ConSurfDB vs. ConSurf

From Proteopedia

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Evolutionary Conservation is introduced at Introduction to Evolutionary Conservation, and treated in somewhat greater depth in the article Conservation, Evolutionary. These describe how conservation patterns in 3D can help to identify functional sites in proteins. Proteopedia displays conservation patterns pre-calculated by ConSurfDB, when available. These are usually based on broad protein families that include sequences of proteins with multiple functions. Consequently, they usually obscure conservation present in a family of proteins with a single function (see Caveats). The present article describes the mechanisms utilized by the ConSurfDB and ConSurf servers, and how to use the latter to reveal conservation within a family of proteins with a single function.

Contents 1 The Two ConSurf Servers 2 Examining Functions of Proteins in ConSurf-DB's MSA 2.1 Example With Multiple Functions 3 Limiting ConSurf Analysis to Proteins of a Single Function 3.1 APD Is Too High 3.2 APD Is Too Low 3.3 Too Few Sequences 3.4 Too Many Yellow Residues 4 The ConSurf-DB Mechanism 4.1 ConSurf-DB Process 4.2 Filtering 4.3 Average Pairwise Distance 5 The ConSurf Server 6 Examples 7 References

The Two ConSurf Servers

There are two ConSurf servers:

• ConSurfDB:
  • Since 2022, ConSurfDB results are NOT compatible with FirstGlance in Jmol. Visualizing and analyzing conservation results in FirstGlance has many advantages. Use the ConSurf Server for compatibility with FirstGlance.
  • In July, 2024, ConSurfDB had not been updated with new entries in the Protein Data Bank since mid-2022. To look for more recent updates, at ConSurfDB Home, click More.
  • Has pre-calculated results for every chain in the PDB.
  • Proteopedia's Evolutionary Conservation resource displays results from ConSurfDB.
  • Results typically obscure some conservation related to a protein's function because the analysis typically included proteins of multiple functions (see ConSurfDB Often Obscures Some Functional Sites).

• ConSurf Server:
  • Results can be visualized and analyzed in FirstGlance in Jmol, which has many advantages.
  • You submit proteins of interest and wait for the analysis to be completed.
  • Completely automated analysis typically gives excellent results.
  • Optionally, highly flexible with many configurable parameters and several sequence database options.
  • You can upload your own multiple sequence alignment, or phylogenetic tree, for use in the analysis.

Both servers use state-of-the-art methods that are published in peer-reviewed journals. For comparisons with other methods, see Other Evolutionary Conservation Servers.

Both servers permit you to download results. This is a good idea since the continual growth of sequence databases and improvements in analysis algorithms will give at least slightly different results for the same jobs run several months or more apart. Also, results are periodically deleted from the ConSurf server to conserve disk space.

Examining Functions of Proteins in ConSurf-DB's MSA

Since 2022, ConSurfDB results are NOT compatible with FirstGlance in Jmol. Visualizing and analyzing conservation results in FirstGlance has many advantages. Use the ConSurf Server for compatibility with FirstGlance.

In July, 2024, ConSurfDB had not been updated with new entries in the Protein Data Bank since mid-2022. To look for more recent updates, at ConSurfDB Home, click More.

As explained above, ConSurf-DB typically includes proteins with more than one function in its conservation analysis. Before deciding whether to do a ConSurf Server job that limits the analysis to proteins of a single function, you may want to see what proteins ConSurf-DB included in its analysis. Here is how to see the names (which hopefully reveal the functions) of the proteins included in ConSurf-DB's analysis of a protein chain. (The following steps are based on ConSurfDB as of July, 2024.)

1. Go to consurfdb.tau.ac.il (the DB, distinct from the ConSurf Server).
2. Enter the PDB code (PDB ID) for the protein of interest. If you get "ERROR: No chains found" it means that this PDB entry was released after the most recent update of ConSurfDB, so you should use the ConSurf Server.
3. Select the chain of interest. If you're not sure, get familiar with the structure using FirstGlance.
4. Click the button Apply, and wait for the results to load.
5. Scroll down and click Homologues, Alignment, and Phylogeny.
6. Notice the number of sequences in the MSA (the number of "hits" upon which "calculations were conducted").
7. To view the list of sequences in the multiple sequence alignment (MSA) from which the conservation pattern was determined, click on the count of hits upon which the calculations were conducted. (If fewer than 50 hits were used, it will probably be useful to do another run using a larger database: UniProt and NR are larger than the default UniProt90. Use the button "Select Run Parameters Manually".
8. Get the Average Pairwise Distance (APD) by clicking "View Alignment Details". A value > ~1.1 suggests that proteins with multiple functions were included in the multiple sequence alignment, which obscures conservation related to the function of your query protein. See Interpreting ConSurf Results, where the APD is explained.

If the list includes proteins of functions that differ from that of the protein of interest, that tends to obscure patches of conservation that exist among proteins with the same function as the query protein of interest.

Example With Multiple Functions

2vaa is a major histocompatibility complex class I protein (MHC I). ConSurfDB used 300 sequences in its calculation, and the MSA had an APD of 1.63. Starting about halfway down the list of sequence is the first MHC II protein, which has a different function. Early in the list is the first of 14 sequences for hemochromatosis proteins (unrelated in function). The list includes 62 "Ig-like domain-containing" proteins of unknown function. The inclusion of these and many other sequences for proteins with functions that differ from the query will obscure conservation of residues critical for the specific functions of MHC I proteins. Therefore, you may prefer to run a ConSurf job in which you limit the APD to a value below 1.0. See such examples below.

Limiting ConSurf Analysis to Proteins of a Single Function

This section was updated in July, 2024 to correspond to changes in the ConSurf Server.

As explained above, ConSurfDB results, and the ConSurf-DB Evolutionary Conservation scene available in Proteopedia often includes proteins with multiple functions. However, the best way to find all functional sites by conservation analysis is to limit the analysis to proteins with a single function.

When the Average Pairwise Distance (APD) in the multiple sequence alignment (MSA) approaches or exceeds approximately 1.0, it is likely that proteins with multiple functions have been included in the MSA. To see conservation that reflects the function of the query protein, it is best to use an MSA with an APD in roughly the range 0.3-0.6. Sometimes, the ConSurf Server's result with default settings may give such a result. If not, you may wish to do additional ConSurf runs with the goal of reducing the APD.

Prior to 2022, the ConSurf Server enabled manual selection of sequences. Unfortunately, after a 2022 update to the ConSurf Server, this is no longer practical. Hence we are limited to adjusting run parameters "in the dark".

1. Go to consurf.tau.ac.il, the ConSurf Server (distinct from ConSurf-DB).
2. Enter your PDB ID in the slot (or upload your PDB file).
3. IGNORE the "pre-calculated ConSurfDB analysis" if offered. (Presumably you've already decided it has an APD value too high, and it will not work in FirstGlance in Jmol.)
4. Enter a title. Best if the title includes abbreviations for the custom settings you plan for this run. For example, if you are running 2vaa chain A with the UniRef90 database, 150 sequences sampled from the unique hits, with maximal %ID 99% and minimal %ID 60%, the title might be "2vaa u90 150s 99-60".
5. Enter your email address.
6. Click Select Run Parameters Manually.

Below are suggestions for selecting the run parameters.

APD Is Too High

As explained above and under Interpreting ConSurf Results, when the APD approaches or exceeds 1.0, conservation related to the function of the query protein may be obscured due to inclusion in the MSA of proteins with multiple functions. There are several ways to get less diversity in the MSA:

1. Increase the "Minimal %ID" from the default 35% to perhaps 60%-75%. This will exclude the more distantly related sequences.
2. Increase the "Maximal %ID" from the default 95% to perhaps 98%. This will include more sequences closely related to the query.

The above two actions are usually sufficient. Actions with larger effects are:

1. The default is to choose the 150 sequences in the MSA by uniform sampling of the unique hits. This sample includes both the closest and farthest sequences from the query. Change "sampling" to closest to the unique hits. This typically makes a large drop in the APD. You could reduce the drop by increasing the number of sequences used from 150 to perhaps 250. The more sequences you use, the longer the job will take to complete.
2. Try getting sequences from SwissProt instead of UniRef90. SwissProt has roughly 100-fold fewer sequences than does UniRef90. This tends to reduce the APD, but you may have difficulty getting enough sequences to avoid a large number of residues with "insufficient data" (colored yellow).

APD Is Too Low

When the APD drops close to, or falls below, about 0.2, conservation will be exaggerated because of limited diversity in the MSA. You could increase diversity:

1. Decrease the "Maximal %ID" below the default of 95%, perhaps to 80% or 60%. This will exclude the most closely related sequences.
2. Search a larger database of sequences. UniProt has more sequences than the default of UniRef90, although the additional sequences may not add much diversity. The NR (Non-Redundant) database is the largest (twice as many sequences as UniProt), but contains more errors.

Too Few Sequences

If the default search for sequence homologs fails to find the minimum of 5 sequences, or if you have more than a few yellow residues (yellow means insufficient data to assign a meaningful conservation value):

• Under "Choose parameters for homolog search algorithm", change the Protein Database to UniProt or NR (larger databases than the default Uniref90).
• Increase the "Maximal %ID between sequences" from the default of 95% to perhaps 98%.

If the larger database does not give enough sequences, you can use other options to widen the search for sequences, knowing that you will be retrieving sequences less related to the query sequence, likely including proteins with functions differing from that of the query:

• Increase the number of iterations in the search to more than the default of one. Each iteration generates a sequence profile that is used as a query for the next iteration.
• Increase the default E cutoff of 0.0001, for example, to 0.001 or 0.01.

Too Many Yellow Residues

If more than a few residues are yellow, it means that the MSA had insufficient data to assign meaningful conservation values to the yellow residues. Try to increase the number of sequences in the MSA: See #Too Few Sequences.

If an entire domain of your query protein is   yellow (insufficient data)  , it is because the multiple sequence alignment (MSA) has poor coverage of the yellow domain. In this case, it is best to do separate ConSurf runs for each domain in your protein.

1. Determine sequence numbers for the linkers between domains. This can be done easily by inspection in FirstGlance in Jmol.
2. Using a plain text editor, delete everything in your PDB file except the lines that begin with ATOM or HETATM. (Its OK to leave the lines ANISOU if present.)
3. Based on sequence numbers, separate the domains into different PDB files.
4. Upload each domain's PDB file to ConSurf as a separate job.

The ConSurf-DB Mechanism

In January 2018: ConSurfDB had not been updated with new entries in the Protein Data Bank since January, 2013.

Because results from the ConSurf DataBase server, ConSurf-DB^[1] are displayed within Proteopedia as Evolutionary Conservation, an overview of its methods is provided here. ConSurf-DB pre-calculates conservation levels for each amino acid in every protein chain in the Protein Data Bank. It went into service in 2008. It uses state-of-the-art methods, all published in peer-reviewed journals^[1].

ConSurf-DB Process

1. A list of unique protein chains is extracted from the Protein Data Bank. Chains shorter than 30 amino acids are not processed because they do not contain enough information for reliable phylogenetic tree construction. Certain non-standard residues are converted to the closest standard amino acids, for example, selenomethionine MSE is converted to MET. Chains that still have more than 15% non-standard residues are not processed. Chains that could not be processed are colored gray in Proteopedia -- see the color key at the top of this page.
2. The amino acid sequence of each protein chain is submitted to HMMER for collection of related sequences from the UniRef90 database. By default, one iteration is performed using an expectation value^[2] cutoff of 10^-4.
3. The found sequences are then filtered (see below) using a scheme that attempts a balance between limiting the sequences to close homologues, and including distant sequences that do not share structure or function.
4. The filtered sequence set is multiply aligned with MAFFT (a multiple sequence alignment algorithm that out-performs older algorithms such as MUSCLE and CLUSTALW).
5. A phylogenetic tree is constructed from the multiple sequence alignment (MSA) using the Rate4Site program developed by the ConSurf team.
6. Rate4Site then calculates an evolutionary rate for each position in the MSA using a Bayesian approach shown by the ConSurf team to be superior^[3]. "The amino acid evolution is traced using the JTT^[4] substitution model. High evolutionary rate represents a variable position while low rate represents an evolutionarily conserved position."^[1]
7. "The conservation scores are normalized so that the average over all residues is zero, and the standard deviation is one."^[1] Thus, conservation scores are relative, not absolute and comparing them between different protein families might be misleading (see Caveat above).
8. The normalized conservation scores are then divided into nine levels from 1 (highly variable) to 9 (highly conserved).
9. Colors mapped to the nine conservation levels, from turquoise (1) to burgandy (9) are applied to the 3D protein structure visualized in FirstGlance in Jmol. A coloring script for RasMol is also provided.

1. A confidence interval for the conservation level is calculated for each amino acid position in the MSA. When this indicates low reliability, the position is colored yellow, signifying that the data were insufficient to assign a meaningful conservation level.

11. An Average Pairwise Distance (APD) is calculated to describe the diversity of sequences in the MSA (see below).

The results of each stage of the above process may be viewed for each chain at ConSurf-DB. In the initial run (February 2008), roughly 100 computer CPU's were utilized concurrently via a distributed computing system. Processing of the 30,918 unique protein chains in the PDB took about five days, or an average of roughly 30 minutes per chain.

Filtering

Filtering of the sequences gathered for each protein chain is crucial to making the ConSurfDB results maximally informative. Filtering consists of the following steps.

1. Sequences with more than 95% sequence identity to the query sequence are discarded.
2. Sequences shorter than 60% of the query sequence are discarded.
3. Locally aligned sequence fragments that overlap by over 10% are discarded.
4. Redundant sequences (>95% identical) are removed using CD-HIT^[5].
5. A maximum of 300 sequences meeting the above criteria is used (the 300 with the lowest expectation values^[2], that is, most closely related to the query sequence).
6. If the above process yields fewer than 5 sequence homologs, no calculation is performed due to insufficient data. In February, 2008, this occurred for 1,348 chains out of 30,918 (4%).

Average Pairwise Distance

The Average Pairwise Distance (APD) is an important measure of the diversity in the multiple sequence alignment. To learn how to interpret it, please see above, and Interpreting ConSurf Results.

The ConSurf Server

The ConSurf Server, first available in 2001^[6][7][8] with many subsequent enhancements, can calculate and display the conservation pattern for 3D structures completely automatically. It should be used whenever the pre-calculated result at the ConSurf-DB needs improvement (for example, see above), or if you have your own multiple sequence alignment (MSA) that you wish to use. The default settings of ConSurf may need to be adjusted in order to get an optimally informative result. The main adjustment needed is to gather an adequate number of sequences for proteins of the same function as your protein of interest (see above).

Like ConSurf-DB, the ConSurf Server uses the same state-of-the-art methods, all of which are published in peer-reviewed journal articles. Unlike ConSurf-DB's pre-calculated results the ConSurf Server permits considerable customization. For example, the user may specify the number of sequences to use, choose the database from which sequences are obtained, set the Expectation cutoff^[2], set the number of HMMER iterations, or submit their own multiple sequence alignment, or phylogenetic tree. Also you can upload your own PDB file, which enables you to process unpublished data, theoretical models, or "trimmed" chains, e.g. a domain of interest from a multiple-domain chain.

In brief, the ConSurf Server uses the following process by default:

1. Obtains the protein sequence for the specified PDB code (or uploaded PDB file) and chain.
2. Gathers closely related sequences from UNIREF90 (or another database that you specify) with an HMMER search (or other algorithm that you specify). E value cutoff^[2], number of iterations, and number of sequences to use are configurable.
3. Filters the sequences, by default eliminating those redundant at 95% or higher identity with each other, and those with less than 35% sequence identity to the query sequence. These percentages are adjustable.
4. Does a multiple sequence alignment with MAFFT. (Or you can choose a different algorithm or upload your own MSA.)
5. Constructs a phylogenetic tree using neighbor joining with ML distance. (Or you can choose a different algorithm or upload your own tree.)
6. Calculates a conservation score with confidence interval for each amino acid. Classifies the conservation scores into nine levels, and maps them to standard conservation level colors (see color key at the top of this page). Marks residues for which the conservation score confidence interval is too large, hence the conservation score is unreliable ("insufficient data").
7. Displays the protein, colored by conservation, in interactive 3D, using the NGL Viewer, FirstGlance in Jmol, Chimera, or PyMOL.

Examples

Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image With default parameters, the ConSurf Server results have an average #Average Pairwise Distance (APD) of 1.00[9], and an average of only a few "yellow" residues with insufficient data.[10] Case #1: MHC The alpha chain of Major Histocompatibility Complex (MHC) protein has a groove that binds a wide range of peptides, and a small loop that binds CD8. Our example is 2vaa (mouse H-2Kb). 2vaa contains three chains. Here, ConSurf colors are applied only to the alpha chain (chain A), while the beta chain (chain B) and the peptide (chain P) are shown as gray backbone traces. See also How to Insert a ConSurf Result Into a Proteopedia Green Link. ConSurfDB ConSurfDB has a result (NOT SHOWN) with an APD of 1.63. Conservation of important residues in the groove is obscured by inclusion in the MSA of proteins with different functions (see analysis above). The sides of the groove are variable, as expected (enabling it to bind a wide range of peptide sequences). The only groove residue that is conserved at greater than level 6 is Leu5 (level 8), part of a hydrophobic pocket that binds a peptide "anchor residue". Only a handful of surface residues are highly conserved (level 9), including Trp274 involved in binding CD8. UPDATE IN PROGRESS: Eric Martz 21:24, 23 July 2024 (UTC) Examples of conserved patches on other proteins, revealed by ConSurf, will be found in the articles on Lac repressor Avian Influenza Neuraminidase, Tamiflu and Relenza Mechanosensitive channels: opening and closing

References