What is Quinores3D web server?

Quinores3D web server was developed primarly for scientists to help in indentifying and characterizing point mutations occuring in the quinolone target proteins of Streptococcus pneumoniae. It is a collection of different web tools comprising:

Quinores3D finder for the identification of point mutation

Quinores3D database which contains information about mutations associated with quinolone resistance

Quinores3D primers for the design of DNA primers using primer3tool

Quinores3D finder

Quinores3D finder - Inputs and user selection

Quinores3D finder requires a nucleotide or protein sequence in order to perform the analysis. Users can upload a complete genome (for example in genbank format, or an assemblied one obtained from sequencing), the gene sequence of interest, the protein sequence, or a plain file containing a list of mutations to analyze. User must specify the input type (using the dropdown menu) and the protein of interest ('gyrA/gyrB/parC/parE').
Examples of input files can be downloaded here: input_examples.zip

Quinores3D finder -Homology modelling

In order to perform the structural analysis, a three dimensional model of the protein is created by homology modelling using MODELLER and as a template the corrispective protein from S.pneumoniae.

Quinores3D finder -Identification of point mutations

All mutations are identified through sequence alignment with the corrispective reference protein sequence using blastp.

If input is a nucleotide sequence ('genome' or 'gene') it is firstly converted into aminoacidic sequence with blastx.

Protein sequence is aligned with the corrispective reference protein sequence and the result is parsed with a Biopython script

If input is 'Mutation', reference protein sequence is mutated in silico using a script developed in Biopython

Mutations identified are mapped on the three dimensional protein model with chimera

All aminoacid variations found are compared with the Quinores3D database in order to find the quinolone resistance associated ones. For more information about references used for the analysis, pleas read 'Materials and Methods' section.

Quinores3D finder -Structural analysis

Stuctural analysis is performed using different tools such as Chimera,PDB2PQR,APBS,DSSP.

Binding site analysis

Analysis of the binding site is done using Chimera. This analysis is applied only for the mutation falling in the quinolone binding region, both for known mutation/position associated with drug resistance and for mutation identified near (< 5 Å ) to drug or QRDR aminoacid. First, residues at 5 Å are selected, then hbonds, favorable and unfavorable contacts are calculated using the 'findhbond' and 'findclash' functions both for the wild type and the mutated aminoacid (after side chain replacement). 'Hbond_drug' refers to the possible hbonds between drug and the residue of interest , while 'Hbond_other' refer to hbonds between the residue and other residues nearby. Similar for contact and clash. According to Chimera definition: "clashes - unfavorable interactions where atoms are too close together; close contacts contacts - all kinds of direct interactions: polar and nonpolar, favorable and unfavorable (including clashes)". For more information please refer to Chimera documentation: hbonds,findclash.

Electrostatic analysis

Electrostatic interactions can play a pivotal role in drug binding. Moreover, some mutations in the QRDR act by disrupting the magnesium -water- quinolone bridge. For these reasons, we calculated the variation of net charge, electrostatic potential, and charge distribution variation after in silico mutagenesis. The protein is mutated in silico with Chimera, then all the residues at 5 Å are selected from the protein. Charges are assigned with PDB2PQR while electrostatic potential and charges distribution are calculated with APBS.

Solvent accessible surface analysis

Aminoacidic changes can modify the solvent accessible surface area(SASA or ASA) of a protein and therefore interfere with drug binding. The relative surface area is calculated for the mutation with the open source tool DSSP.

Quinores3D finder -Results

After job is completed, you're brought to the main result page. It contains:

Sequence alignment between your query (indicated as '_query') and the protein of reference ('_ref') plus blast output (for more information about 'bitscore','identity,'evalue','gaps','positives' please refer to Blast documentation)

Summary table of mutations found, with a 'profile' indicating if the mutation is known to be associated with drug resistance or not and a link to specific analysis for the mutation.

A viewer developed in WebGL, with the mutations found mapped onto the protein structure. For each mutation you can visualize additional information.