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| Original file line number | Diff line number | Diff line change |
|---|---|---|
| @@ -0,0 +1,274 @@ | ||
| <!--#include virtual="./head.shtml" --> | ||
|
|
||
| <h2>How to use TOPOLINK</h2> | ||
| <br> | ||
| The package is accompanied by an example input file, which is found in | ||
| the <code>topolink/input</code> directory. It is mostly | ||
| self-explicative. Possibly the best way to get started is to open | ||
| the input file and look at it. An examle is available | ||
| <a target=_blank_ href=./example.txt>[here]</a>. | ||
| <br><br> | ||
| The executable must be run with: | ||
| <pre> | ||
| topolink inputfile.inp > topolink.log | ||
| </pre> | ||
| Alternatively, the PDB file of the structure can be provided on the | ||
| command line as the second argument, overwriting the definition | ||
| given in the input file: | ||
| <pre> | ||
| topolink inputfile.inp model.pdb > topolink.log | ||
| </pre> | ||
| <br> | ||
| <table align=center width=80%> | ||
| <tr><td colspan=2 class=tdclean><h3> Sections: </h3></td</tr> | ||
| <tr><td width=50% class=tdclean bgcolor=orange align=center><a href=./usage.shtml><b>INPUT</b></a></td> | ||
| <td width=50% class=tdclean bgcolor=orange align=center><a href=./output.shtml><b>OUTPUT</b></a></td></tr> | ||
| </table> | ||
| </center> | ||
| <br><br> | ||
| <br> | ||
| <h3>The output of TopoLink</h3> | ||
|
|
||
| The ouput file generated by TopoLink contains two sections which are of | ||
| interest of the investigator: | ||
| <list> | ||
| 1. The result of the search of each topological distance. | ||
| 2. The statistics of the links for each experiment. | ||
| 3. The overall statistics of the links in the model. | ||
| </list> | ||
| <br> | ||
| Before these sections, the output file contains several input file | ||
| checking prints, which can be ignored. The package also outputs errors | ||
| and stops when inconsistencies in the input file are found, such as: | ||
| duplicate definition of observations, observations that are not | ||
| consistent with the defined reactivity, and others. | ||
| <br><br> | ||
| <b> 1. Result for the topological distance of each link </b> | ||
| <br><br> | ||
| At some point, the output file of TopoLink will contain specific link | ||
| information, in the following form: | ||
| <pre2> | ||
| ------------------------------------------------------------------------------------------------------------------------- | ||
| RESIDUE1 ATOM1 RESIDUE2 ATOM2 EUCLDIST TOPODIST OBSERVED DMIN DMAX RESULT OBSRES REACRES RA AA | ||
| ------------------------------------------------------------------------------------------------------------------------- | ||
| LINK: MET A 1 N LYS A 17 CB 20.647 >16.900 YES 0.000 16.900 BAD: EUCL 0/ 1 0/ 1 YN YN | ||
| LINK: MET A 1 N LYS A 113 CB 8.955 9.058 YES 0.000 16.900 OK: FOUND 1/ 1 1/ 1 YY YY | ||
| LINK: GLU A 4 CB ASP A 22 CB 29.619 >15.600 NO >15.600 >15.600 OK: EUCL 0/ 1 0/ 1 YY YY | ||
| ... | ||
| </pre2><br> | ||
| This is the result for the search of a topological distance for each | ||
| link. the results are to be intepreted in the following manner: First, | ||
| there is the definition of the atoms forming the possible link. | ||
| <br><br> | ||
| The 10th column contain the euclidean distance between these atoms. | ||
| <br><br> | ||
| The 11th column contain the topological distance, reported with the | ||
| following details: if an upper bound is reported, for example | ||
| <code>>16.900</code>, that means that TopoLink could not find any | ||
| topological distance smaller than that value. That upper bound is the | ||
| linker length of the largest linker that can bind the two atoms | ||
| involved, according to link type definitions. | ||
| Therefore, there is no topological distance shorter than the | ||
| maximum linker length which could bind these atoms. The link should not | ||
| be observed in this case, according to the model. | ||
| <br><br> | ||
| The 12th column reports if that particular link was observed in some | ||
| experiment. In this case, the first link was observed. | ||
| <br><br> | ||
| The 13th and 14th columns contain the experimental maximum and minimum | ||
| distances for that linker. That is, if the link was observed with a | ||
| linker of <code>16.900</code> length, the distance between the atoms | ||
| must be shorter than that, defining a range <code>[0.,16.9]</code>. If, | ||
| additionally, the link was NOT observed for a shorter link of, lets say, | ||
| <code>12.0</code> length, that defines an lower bound for the distance, | ||
| and the range would be <code>[12.0,16.9]</code>. In the first link | ||
| example, the link was observed using a link of length 16.9. | ||
| <br><br> | ||
| The 15th column contains the specification of the overall result. | ||
| <code>BAD</code> specifiers indicate that the topological distance | ||
| obtained is not consistent with the observations. <code>OK</code> | ||
| indicates that it is consistent. The possible results are: | ||
| <table align=center width=80%> | ||
| <tr bgcolor=lightgrey><td class=tdclean width=120px><b>Code</b></td> | ||
| <td class=tdclean><b>Description</b></td></tr> <tr> | ||
| <td class=tdclean><code>OK: FOUND</code> </td> | ||
| <td class=tdclean>A valid topological distance was found, and it is | ||
| consistent with all observations.</td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean> <code>OK: LONG</code> </td> | ||
| <td class=tdclean>A valid topological distance was found, is it longer | ||
| than the linker length, and the link was NOT observed, thus the result | ||
| is consistent with observations. </td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>OK: EUCL</code></td> | ||
| <td class=tdclean>The euclidean distance is already too long, and the | ||
| link was NOT observed, thus the result is consistent with observations.</td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>OK: NOTFOUND</code></td> | ||
| <td class=tdclean>No valid topological distance was found, but | ||
| the euclidean distance is shorter than the linker | ||
| length. The link was also | ||
| NOT observed, such that the result is consistent with observations.</td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>BAD: SHORT</code></td> | ||
| <td class=tdclean>A topological distance was found, which is shorter | ||
| than a linker length for which the link was NOT observed. Since it was | ||
| NOT observed, that linker should be a lower bound to the distance, which | ||
| is violated. </td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>BAD: LONG</code></td> | ||
| <td class=tdclean>A topological distance was found, and the link was | ||
| observed. However, the distance is too long for the linker length. The | ||
| link, therefore, is not consistent with observations. </td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>BAD: EUCL</code></td> | ||
| <td class=tdclean>The link was observed, but the euclidean distance in | ||
| the model is already too long to be consistent with that observation.</td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>BAD: NOTFOUND</code></td> | ||
| <td class=tdclean>The link was observed, the euclidean distance is fine, | ||
| but no valid topological distance was found. Thus, the link is not | ||
| consistent with the observation.</td> | ||
| </tr> | ||
| <tr> | ||
| <td class=tdclean><code>BAD: MISSING</code></td> | ||
| <td class=tdclean>The link was NOT observed, and the topological | ||
| distance is such that it should have been.</td> | ||
| </tr> | ||
| <tr> | ||
| <tr><td colspan=2 class=tdclean><hr></td></tr> | ||
| </table> | ||
| </list> | ||
| <br><br> | ||
| The 16th column contains the ratio between consistent observations and | ||
| expected observations, considering that reactive atoms are only those which | ||
| were experimentally observed to react (at a crosslink or deadend). | ||
| That is, <code>1/3</code> means that the link was observed in one | ||
| experiment, but it should have been observed in three, according to the | ||
| linkers used and <i>observed</i> reactivity. | ||
| <br><br> | ||
| The 17th column contains the ratio between consistent observations and | ||
| expected observations, considering that reactive atoms are given by | ||
| residue types. That is, if the linker is expected to bind two Lysine | ||
| residues, all pairs of Lysine residues with consistent topological | ||
| distances should be observed to crosslink. The ratio reported is the number of actual | ||
| observations relative to the number of expected observations. | ||
| <br><br> | ||
| The 18th and 19th columns contain the information about solvent | ||
| accessibilities of residues (RA - Residue Accessibility) and specific | ||
| atoms (AA - Atom Accessibility). A "Y" indicates that a residue or atom | ||
| is accessible to solvent, and a "N" indicate it is not accessible. A | ||
| residue is considered accessible to solvent if ANY of its atoms is | ||
| accessible to solvent. Each pair of "YY" or "YN", etc, refers to the | ||
| first and second residue or atom, respectively. Therefore a "YN" in the | ||
| "RA" column indicates that the first residue is accessible to solvent, | ||
| but the second residue is not. | ||
| <br><br> | ||
| <b> 2. Statistics of the links for each experiment</b> | ||
| <br><br> | ||
| For each experiment, a detailed account of the links is provided, in the | ||
| following form: | ||
| <pre> | ||
| ------------------------------------------------------------------------------- | ||
|
|
||
| Experiment: DSS | ||
|
|
||
| Number of type-reactive pairs of atoms: 105 | ||
|
|
||
| Number of observed-reactive pairs of atoms: 21 | ||
| Number of observed-reactive pairs of atoms within linker reach: 5 | ||
| Number of observed-reactive pairs of atoms outside linker reach: 16 | ||
| Missing links, according to the structure and observed-reactivity: 0 | ||
|
|
||
| Number of observed links: 7 | ||
| Number of observed links consistent with the structure: 5 | ||
| Number of observed links NOT consistent with the structure: 2 | ||
|
|
||
| Sum of scores of observed links: 0.0000000 | ||
|
|
||
| Sensitivity of the cross-linking reaction: 1.0000000 | ||
| False-assignment probability: 0.0190476 | ||
|
|
||
| Likelihood of the experimental result: 0.9999953 | ||
| Log-likelihood of the experimental result: -0.0000047 | ||
|
|
||
| Likelihood using user-defined pbad and pgood: 0.9996471 | ||
| Log-likelihood using user-defined pbad and pgood: -0.0003530 | ||
| Using: pgood = 0.700; pbad = 0.010 | ||
|
|
||
| ------------------------------------------------------------------------------- | ||
| </pre> | ||
| Important here is to understand what is the "observed-reactivity": | ||
| observed reactivity is defined by the experiment. If a residue is | ||
| observed, experimentally, to participate in a crosslink or form a | ||
| dead-end, it is a "observed-reactive" residue, meaning that one is sure | ||
| that it is solvent exposed and available for reaction. Type-reactivity, | ||
| on the other side, is simply the expected reactivity as a function of | ||
| the chemical nature of the residue and of the linker. | ||
| <br><br> | ||
| The <code>sensitivity</code> of the crosslinking reaction is defined, | ||
| here as the fraction of links that were observed relative to the links | ||
| that were expected to be formed from the reactivity and structure. In | ||
| the example above, 5 pairs of observed-reactive pairs of atoms were | ||
| found to be within linker reach, and all of these links were | ||
| experimentally observed, thus the sensitivity is 1.00. | ||
| <br><br> | ||
| The <code>false-assignment probability</code> is the ratio between the | ||
| number of links experimentally observed and the total number of | ||
| type-reactive pairs of atoms. That is, the probability of any pair of | ||
| type-reactive atoms be reported as forming a link, incorrectly. In the | ||
| example above, 2/105=0.019. | ||
| <br><br> | ||
| The results above allow for the estimation of a likelihood for the | ||
| model, which is reported. The likelihood computed using user-defined | ||
| sensitivities and false-positive probabilities is also reported. | ||
| <br><br> | ||
| <b> 3. Overall crosslink statistics of the model</b> | ||
| <br><br> | ||
| The final result of TopoLink is reported as follows: | ||
| <pre> | ||
| ------------------------------------------------------------------------------------ | ||
|
|
||
| FINAL RESULTS: | ||
|
|
||
| RESULT0: 12 : Number of observations that are consistent with the structure. | ||
|
|
||
| RESULT1: 12 : Number of topological distances consistent with all observations. | ||
| RESULT2: 16 : Number of topological distances NOT consistent with observations. | ||
| RESULT3: 0 : Number of links with missing observations. | ||
|
|
||
| RESULT4: 0.00000 : Sum of scores of observed links of all experiments. | ||
|
|
||
| RESULT5: 0.99980 : Likelihood of the set of experimental results. | ||
| RESULT6: -0.00020 : Log-likelihood of the set of experimental results. | ||
|
|
||
| Using: pgood = 0.700; pbad = 0.010 | ||
| RESULT7: 0.99965 : Likelihood of the set of experimental results. | ||
| RESULT8: -0.00035 : Log-likelihood of the set of experimental results. | ||
|
|
||
| ------------------------------------------------------------------------------------ | ||
| </pre> | ||
| The results are reported with flags "<code>RESULT0/8</code>" to | ||
| facilitate greping the data for multiple model evaluation. The results | ||
| are self explicative, but must be clarified in some points: | ||
| <br><br> | ||
| The number of observations that are consistent with the structure is not | ||
| the same as the number of topological distances which are consistent | ||
| with observations. For a topological distance be consistent with | ||
| observations, it must be consistent with <u>all</u> observations at the | ||
| same time. This is particularly important if different linkers that can | ||
| react with the same pairs of atoms were used in different experiments. | ||
| <br><br> | ||
| A topological distance is said to be NOT consistent with the | ||
| observations if it is not consistent with a single observation. | ||
| <!--#include virtual="./tail.shtml" --> | ||
|
|
||
|
|
||
|
|
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