SRide help page

Stabilizing Residues in known tertiary protein structures are identified using the properties surrounding hydrophobicity, long-range order, stabilization center and conservation score. This concept was first presented for TIM-barrel proteins in Magyar et al. (2005) Nucleic Acids Research 33:W303. In this consensus approach we identified Stabilizing Residues according the following conditions:
(i) Hp ≥ 20 kcal/mol (ii) LRO ≥ 0.02; (iii) SC ≥ 1 and (iv) Conservation score ≥ 6.
These are the default values for the SRide server, however the user now has the choice of changing these values.
The run time is in the range from 5 seconds (4pti, 10 sequences) up to 10 minutes for large proteins with 50 sequences.

Submission:

Select protein structure: Proteins with known three dimensional structures can be submitted to the server. The server takes a four letter PDB code or an uploaded coordinate file in PDB format as input. The server can process files containing the atomic coordinates only (ATOM records). There is no default value.

Select protein chain: The calulations are carried out for a single protein chain. The user has to specify a valid one letter chain identifier present in the structure. of polypeptide chain. If the sturcture contains only a single chain, or the first chain is of interest, user can leave the default value “*”.

Threshold values: All relevant parameters can be set by the user.

E-value: The first step of the procedure is a PSI-BLAST search for homologous sequences in the Swissprot database. The E-value criteria of this search can be set here. If there are only a few homologous sequences in the database and the E-value is set to a strict (low) value, the number of homologous sequences found by the BLAST search will be too low. In such cases relaxing (increasing) the threshold can result in more hits. If the number of sequences is too low, the calculated conservation scores would not be reliable. Default value: 0.001

N_max.seq: The maximum number of homologues, from those found by PSI-BLAST (with the given E-value), which are aligned with the ClustalW program. A minimum value of 10 is recommended. Default value: 50

Cons score: The conservation score is transformed into discrete values. If a residue has a higher score than this Cons score threshold, it is accepted as a potential Stabilizing Residue (SR). If the zero value is choosen, the calculation is still done, but all residues are selected for further processing. Default value: 6

LRO: The Long-range order (LRO) of residue 'i' is the number of long range contacts divided by the total number of residues in the polypeptide chain. Two residues are in long range contact if they are at least 12 position away in the sequence, but their alpha-carbon distance less than 8Å. If a residue has a higher LRO value than this thershold it is accepted as a potential SR. . If the zero value is choosen, the calculation is still done, but all residues are selected for further processing. Default value: 0.02

H_P: Surrounding hydrophobicity H_P(i) of residue 'i' is calculated as the sum of hydrophobic indices, obtained from thermodynamic transfer experiments, of residues whose alpha carbon atoms are within the distance of 8Å from the alpha carbon atom of residue i. If a residue has a higher surrounding hydrophobicity value than this threshold it is selected as a potential SR. If the zero value is choosen, the calculation is still done, but all residues are all residues are selected for further processing. Default value: 20

SC check: The definition of stabilization centers is the following: Two residues are in contact if there is at least at least one heavy atom-atom distance smaller than the sum of their van der Waals radii plus 1Å. A contact is recognizied as "long-range" interaction if the interacting residues are at least 10 amino acids apart. Two residues are involved in a stabilization center if they are in long-range interaction, and if it is possible to select one-one residues from both flanking tetrapeptides of these two residues that make at least seven contacts out of the possible 9 between these two triplets. If SC check value “yes” is selected, only residues which are involved in stabilization centers are selected as potential SRs, while using the value “no” the calculation is done, but all residues are selected. If a residue in involved in a Stabilization Center, its SC value becomes 1, and 0 otherwise. Default value: “yes”.

E-mail address: Since the conservation score calculation can be a time consuming step depending on the size of the protein chain and the number of homologous sequences, results are submitted via e-mail. To avoid superflous calculations with non existent e-mail addresses, only registered e-mail addresses are accepted. During the registration procedure a code is sent to the address. The e-mail adress becames registered, when this “registration” code is copied back to the registration page.

Output:

The sequences of the queried polypeptide chain is printed.

The Swiss-Prot Primary Accession Number of all homologs are outputted from the PSI-BLAST run, which fullfill the E-value and N_MAX.SEQ citeria.

The method of submission (PDB code, or upload) is printed together with the selected chain identifier.

The selected threshold values are printed.

The list of residues which are in agreement with the given thresholds is printed, together with the calculated conservation score, LRO, and Hp values. Under SC “1” is printed if the given residues in involved into an SC formation, or “0” when it is not. The values are TAB separated. Data for all residues can be listed when threshold zero is choosen for Cons score, LRO, H_P and “no” for SC.

Important note: If you try to compare the results published in our paper about TIM barrel proteins with the output of this server you may find some differences in the list of SRs. These differences can be accounted for the following facts:
(1) In the SRide server more accurate Van der Waals atom radii are used. This influences if a residues is classified as SC, or not.
(2) The conservation scores calculated with our server slightly differ from the scores calculated with the Consurf server. This is probably due to the fact that we use a more current version of ther ClustalW alignment program.

Abbreviations:
LRO: Long Range Order
SC: Stabilization Center
SR: Stabilizing Residue
H_p: Surrounding hydrophobicity

Flow chart:

1) PSI-BLAST search for homologous sequences in the Swiss-Prot database
2) ClustalW alignment of sequences
3) Calculation of conservation scores with the Rate4Site aligorithm
4) Surrounding hydrophobicity calulation
5) LRO calculation
6) Identification of SC elements
7) Selection of residues according the threshold values
Steps 1-3 are taken from Consurf

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