View source for FirstGlance/Visualizing Conservation

[[FirstGlance/Index|FirstGlance in Jmol]] is the easiest-to-use tool for visualizing, understanding, and sharing protein molecular structure. It offers guided exploration with extensive explanations and examples, yet provides ample power for advanced users. With a few mouse clicks, any molecular scene can be saved as an '''animation (GIF file) ready to drop into a Powerpoint Slide''' -- see examples below, and [http://tinyurl.com/movingmolecules tinyurl.com/movingmolecules].

Among many conveniences offered by FirstGlance, it is easy to see the levels of [[ConSurf/Index|evolutionary conservation determined by the ConSurf Server]] for '''amino acids of special interest'''. Below is an overview with some illustrated examples. Here are [[ConSurf/Index|instructions for running the ConSurf Server]] on proteins of interest. ''Note that you '''cannot''' use FirstGlance to visualize results from the ConSurf Database, ConSurfDB. Unfortunately, the interface to FirstGlance was removed from ConSurfDB PDB files in 2022.''

There are 2 ways to analyzie your own ConSurf Server results in FirstGlance:
:1. At the ConSurf Server results page, open ''High Resolution Figures and PDB Files'', and then click ''Download ConSurf PDB File for FirstGlance in Jmol''. Now, you can [http://firstglance.jmol.org/where.htm#u upload the downloaded file to FirstGlance]. By downloading the PDB file, you will have it after the results '''disappear''' from the ConSurf Server.

:2. At the ConSurf results page, '''Right''' click the link ''Download ConSurf PDB File for FirstGlance in Jmol'', and select '''Copy Link'''. Then, at FirstGlance, click [http://firstglance.jmol.org/where.htm#u enter a molecule's URL], paste the address into the slot, and click Submit.


==Initial View of Conservation==
Initially, FirstGlance colors each amino acid in the protein chain processed by ConSurf according to its level of conservation. This gives you an overview, enabling you to see patches of highly conserved residues (functional sites).

===Biological Unit 1 Shown Initially===

FirstGlance automatically constructs and displays [[Biological Unit]] 1 (believed to be the major functional quaternary assembly) in its initial view. If the protein chain processed by ConSurf is duplicated, you
will have the option of applying conservation colors to all chains that are identical in sequence with
the ConSurf-processed chain (see snapshot in next section).

To find out whether any other biological units that are specified, and to display each, or to display the [[asymmetric unit]], you will need to go to the FirstGlance Control Panel: see [[#Go_To_The_FirstGlance_Control_Panel|instructions below]].

===Multiple Chains===

If the model has more than one copy of the chain processed by ConSurf, you can apply conservation colors to all of those sequence-identical chains. Here, for example, is a potassium channel homotetramer:
<br>
<table class="wikitable"><tr><td>
[[Image:1bl8-fg-consurf-chains.gif]]
</td></tr></table>

===Optimization===
Before going further, it is good to decide whether the results are satisfactory, or need optimization. Lets consider [[7bak]], a drug-target protease of SARS-CoV-2 (see [https://pdb101.rcsb.org/motm/242 Coronavirus Proteases]).

:[http://firstglance.jmol.org/fg.htm?mol=7bak_consurf1641000454_pipe.pdb Display 7BAK ConSurf Result in FirstGlance]

A ConSurf run with all default settings left 10% of the residues with insufficient data<ref name="insufficientdata">When the confidence interval for a conservation value of an amino acid is too large, ConSurf reports "insufficient data" (uncertainty: yellow color). See [https://consurf.tau.ac.il/quick_help.php#RELIABILITY Position Specific Quality] and [https://consurf.tau.ac.il/overview.php#_Toc311131298 Confidence Interval] in the ConSurf documentation.</ref> (yellow color), and the diversity in the multiple sequence alignment (MSA) was too low ([[Interpreting_ConSurf_Results#Average_Pairwise_Distance|average pairwise distance]] 0.34), leaving conservation grades 2 and 3 almost unoccupied. These inadequacies are highlighted by a distribution report displayed by FirstGlance.
<br>
[[Image:7bak-consurf-dist-defaults.png]]

To improve the results, a new ConSurf job for 7bak was submitted, increasing the HMMER search iterations from 1 to 2, and increasing the maximum number of sequences in the MSA from 150 to 300. The results were satisfactory, with a more reasonable APD of 0.77.
<br>
[[Image:7bak-consurf-dist-2its-300max.png]]

For more examples, see [[Interpreting ConSurf Results]]

==Initial View: Conserved Face==
The initial view utilizes the &quot;ConSurf Control Panel&quot; (labeled ''ConSurf View''). One side of the molecule is more conserved than the opposite side. The more conserved face includes the catalytic site.
<br>
<table class="wikitable"><tr><td>
[[Image:7bak-consurf-view.png]]
</td><td>
[[Image:7bak-consurf.gif]]
</td></tr></table>

===Animations for Slides===
It is very easy to save any molecular view in FirstGlance as a GIF animation, ready to drop into a slide in Powerpoint (or other presentation software): [http://firstglance.jmol.org/slides.htm Instructions]. The animation above is one example. More examples: [http://tinyurl.com/movingmolecules tinyurl.com/MovingMolecules].

==Go To The FirstGlance Control Panel==
<table><tr><td valign="top">
To further explore the pattern of conservation, we need to change from the ''ConSurf View'' to the ''FirstGlance Control Panel''. Click the link indicated by the red arrow in the above snapshot.

The ''FirstGlance Control Panel'' has 5 tabs (see snapshot at right). The ''Molecule Information Tab'' and the ''Views'' tab each provide a wealth of information.

Notice that you can move freely back and forth between the ''ConSurf Controls'' and the ''FirstGlance Controls'' using the link '''ConSurf Controls''' in the middle of the left side of FirstGlance.

===Biological Units===

Note the link ''Showing Biological Unit 1'' in the ''Molecule Information Tab''. Clicking this will show a report in the lower left panel of FirstGlance listing any other biological units that are specified. Links are provided to display any of these, or the asymmetric unit, in a separate section.

Example: Bacterial gas vesicle [[7r1c]].<br>
[http://firstglance.jmol.org/fg.htm?mol=7r1c_N_consurf_APD0.59.pdb Display ConSurf-Processed 7r1c in FirstGlance].
</td><td>
[[Image:7r1c-consurf-fg-biounits.png]]
</td></tr></table>

==Conservation of Salt Bridges==
===Show Salt Bridges===
Click the Tools tab, and there click ''Salt Bridges/Cation-Pi..'' (red arrow below). This shows all the protein-protein salt bridges in the model where <span style="color:red;font-weight:bold;">Negative (Asp, Glu)</span> charges are within 4.0 &Aring; of <span style="color:blue;font-weight:bold;">Positive (Arg, Lys)</span> charges.
<br>
<table class="wikitable"><tr><td>
[[Image:7bak-fgij-salt-bridges.png]]
</td><td>
[[Image:7bak-salt-bridges.png]]
</td></tr></table>

===Color by Conservation===
To see how well the salt-bridged residues are conserved, click the checkbox ''ConSurf Colors'' (red arrow below). Declutter the view by unchecking ''Show Backbones'' (green arrow below). Scroll down in the lower left panel and click {{Yelspan|List All}} to get a spreadsheet-ready list that includes conservation values for each pair of salt-bridged atoms.
<br>
<table class="wikitable"><tr><td>
[[Image:7bak-fgij-salt-bridges-consurf-colors.png]]
</td><td>
[[Image:7bak-salt-bridges-nobb.png]]
</td></tr></table>

===Label All Residues===

You can identify residues by touching them (hold the mouse still, and a tooltip appears), or clicking on them. But it may be more convenient to label all salt bridge residues. Scroll down to the ''Focus Box'' in the upper left panel. Here you can check ''Sequence Numbers'' and ''Residue Names'' (red arrows below). If you wish, you can hide the '''S-''' labels that designate [http://firstglance.jmol.org/notes.htm#icsc incomplete sidechains] by unchecking the checkbox ''Labels, Show'' (green arrow below).
<table class="wikitable"><tr><td>
[[Image:7bak-fgij-focus-box.png]]
</td><td>
[[Image:7bak-salt-bridges-consurf-colored.png]]
</td></tr></table>

===Conclusions for Salt Bridges===
Conservation of residues engaged in salt bridges varies over the entire range of conservation grades from 1 to 9. In some bridges, both residues are highly conserved (grades 8 or 9). In others, one partner is highly conserved, while the other is not. In some pairs, neither partner is conserved.

==Conservation of Cation-Pi Interactions==
This can be done the same way as Salt Bridges. Simply click the radio button to switch from Salt Bridges to Cation-Pi interactions (see snapshots above).

==Conservation of Residues Binding Ligand/Substrate==
FirstGlance provides an easy and powerful tool to explore residues contacting any specified portion of the model. For this example, we'll use [[acetylcholinesterase]] complexed to the [https://en.wikipedia.org/wiki/VX_(nerve_agent) inhibitor VX], crystal structure [[6cqz]].

:[http://firstglance.jmol.org/fg.htm?mol=6cqz_consurf1641072297_pipe.pdb Display 6CQZ ConSurf Result in FirstGlance]

We'll look at the conservation of the residues that contact the ligand/inhibitor VX. From the ''ConSurf View'', go to the FirstGlance Control Panel. 
<br>
<table class="wikitable"><tr><td width="300">
#Click '''Contacts & Non-Covalent Interactions..''' in the '''Tools tab'''.
#Click Cartoon in order to see the ligand VX buried in the protein.
#Click Residues/Groups.
#Click on the green buried ligand to select it as the "target". (The larger green mass on the surface is glycosylation.)
#Click ''Show Atoms Contacting Target''.
</td><td>
[[Image:6cqz-consurf-contacts-ligand-selection.png]]
</td></tr></table>

The atoms that are likely bonded, covalently or non-covalently, to the target ligand VX are now shown as balls.

<table class="wikitable" ><tr><td width="300">
1. Click '''Center Contacts''' and zoom in (mouse wheel).

2. Click the '''Label Contacts''' checkbox.

3. There are 4 "snapshots" at the lower left. Clicking each one renders the contacts differently. The most detailed view is given after you '''click the 4th snapshot'''.

4. Uncheck ''Color Target Gray''. VX is now colored by chemical element:
{{Template:ColorKey_Element_C}}
{{Template:ColorKey_Element_O}}
{{Template:ColorKey_Element_P}}

5. Uncheck ''Backbones'' to declutter the view.

6. Toggle the ''Background'' (in the gray Button Box) as needed to see light or dark labels.

7. Scroll down in the ''Contacts Shown'' (lower left) panel to see the color key and the paragraph marked &dagger; which explains this view.

8. '''ConSurf Colors''' is checked by default, but you can uncheck it to color by chemical element.

</td><td>
[[Image:6cqz-consurf-vx-contacts-shown.png]]
</td><td>
[[Image:6cqz-consurf-vx-contacts-shown.gif]]
</td></tr></table>

'''Conclusions:''' Ser203 is covalently bonded to the phosphorus in VX. His447 (3.4 &Aring;) and Gly121 (2.9 &Aring;) are likely [[Hydrogen bonds|hydrogen-bonded]] to oxygens in VX.

For instructions on how to measure a distance, click ''Distances/Angles'' in the Tools tab. You can later return to the Contacts Shown controls by clicking ''Return to Contacts'' which will appear in the middle left next to ''ConSurf Colors''.

In human acetylcholinesterase, the catalytic triad residues<ref>PMID: 24900610</ref> are Ser203, His447, and Glu334. We can see that the first two are highly conserved, but what about Glu334 which is not contacting VX?

==Conservation of Specified Residues==
This section explains how to visualize the conservation of any residues that you specify. The snapshot below is a continuation of the previous section, but this procedure works for any specified residues in any ConSurf result.

===Glu334 in 6CQZ===

In the previous section, we remained curious about the conservation level of Glu334 in 6CQZ, a member of the catalytic triad of human acetylcholinesterase.

<table class="wikitable"><tr><td width="300">
1. Click '''Find..''' (in the ''Focus Box'').

2. In the ''Find'' slot, type
<br>
<span style="font-family:monospace;font-size:120%;">Glu334 and chain=A</span>
<br>
and press Enter. (Specifying chain A excludes the Glu334 in chain B. Scroll down below the slot to see instructions on how to find other things.) Yellow halos now mark the positions of the atoms found, even when those atoms are not rendered as balls or sticks.

3. Click '''Center Atoms with Halos'''.
</td><td>
[[Image:6cqz-consurf-fg-find.png]]
</td></tr></table>

<table class="wikitable"><tr><td width="300">
4. Click the ''Views'' tab at the very top.

5. Click '''Vines/Sticks'''.

6. Depress the '''Slab''' button to declutter the view. (Adjust the slab thickness if needed.)

7. Check '''ConSurf Colors'''.

<b>Conclusion:</b> Now we can see that Glu334, the residue with yellow halos, is highly conserved. To confirm its identity, touch it to see a tooltip, or click on it. Or you can label all residues as shown in the [[#Label All Residues|example above]].
</td><td>
[[Image:6cqz-consurf-fg-glu334.png]]
</td></tr></table>

===All Tryptophans===

Tryptophans are known to orient at, and parallel to, the apolar-polar interface in transmembrane channels such as the potassium channel<ref name="koeppe2008">PMID: 18550546</ref><ref name="allen2013">PMID: 22989724</ref><ref name="walrant">[https://www.mdpi.com/2073-4352/11/9/1032/htm  Tryptophan, an Amino-Acid Endowed with Unique Properties and Its Many Roles in Membrane Proteins], Sonia Khemaissa, Sandrine Sagan,
and Astrid Walrant. ''Crystals'', 2021, '''11'''(9), 1032; [https://doi.org/10.3390/cryst11091032 doi.org/10.3390/cryst11091032].</ref>. Let's see which Trps are conserved in the potassium channel [[1bl8]] (a [[Nobel_Prizes_for_3D_Molecular_Structure#2000-2009|Nobel Prize-winning structure]]!).

:[http://firstglance.jmol.org/fg.htm?mol=1bl8_consurf1640894833_pipe.pdb Display 1BL8 ConSurf Result FirstGlance]

<table class="wikitable"><tr><td colspan=3><center>
Potassium Channel [[1bl8]]
</center></td></tr><tr><td>
[[Image:1bl8-chains.gif]]

</td><td width="270">
[[Image:1bl8-opm.gif]]

</td><td width="270">
[[Image:1bl8-trp.gif]]

</td></tr><tr><td><center>
4 chains with <span style="color:purple;font-weight:bold;">K+</span>.
<br>
FirstGlance:
<br>
''Cartoon'' (Views tab),
<br>
Toggle ''Ligands+'' button twice (Button box).
</center></td><td><center>
{{Template:ColorKey_Hydrophobic}},  {{Template:ColorKey_Polar}}.
<br>
<b>Lipid bilayer boundaries:</b>
<br>
<span style="color:red;font-weight:bold;">Extracellular</span>, <span style="color:blue;font-weight:bold;">Cytoplasmic</span>.
<br>
FirstGlance: Resources tab,
<br>
''Show Lipid Bilayer Boundaries''*.

</center></td><td><center>
<span style="color:yellow;background-color:black;font-weight:bold;padding:3px 4px 1px 4px;">Trp positions</span>.
<br>
FirstGlance:
<br>Views tab, ''Thin Backbones''.
<br>Find (Focus Box), enter "Trp".

</center></td></tr></table>
<nowiki>*</nowiki> FirstGlance uses the [https://opm.phar.umich.edu/ Orientations of Proteins in Membranes] (OPM) Server at the University of Michigan.

====Conservation of Trp====

<table class="wikitable"><tr><td>
After entering "Trp" in the ''Find'' slot:

1. Views tab: ''Vines/Sticks''.

2. Check ''ConSurf Colors''.

3. Isolate: ''Atoms with Halos''.

4. Find: ''Clear Halos''.

5. [[#Label All Residues|Label the Tryptophans]].
</td><td>
[[Image:1bl8-consurf-fg-isolate-trp.png]]
</td><td>
[[Image:1bl8-trp-conservation.gif]]
<br><br>
{{Template:ColorKey_ConSurf_NoYellow_NoGray}}
</td></tr></table>

====Conclusions====
Only Trp67 and Trp68, near the extracellular opening of the channel, are highly conserved.

==Conservation of Protein Crosslinks==
FirstGlance automatically detects [[Protein crosslinks|covalent protein crosslinks of six types]], plus disulfide bonds. FirstGlance has tools that make it easy to examine each such crosslink in detail. If the PDB file has been processed by [[ConSurf/Index|ConSurf]], it is easy to see the levels of conservation of the crosslinked residues.

===Isopeptide-Linked Ubiquitin Trimer===

An example is isopeptide bond-linked chains of ubiquitin. [[7cap]] is an isopeptide-linked trimer of ubiquitin. The carboxy-terminal Gly76 forms an isopeptide bond with Lys48. Here is one ConSurf result (300 sequences in the multiple sequence alignment (MSA), sampled from 10,122 unique similar sequences. The MSA had an [[Interpreting_ConSurf_Results#Average_Pairwise_Distance|average pairwise distance]] of 1.15):

:[http://firstglance.jmol.org/fg.htm?mol=7cap_consurf1639680092_pipe.pdb 7CAP Conservation in FirstGlance]

First, follow the [[FirstGlance/Evaluating_Protein_Crosslinks#Instructions_for_an_Isopeptide_Bond|instructions for getting a list of the isopeptide crosslinks]].

Then, proceed as follows:

<table class="wikitable"><tr><td width="300">
1. Click on one of the crosslink bonds (bottom red arrow).

2. Check the box to apply ConSurf Colors (middle red arrow).

3. Touch a residue (hold the mouse still) to pop up a tooltip reporting its ConSurf conservation Grade, as shown in the yellow box on Lys48.


<b>Conclusions:</b> The C-terminal Gly76 is highly conserved, while Lys48 is less conserved. (Grade 7 in this particular ConSurf job. Grades 5 or 6 in other jobs with fewer sequences in the MSA.)
</td><td>
[[Image:7cap-isopeptide-conservation.png]]
</td></tr></table>

==Other Crosslink Examples==
Other examples of covalent protein crosslinks colored by evolutionary conservation are:
*[[FirstGlance/Evaluating_Protein_Crosslinks#Conservation_of_Crosslinking_Residues|A thioether crosslink in catalase]].
*[[Interpreting_ConSurf_Results#More_Sequences_Needed|An isopeptide in crosslinked lamprey fibrin]].

==See Also==
*[[ConSurf/Index]]: List of Proteopedia pages about ConSurf.
*[[FirstGlance/Index]]: List of Proteopedia pages about FirstGlance in Jmol.

== References ==
<references/>