How to use PremPDI Server?

Step 1 - Provide the structure of a protein-DNA complex

You can either input the PDB code, the structure will be retrieved from the Protein Data Bank, or upload your own structure file, which must comply with the PDB format.

In either case, the structure file should contain at least two distinct protein or nucleic acid chains. Please note that:

• Bioassembly (biological unit) is the default structure. You can choose all biological assemblies for each protein-DNA complex and the first bioassembly (.pdb1) will be used as default.
• If the structure file includes multiple NMR models, only the first one will be used in calculations.
• If the uploaded structure file includes multiple protein-DNA complexes, only the first one will be considered.
• Non-protein or nucleic acid molecules and non-standard residues are not taken into account during the calculations.
• PremPDI chooses the first coordinate for the atom if several different coordinates are provided in the PDB file.

If a valid PDB code is input, the title of the PDB will be shown.

Step 2 - Define two partners for a protein-DNA interaction

A user can define interaction partners by clicking on the button of any chain thumbnail. Please note Partner 1 and Partner 2 is restricted to Protein and Nucleic acid respectively, so the selected protein/DNA chain will be put into the box of Partner1/Partner2 automatically.

• PremPDI provides a 3D view of the complex colored by chains. Each chain is listed with the corresponding name of the protein or nucleic acid.
• One can assign one chain or multiple chains to either Partner 1 or Partner 2, but both partners should include at least one chain; only the selected chains will be taken into account during the calculation.
• For bioassemblies if any protein or nucleic acid/chain in the structure were generated by applying symmetry operations, they are depicted with the labels of alphanumeric combinations (for example, ‘A_1’) indicating the source molecule/chain from which they were generated.

Step 3 - Select mutations to estimate their effects on protein-DNA interaction

One can select several mutations (only for amino acid substitutions on chains of Partner 1) on this page but only one mutation will be introduced to the complex each time (the maximum number of single mutations is 16). Please note that when more than one mutations are selected, each of them will be treated independently.

After a user makes the selection of a chain and a position to be mutated, they can visualize the location of the mutated (substituted) residue in the wild-type complex in the 3D viewer.

Results

Upon the completion of the third step and submission of the job, PremPDI provides a job identifier for checking the job status. If an e-mail address is provided, the link to the results page will be sent via e-mail. The results will be available on this page as soon as the calculations are done and they will remain on the webserver for two weeks. The example below shows precalculated results for job 2018090501593452439163653.

For each protein/mutation, Mutabind provides the following results:

• ΔΔGbind(kcal mol-1) is the predicted change in binding affinity induced by a mutation. A positive and negative signs correspond to destabilizing and stabilizing mutations predicted to decrease and increase binding affinity correspondingly.
• Interface (yes/no), PremPDI defines a residue to be located on a protein-DNA interface if residue solvent accessibility in the complex is lower than in the corresponding unbound partner.
• Deleterious (yes/no), PremPDI server classifies a mutation as deleterious if ΔΔGbind is higher or equal to 1.10 kcal mol-1. This threshold corresponds to 14% FPR and 77% TPR which minimizes the value of √(1-TPR)2+ FPR2)  to compensate sensitivity and specificity of retrieval.
• Mutant PDB, MutaBind provides the minimized mutant structure for users to download.

Results can be viewed directly on the browser or downloaded as a plain text file.

Homologous binding sites

PremPDI allows exploring homologous binding sites for each protein chain and amino acid residue that was tested. This option can be accessed by clicking on "Explore" link in the results table.

The binding sites are download from Inferred Biomolecular Interactions Server at NCBI (IBIS, www.ncbi.nlm.nih.gov/Structure/ibis/ibis.cgi). It may take several seconds before the results are retrieved. IBIS server identifies protein-DNA/RNA complexes and binding sites homologous to the query (protein chain that was mutated) and include the mutated site.

Note that if there is no binding site defined for the protein chain, or if the mutated residue is not part of any binding site in IBIS no results will be displayed. Interactions with different partners are shown separately; see IBIS for partner description. In some cases, as in the example above, IBIS does not identify any homologs, thus only the binding site for query protein is shown. Otherwise, see another example below (open precalculated job), the table will show alignments of binding site residues colored according to their conservation (red denoting highly conserved positions). Mutated site is highlighted in yellow. One can click on a residue in a homologous structure to check it in PremPDI, which will require submitting a new job for calculation.