm Beach Location is Ideal
The Holiday Inn® Palm Beach - Airport Conference Center hotel offers excellent amenities and an ideal location. Our guests value being a half-mile from Palm Beach International Airport (PBI) and close to area attractions.
Business travelers appreciate our modern facilities, which feature a Business and Fitness Center. Our conference facilities feature 9 meeting rooms with over 7,000 square feet for the perfect board meeting, seminar or sales presentations. Cemex, FlightSafety, Watchtower Conference Center and Thermo Fisher Scientific, as well as the Palm Beach County Convention Center, are also located here in West Palm Beach.
Our hotel's proximity to the South Florida Fairgrounds makes it easy to tour Yesteryear Village. Events at the Coral Sky Amphitheatre are a favorite destination. Other area attractions include Kravis Center for Performing Arts, a mecca for Broadway-style performances. We suggest visiting downtown West Palm Beach, home of Sunfest and CityPlace. Families enjoy a trip to the South Florida Science Museum and Lion County Safari.
While here, be sure to stop by The Spot Eats & Drinks after a full day in West Palm Beach, FL. Our hotel is also pleased to provide a complimentary shuttle to the airport to make your stay as stress-free as possible.
Kids Stay and Eat Free
Kids ages 18 and under stay for free when sharing their parents' room. Up to four kids ages 11 and under eat free any time of the day in any Holiday Inn® on-site restaurant.
|Kids Stay Free
|Kids Eat Free
|United States, Canada, Central America, South America, Mexico, Caribbean
|18 and Under
|11 and Under
Free High-Speed Internet
Standard Wireless Internet service is included for all guests.
Shuttle/ Parking
- Car Parking Available
- Complimentary Daily Self-Parking is available
- Self-Parking Fee: 0 USD
- Ample, open air parking is offered on property. A large area is available to accomodate motorcoaches.
Pet Policy
- No pets allowed
- Service Animals Allowed
- No pets allowed. Service animals only please.
Languages Spoken by Staff
Credit Cards
- American Express
- Visa
- MasterCard
- Diners Club
- Carte Blanche
- Discover
- Number of Floors: 11
- Total Number of Rooms: 199
- Number of Suites: 1
- Number of Non-Smoking Rooms: 188
- Number of Accessible Rooms: 10
- Connecting Rooms Available
- Public Spaces Renovated in: 2019
- Guest Rooms Renovated in: 2019
- Public Entrance
- Guest Rooms
- Elevators
- Public Restrooms
- Registration Desk
- Exercise Facility
- Business Center
- Pool
- Number of Accessible Meeting Rooms: 7
Accessible Hotel Areas
- Registration Area
- Accessible Guest Rooms
- Business Center
- Meeting Room
- Exercise Facility
- Pool
Hotel Areas with Accessible Routes from Accessible Public Entrance
- Accessible Self-Parking
- Van Accessible Self Parking
- Ramp Access
- Portable Bathtub Seats
- Closed Caption TVs
- Telephone with TDD
- Visual Alarm/Alert in Guest Rooms
- Visual Alarm/Alert in Public Spaces
- Communication Kits with Visual Alarms
Provided by Hotel


text:
cleveland boudoir photography
What is cleveland boudoir photography and why is it so popular? Everyone seems to have their own ideas about what boudoir photography is. Well, let's start with what it isn't. It isn't sleazy, it isn't degrading, it doesn't mean you have to be nude and it doesn't mean the photographer is creepy.
So what is it all about? It's about you and who you are on your more personal side. It's about your own special beauty and sensuality. It's about the experience and how it can make you feel about yourself. A boudoir session can be very empowering and FUN while preserving your experience for years to come.
Boudoir sessions have become very popular and fashionable because every woman wants or needs to know she is beautiful in her own way.
Sensual or flirty images and small books of these images are now often given as gifts for those special someone's. Women are now treating themselves with this experience after a milestone achievement, weight loss or recovering from an illness or even divorce. It is also common now for brides to surprise their future husbands with a boudoir book on their wedding day.
Now what to wear? Well that will depend on your personality. Basically, what are you comfortable wearing? A sexy robe, lingerie, boy shorts, thongs, corsets, fancy bras, stockings, heels, boots or maybe even your guy's favorite team tee. It's all about you and your personality really so we will talk and decide on what works best for you.
Who goes for boudoir photography? There's a number of reasons to have these done.
1. A gift. Boudoir photos are a unique and personal gift for someone special in our life. Whether it be a bridal gift to your new husband or a Valentines gift. I assure you it's not like any other gift he's gotten before! Or maybe you've been married for awhile and need a boost. A boudoir album will for sure do the trick.
2. Just because. Maybe you just want to capture your beauty forever. Or maybe you just want to flaunt your hotness and show off that inner vixen in you that's been dying to get out! No matter what size you are, 0-100, you're sexy in your own way. So why not gift yourself with a boudoir session. You'll leave feeling beautiful and more confident.
3. Celebrate. Maybe you recently lost weight, got a job promotion, or you reached some goal you've been working so hard at. Boudoir photography will assure you what an awesome, beautiful woman you are.
So, what does boudoir photography mean to you?


text:
There are no products to list in this category.
Your shopping cart is empty!
These suppliers are looking for exclusive distributor in your country for their easier management, while you can sell them with confidence.
These products are offered by Thai suppliers who are willing to ship their products directly to your customers on your behalf.
OEM or custom package under your own brand is welcome for these products, which are offering by Thai factories and small producers only.
MARKET are wholesale B2B marketplace where Thai suppliers offer their products for export. We do preliminary check to screen the qualified, while you can contact them directly.
DISCOVER Thai products to help you explore new business opportunities and develop into business conveniently. Get samples and quotes from multiple & qualified Thai suppliers. !


text:
Female swimmers shine on final day of 2012 U.S. Paralympics Swimming Trials
BISMARCK, N.D. – Five more world records fell on Saturday, the final day of competition at the 2012 U.S. Paralympics Swimming Trials. At the conclusion of three days of competition, a total of 99 American, 37 PanAm and 12 world records were set at the Bismarck State College Aquatic and Wellness Center.
Standout Jessica Long (Baltimore, Md.) set her fifth world record of the meet in the women's 200m IM (S8) in 2:37.71 in the morning prelims. During evening finals competition, Long shaved more than a second off her time finishing in 2:36.00 with her sixth world record of the meet.
"It's exciting, I just feel really blessed and love what I do," said Long.
Mallory Weggemann (Eagan, Minn.), who is looking to make her Games debut in London, also set a world record in the women's 50m butterfly with a time of 33.81.
"Swimming has really saved me," said Weggemann. "It wasn't long after I became paralyzed that I remember sitting at the trials before Beijing and watching the competition with my sister and wondering what would lie ahead for me. It's been an amazing journey, and I'm excited for that next step – to find out the team for London."
Paralympic Swimming Resident Team member Ileana Rodriguez (Miami, Fla.), who lives and trains at the U.S. Olympic Training Center in Colorado Springs, Colo., was also excited about her performance at trials.
"In all of my events, I swam my best times yet," said an excited Rodriguez. "That's the best a swimmer can ask for – to continue improving."
She's especially excited about tomorrow's team announcement for London. Rodriguez, a U.S. naturalized citizen originally from Cuba, hopes to make her Paralympic Games debut wearing the red, white and blue.
"I lived in Cuba for 13 years, and just as long in the U.S., so I consider it home. It's a dream of mine to make the team and swim for the U.S. in London."
Among the standout performers emerged a few, new faces. Like Victoria Arlen (Exeter, N.H.). Arlen, who set a world record on day one of the competition in the 400m freestyle (S6), racked up a second new record in tonight's women's 100m freestyle (S6) in 1:14.74.
"Both were totally unexpected," remarked Arlen. "I just swam the best I could and was so excited to see my time. "
Sixteen-year-old Reilly Boyt (Fort Collins, Colo.) also showed marked improvement in all of her events including tonight's 200m IM (S6) finals. Boyt and U.S. Paralympian Miranda Uhl (Gainesville, Fla.) battled to the finish line, with Boyt narrowly missing the first place finish. Uhl won in 3:28.16, and Boyt was right on her heels in 3:28.71.
In men's competition, Rudy Garcia-Tolson (Bloomington, Calif.), who holds the world record in the 200 IM (SM7), turned in a strong performance in tonight's finals finishing in 2:36.40, just off of his world mark of 2:35.92.
Another newcomer to Paralympic swimming is Lt. Brad Snyder (St. Petersburg, Fla.), U.S. Navy, who lost his vision as the result of an IED blast while deployed in Afghanistan in Sept. 2011. In the prelims of the men's 200m IM (S11), posted a time of 2:36.84. A few hours later in finals, he went 2:32.31, ranking him No. 6 in the world in the event.
Snyder also earned a No. 1 ranking in the world in the men's 100m free (S11) with a time of 57.75.
Sixteen-year-old Ian Silverman (Baltimore, Md.) also had an impressive showing at trials. Silverman, who is new to Paralympic swimming won the men's 200m IM (S10) race with a time of 2:21.18.
Evan Austin (Terre Haute, Ind.) also made his presence known at trials. Austin took first place in the men's 200m IM finals (SM8) in 2:38.17.
The 2012 U.S. Paralympic Swimming Team will be nominated on Sunday; 20 female and 14 male athletes will earn their tickets to the 2012 Paralympic Games. More than 4,000 athletes with a physical or visual disability from more than 160 countries are expected to compete.
Complete competition results can be found online at www.bismarckaquatics.com/hytek/results/evtindex.htm.
For more information, please contact Beth Bourgeois, U.S. Paralympics, at 719-306-5639 or email@example.com.


text:
Hollywood events are more conscious in creating a special time for the guests through awareness and fun when mixing art, music, food and other fabulous highlights; but when you can also find a bigger purpose than just another great event to go to, it becomes a bigger night for many others, especially for those in need.
On the evening of Wednesday, December 5th, eclectic conscious attendees enjoyed The Conscience Cocktails' Holiday Mixer benefitting The Seany Foundation. Guests mingled at the luxurious Daniel Maltzman Gallery in Beverly Hills for an evening of socializing while creating awareness for this important cause.
The Seany Foundation is a 501(c)(3) non-profit organization dedicated to funding research to help find cures for pediatric cancers while improving the quality of life for children, teens, and young adults battling cancer.Every day, 35 children and teens are diagnosed with cancer, 7 will die and 17 of the 28 survivors will suffer devastating life-long side effects. The foundation it's working to ensure that research programs are devoted to rare childhood cancers to get the funding they desperately need.
Mitch Robins, father of Sean Lewis Robins, spoke to the audience about his son's fight with Ewing Sarcoma ( ES-a rare bone cancer ) and how The Seany Foundation was founded in memory of their son. Sean fought Ewing Sarcoma for nearly 7 years, until his death in 2006; he was 22.
It was a very special night with a clear understanding of how important is to get involved and support our future finding cures at an early stage of cancer. My favorite moment of the night was when a video was playing showing pictures and footage of Sean Lewis along with other children suffering from the same cause. Their faces expressed joy, hope and dreams. I'm an artist, so I understand how important creativity is during healing process from any cause. I was inspired to come on board and help out however I can. I want to make a difference while creating through my passions, so starting next year I will be part of someone's healing progress by sharing my passion for the arts with The Seany Foundation Family. This foundation is responsible for the light of this children's dreams by showing them fun times while getting treated. Thanks to Sean Lewis' goals, many children and young adults are enjoying fun creative activities during intense care.
Guests had the chance to enter the raffle with fabulous prizes such as a Randy Kirk Original Artwork valued at $4,000 benefitting 100% The Seany Foundation.
Open bar sponsored by Akvinta Vodka, Dirty Tequila, IZZE and Aro Pistachios along with food stations sponsored by Hollywood Pasta Company. Music sets by DJ Frackture and included live musical performances by cancer survivor Urbalist, and up and coming sensation Ruby Summer.
The mission of Conscience Cocktails is to expose Angelenos to our city's wonderful charities and the people behind them by aligning with a different non-profit organization at each event and giving a platform to introduce their cause to a captive audience. These networking nights will be held bi-monthly at L.A.'s hot spots, so stay tuned!
For more info, please visit www.theseanyfoundation.org
Come out and make a difference today while mingling at LA's hottest places.
See you there!


text:
United States of America Hotels
Find a place to stay
See on a map
- Limerick Hotels, United States of America
- Adirondack Region Hotels, United States of America
- Yarmouth Hotels, United States of America
- East Las Vegas Hotels, United States of America
- Maui Hotels, United States of America
- South Bay Hotels, United States of America
- Long Island Hotels, United States of America
- Cape Cod Hotels, United States of America
- Oahu Hotels, United States of America
- Kauai Hotels, United States of America
Hotels in United States of America
From the northern sophistication of New York City or Chicago to the tropical flair of Miami or Los Angeles, Hotels.com makes choosing USA hotels easy. With Hotels.com, travelers get real reviews from previous customers for a reliable rating of US hotels they can trust along with hundreds of options and an easy online booking process. The USA covers a vast territory, comprising over 2500 miles in width from east to west and includes a wide variety of terrains and urban experiences for travelers, from the vibrant and cultured cities of the east coast to the laid back vibe of the west coast and the vast and diverse rural states inbetween. The number of hotel options can be confusing - Hotels.com takes the guesswork out of choosing the right accommodations with recommendations backed up by real life reviewers.
What's the USA Like?
The east coast of America includes some of the most famous locations, such as New York City, with its world famous cultural landmarks like the Guggenheim Museum or Museum of Modern Art (MoMA) and the Wall Street stock exchange. Boston, meanwhile, is one of the oldest cities in the United States and home to Harvard University, MIT and other prestigious institutions. The cities of the west coast feature the coffee houses of Seattle in the north and the sunny beaches of California along with the dream factory that is Hollywood to the south. The glittering city of Las Vegas lures travelers into the southwest desert area to gamble in stunning casinos and take in world class entertainment, while Florida offers a unique combination of semi-tropical weather and stylish sophistication. The mountains of the western states offer skiing holidays and there countless opportunities to enjoy fishing or swimming with coast lines to the east and west and a vast array of fresh water lakes. Travelers can expect a similar variety in USA hotels from luxurious resorts to sleek urban accommodations with cutting edge style.
Tips for Getting Along with Locals in the USA
Americans are known simultaneously as among the friendliest of societies and the most abrasive and many of the differences are regional. New Yorkers are renowned for their rudeness while southerners are known for their relaxed charm - although New Yorkers can also surprise visitors with their open friendliness. Strangers may complain at tourists who walk slowly on the sidewalk in Chicago during rush hour while a slow stroll is the only way the locals move in the Florida Keys or in the deep South. A visitor to the states will experience all types of people wherever he travels so it's best to keep open minded as if you move around a lot. With hundreds of options available, Hotels.com offers the voice of experience to make your choice of hotels in the USA reliable and secure.
United States of America travel guides
Nightlife & Entertainment in Las Vegas
Las Vegas is surely the USA's glittery, neon-washed capital of lavish nightlife and entertainment. You...
Top 15 Fun Facts About New Orleans
Located in the state of Louisiana, New Orleans is the largest city and metropolitan area of the state,...
10 Fun Facts about the Statue of Liberty
The Statue of Liberty is one well-known landmark, but how well do you really know her? Read our fun...
Where to Stay in Jacksonville - River View Rooms to Luxury Beach Resorts
As the USA's largest mainland city by area, Jacksonville has plenty of accommodation options. Beach...
Where to Stay in Pittsburgh - From International Chains to Mansion-Style Guesthouses
Pittsburgh's accommodation choices run from centrally located, modern chain hotels to quiet bed and...
Save more with Secret Prices
Get instant savings with Secret Prices


text:
iPad mini Smart Cover - Pink Sand
The Smart Cover for iPad mini is constructed from a single piece of polyurethane to protect the front of your device. It automatically wakes your iPad when opened and puts it to sleep when closed. You can easily fold the Smart Cover into different positions to create a stand for reading, viewing, typing or making FaceTime calls. And it's easy to remove, so you can snap it on and off whenever you want.
What's in the Box
iPad mini Smart Cover
Answers from the community
is the back of the cover clear?
- Asked by Tracey O from Troon
- on 7 Jan 2020
How much does the IPad mini 5 Smart Cover weigh?
- Asked by lynn K from edmond
- on 30 Jul 2019
Does this cover fit mini3
- Asked by Karen B from Oro-Medonte
- on 22 Nov 2019
Answer now(Does this cover fit mini3)No answers yet
Does the smart cover from an iPad Mini 4 fit the new iPad Mini gen 5?
- Asked by John F from Melbourne
- on 18 Mar 2019
Best Answer:
It attaches the same and it'll work to protect your iPad Mini 5 screen but it's not a perfect fit. It attaches the same and it'll work to protect your iPad Mini 5 screen but it's not a perfect fit. In the closed position, the Mini 4 Smart Cover overhangs the right edge of the Mini 5 by about 2-3mm. it's noticeable visually and when holding, you feel the edge of the smart cover. The Mini 5 Smart Cover, on the other hand, sits flush with the right edge. When holding, you feel the iPad and not the edge of the cover.
- Answered by Jason C from Issaquah
- on 8 Apr 2019


text:
Help us to improve GuitareTab.comTake our survey!
Indigo Girls – Cold As Ice chords
COLD AS ICE (Amy Ray - Lead vocalist) ----------- (Emily Saliers - Back Vocalist) This is a pretty easy and very cool song from the Indigoes! it's from the 1986 EP. one of the very first recordings from Amy & Emily. about the chords you just have to go through all the song with those same chords : Emajor, Amajor and Dsus4 or whatever it is. anyway the tab for that last one is x00230. practically is a D chord without tapping the High E string. you can also alternate Dsus4 with Dsus2 and D chords. but the more correct one is Dsus4. Enjoy!!!A E Dsus2 AI don't think you understand oohE Dsus2 (go on all this way out for the rest of the song) The rhythm of my heart There's a strong line to mind and soul And I feel it tearing apart. I've been searching for new words To tell you in a different way... When I told you I'd hang on forever, I threw my choice away... Chorus: and it'sE Dsus2 E Dsus2It's cold as ice, I can't let goE Dsus2 A E Dsus2 It's cold as ice, but I need your loooooove.And I wish I could have been the first one Oh, to walk out the door, But the more I find myself to blame, The less there is to come back for. You wish you could have been the last one To tell me how you really feel. When you closed the door, oh, deep inside my heart It still seemed so sincere. Chorus If there's a message to this madness Well it still seems quite unclear... If I really need to save myself, Why can't I just walk out of here??? There's a message in your eyes And you make it perfectly plain, Either I leave your heart behind Or crack beneath this freezing rain. Chorus


text:
Ed Andrieski / AP
Two women share an umbrella to ward off snow as they walk the 16th Street Mall during the noon hour in Denver on Wednesday, April 17, 2013.
Severe thunderstorms, large hail and possible tornadoes menaced a swath of the country from northern Texas to St. Louis, Missouri, on Wednesday, while heavy rains in northern Illinois caused delays at Chicago-area airports and snow made for messy travel in Colorado.
The National Weather Service issued advisories of all types as harsh weather pelted the middle of the country throughout the day.
Much of Oklahoma was under a tornado watch until late Wednesday as intense storms ravaged the Texas-Oklahoma border. That tornado watch extends north through St. Louis and central Illinois.
"There could be really strong storms later tonight, and that's always scary. That could be the case in central Oklahoma," said Carl Parker, a storm specialist for The Weather Channel.
A flash flood warning was in effect in northern Illinois, causing airport delays and cancellations. O'Hare International Airport reported delays averaging almost one hour, with more than 300 flight cancellations due to weather, according to the Chicago Department of Aviation.
At Chicago Midway International Airport, airlines were reporting some delays of 30 minutes or more, with a few flight cancellations.
Meanwhile out west, Colorado was still dealing with the lingering effects of heavy snowfall, which had created messy driving conditions. Multiple accidents Wednesday afternoon led the state's Department of Transportation to shutdown westbound traffic for a portion of Interstate 70.
Inbound flights to Denver International Airport were delayed an average of 2 hours 16 minutes Wednesday evening, according to tracking site FlightAware.com. Outbound flights were experiencing delays of about 45 minutes.
Areas around Denver were expected to receive 3 to 6 inches of new snow by the end of Wednesday.


text:
teckcrew needed a new logo design and created a contest on 99designs.
A winner was selected from 13 designs submitted by 7 freelance designers.
I want that booth image incorporated into the logo. I want a P and W in it as well as a shadow figure like the logo attached. I also want to have it look like light is shining from the back of the box casting the shadow. The colors should match the picture of the illusion.
Magic's Newest Star
Desired Color Scheme:
The colors should match the screenshot of the theme.
Accepted File Formats:
.psd, .gif
Let me know if you have any questions.
Find out how a design contest works or learn more about our logo design services.


text:
- home
- news
- blogs
- calendar
- enewsletters
- people on the move
LEED Comes Home In This
GRAND RAPIDS — In the midst of frenzied publicity for large green-building projects throughout West Michigan, a significant number of local builders have quietly embraced the LEED for Homes green building standard, with five houses slated for this fall's Parade of Homes, and 26 houses under way or completed as part of the standard's ongoing pilot program.
"A lot of this was through the homeowner. They wanted a green-built home with things like geothermal heat, so I suggested the rating system," said Scott Branc of New Urban Home Builders LLC, which is building a LEED-registered home on Burton Street in Grand Rapids. "There are a lot of people looking for this right now. It's what the market is starting to demand."
For several years, New Urban has built all of its homes to Green Built Michigan standards, a certification developed and administered by the Michigan Association of Home Builders similar to — if much less comprehensive than — the U.S. Green Building Council's Leadership in Energy and Environmental Design rating system.
With little fanfare, the Midwest rollout of the council's latest LEED system is being managed locally by the Grand Rapids-based Alliance for Environmental Sustainability. The group currently has 78 projects on the books throughout its seven-state region and has trained hundreds of builders, raters and designers.
Branc was introduced to the LEED system through these training sessions, which qualified him as the home-building field's equivalent of a LEED Accredited Professional. There is no such program currently for the home rating system.
"It's amazing all the projects that are going on right now, and there has been no marketing for it," said Michael Holcomb, principal of Home Inspector General in Byron Center and founder of the Alliance for Environmental Sustainability. "We just had Kiplinger's magazine in East Grand Rapids do a photo shoot on one of our homes; we had Home and Garden at another one in Minneapolis, and Cooking Light at one in Chicago. And it's happening all over the country."
A 20-year-veteran of the green building field, Holcomb has been an Energy Star rater since 1993 and helped launch the Green Built Michigan program in 2000. He launched the nonprofit alliance, today managed by executive director Jeanine Reynolds, as a means of promoting the green-built standard, and is now serving as the chief instructor for its LEED training programs across the Midwest.
The ready adoption of the green standard is a dream come true for Holcomb, who has long railed against the state's weak energy code.
"A LEED home in Michigan is probably 50 to 70 percent more energy efficient," he explained. "Michigan has the weakest energy code of any cold weather state. We're a national embarrassment."
Holcomb was one of four raters dispatched by the state in 2005 to evaluate the costs of building energy-efficient homes as part of a rewrite of the Michigan Uniform Energy Code. The changes were subsequently blocked by an MAHB lawsuit. The current energy code is at least 15 years out of date, Holcomb said, and the first thing owners of a LEED home will notice are the immense energy savings.
Several home builders across the state have already committed to build all of their homes to criteria set by the EPA's Energy Star program or the more stringent Green Built standard. Like Green Built, LEED uses Energy Star as a baseline for its rating system. Points are assigned on top of those basic criteria for concerns ranging from air quality to durability issues.
Ensuring a low concentration of volatile organic compound emissions and an effective heating, ventilation and cooling system are a cornerstone of the program. Toxic products such as pesticides and certain cleaning materials are banned from the structure. Water use and reduction is carefully monitored, as is the creation of waste and how it is disposed. Like all of the LEED certification programs, the occupant is required to complete a lengthy monitoring period before earning certification.
Builders are also required to look at more practical green applications, such as ensuring that the home is not in constant need of materials to keep it in working order, that landscaping minimizes water runoff and maximizes geothermal attributes, and that locally produced materials are used. Other considerations include the site plan, preserving vegetation and the home's proximity to public transportation and services.
And with that, severe penalties are built into the system for large homes with a large amount of open space.
"The larger the home, the fewer number of bedrooms, the more points you're going to have to score," said Holcomb. The reasoning is that larger homes require more materials and resources to build and maintain. The more square footage per occupant, measured by bedrooms, the more wasteful the environmental impact.


text:
Dear Visitors
Welcome to our "Diary of Events" page. This section is a record of the diverse activities, in the various project categories, conducted by Gift of the Givers on a monthly basis. Feeding Schemes, Sarah Fox Hospital Support and Counselling Services which are also monthly projects are hosted in their "own" space and so are our disaster response projects. To get a more comprehensive understanding of the scope of our activities we recommend you navigate the project section elsewhere on this site.


text:
Nuggets could turn to Billups if Arturas Karnisovas goes to Nets
The Nets are in an on-going search for a general manager to replace to Billy King, who was reassigned within the organization, and Nuggets assistant general manager Arturas Karnisovas continues to be a prominent candidate for the opening.
Karnisovas spoke with the Nets earlier this week while the Nuggets were in New York for three days to face the Knicks and then the Nets. He is as hot a candidate as there is, and there a pretty good chance the Nets offer him the job.
If they do, and he accepts, could Chauncey Billups be an option for the Nuggets to get into their front office? He could. It would be up to the Nuggets to make the call. Read more…


text:
A sitemap lists all pages available on a website. The sitemap for web pages on Sharon Blain Hair Education Courses and Products is shown below:
See All
Copyright 2020 Sharon Blain Hair Education Courses and Products. All Rights Reserved.
It's that time of the year again, the release of Modern Hair+Beauty Magazine. …
All prices are in USD.
Winner 2014 Hair Expo Business Performance of the Year, Boot Camp
More Awards »