FirstGlance/Evaluating Protein Crosslinks: Difference between revisions
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Below are step-by-step instructions for evaluating putative [[protein crosslinks]] reported by FirstGlance in Jmol. Genuine crosslink bonds are illustrated, along with [[clashes]] that are irregularities in the model, not crosslink bonds. | Below are step-by-step instructions for evaluating putative [[protein crosslinks]] reported by FirstGlance in Jmol. Genuine crosslink bonds are illustrated, along with [[clashes]] that are irregularities in the model, not crosslink bonds. | ||
==Instructions for an Isopeptide Bond== | ==Instructions for an Isopeptide Bond== | ||
1. | 1. Go to [http://firstglance.jmol.org FirstGlance in Jmol] (right click link, open in new tab), and enter [[PDB code]] <b>2xi9</b>. | ||
<br> | <br> | ||
<table class="wikitable"><tr><td>[[Image:Fgij-xl1.png]]</td></tr></table> | <table class="wikitable"><tr><td>[[Image:Fgij-xl1.png]]</td></tr></table> | ||
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===Conservation of Crosslinking Residues=== | ===Conservation of Crosslinking Residues=== | ||
If the PDB file is pre-processed by [[ConSurf/Index|ConSurf]], after displaying the crosslinked residues, their levels of evolutionary conservation will be shown by checking ''ConSurf Colors'' (in the middle-left of FirstGlance). For more complete instructions and illustrations, see [[Interpreting ConSurf Results]]. | If the PDB file is pre-processed by [[ConSurf/Index|ConSurf]], after displaying the crosslinked residues, their levels of evolutionary conservation will be shown by checking ''ConSurf Colors'' (in the middle-left of FirstGlance). | ||
'''For more complete instructions and illustrations, see [[Interpreting ConSurf Results]]'''. | |||
For example, | For example, below is a thioether covalent crosslink between Cys356 and Tyr379 in catalase [[1sy7]]. (The multiple sequence alignment included 150 sequences sampled from 22,855 unique similar sequences. The [[Interpreting_ConSurf_Results#Average_Pairwise_Distance|average pairwise distance]] was 0.96.) | ||
<table class=" | <table class="wikitable"><tr><td> | ||
[[Image:1sy7-fg-thioether.png]] | |||
</td><td width="350"> | |||
[[Image:1sy7-fg-consurf-thioether.png]] | |||
</td></tr><tr><td> | |||
Thioether crosslink in catalase [[1sy7]]. | |||
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<span style="font-size:160%;"> | |||
{{Template:ColorKey_Element_C}} | |||
{{Template:ColorKey_Element_N}} | |||
{{Template:ColorKey_Element_O}} | |||
{{Template:ColorKey_Element_S}} | |||
</span> | |||
</td><td> | |||
Crosslinking residues: | |||
<br> | |||
{{Template:ColorKey_ConSurf_NoYellow_NoGray}} | |||
<br> | |||
Heme: | |||
<span style="font-size:160%;"> | |||
{{Template:ColorKey_Element_C}} | |||
{{Template:ColorKey_Element_N}} | |||
{{Template:ColorKey_Element_O}} | |||
{{Template:ColorKey_Element_Fe}} | |||
</span> | |||
<br> | |||
Clicking the Cys sulfur atom (green arrow) produced the report at the bottom. | |||
</td></tr></table> | </td></tr></table> | ||
Additional crosslink examples showing conservation: | |||
*[[FirstGlance/Visualizing_Conservation#Conservation_of_Protein_Crosslinks|An isopeptide crosslink in polyubiquitin]]. | |||
*[[Interpreting_ConSurf_Results#More_Sequences_Needed|An isopeptide in crosslinked lamprey fibrin]]. | |||
==Verified Crosslink Examples== | ==Verified Crosslink Examples== | ||
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==Clashes== | ==Clashes== | ||
FirstGlance suggests the presence of a "putative crosslink" based solely on the distances between relevant atoms (click the link '''Detailed Methods''' in FirstGlance). Sometimes, instead of a real covalent bond crosslink, the proximity of the relevant atoms is due to errors in the atomic model that produce [[clashes]]. | FirstGlance suggests the presence of a "putative crosslink" based solely on the distances between relevant atoms (click the link '''Detailed Methods''' in FirstGlance). Sometimes, instead of a real covalent bond crosslink, the proximity of the relevant atoms is due to [[alternate locations]] or small errors in the atomic model that produce [[clashes]]. | ||
For each putative crosslink suggested by FirstGlance, it is the responsibility of the user to evaluate whether it is a real covalent crosslink, or a clash. FirstGlance makes this responsibility clear (see the bottom paragraph in the snapshot under step #4 above). | For each putative crosslink suggested by FirstGlance, it is the responsibility of the user to evaluate whether it is a real covalent crosslink, or a clash. FirstGlance makes this responsibility clear (see the bottom paragraph in the snapshot under step #4 above). | ||
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===Bonds=== | ===Bonds=== | ||
It is important not to take the bonds displayed by FirstGlance/Jmol literally. [[Atomic coordinate files]] from the [[wwPDB]] do not specify most covalent bonds. Rather, Jmol draws covalent "bond" sticks between atoms wherever two atoms are sufficiently close. In the case of carbon, nitrogen and oxygen, this is wherever a pair of those atoms is separated by ≤1.81 Å<ref name="bondlengths">Actual covalent bond lengths are approximately C-C: 1.4 Å; C-N: 1.4 Å; C-O: 1.4 Å; C=O: 1.2 Å C-S: 1.7 Å ([https://en.wikipedia.org/wiki/Covalent_radius Covalent radii in Wikipedia]).</ref>. In perfectly accurate models, this works correctly, but the errors in [[empirical models]] sometimes lead to bonds being drawn incorrectly. Thus, Jmol may show bond sticks where the reality is a clash, not a bond; and Jmol may fail to show bond sticks between atoms that are believed to be covalently bonded, but are too far apart in the model. | It is important not to take the bonds displayed by FirstGlance/Jmol literally. [[Atomic coordinate files]] from the [[wwPDB]] do not specify most covalent bonds. Rather, Jmol draws covalent "bond" sticks between atoms wherever two atoms are sufficiently close. In the case of carbon, nitrogen and oxygen, this is wherever a pair of those atoms is separated by ≤1.81 Å (for actual bond lengths, see footnote<ref name="bondlengths">Actual covalent bond lengths are approximately C-C: 1.4 Å; C-N: 1.4 Å; C-O: 1.4 Å; C=O: 1.2 Å; C-S: 1.7 Å ([https://en.wikipedia.org/wiki/Covalent_radius Covalent radii in Wikipedia]).</ref>). In perfectly accurate models, this works correctly, but the errors in [[empirical models]] sometimes lead to bonds being drawn incorrectly. Thus, Jmol may show bond sticks where the reality is a clash, not a bond; and Jmol may fail to show bond sticks between atoms that are believed to be covalently bonded, but are too far apart in the model. | ||
==Case Illustrations== | ==Case Illustrations== | ||