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Software Tool Article
Revised

ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex

[version 2; peer review: 3 approved]
PUBLISHED 01 Dec 2014
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Abstract

Most physiological processes in living systems are fundamentally regulated by protein–ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are calculated by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide a ranked list of residues to the user in order to obtain loss-of-function mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.

Revised Amendments from Version 1

The 'Workflow' and the 'Validation' section has been revised appropriately in this version of the manuscript to address all the queries raised by the reviewers.

  1. The ABS-Scan workflow now reports the DOPE scores (Discrete Optimized Protein Energy) to assess the quality of the mutant protein structure generated. The details on the evaluation of protein-ligand energetics has been explained in the 'workflow' section.
  2. An option of residue-range has now been provided to deal with complex ligand that contains more than one moiety/residue (Ex: 401-404 for PDB ID: 1J84).
  3. In addition to CSAR dataset, the 'Validation' section has now been updated to include the analysis carried out on the 'PDBbind' dataset to assess the significance of ΔΔG scores reported.
  4. 'Validation' now also includes case studies describing three different examples to evaluate the predictions of ABS-Scan. 
  5. The 'Input' section of the web-server also provides an advanced option to deal with water molecules for protein-ligand energetic calculations. This has been illustrated with an example in manuscript
  6. In addition to citations of 'Modeller' and 'AutoDock Tools' in the manuscript, the source code - 'alanine_scanning_v2.py' in github also has been updated to include all the 'original' citations and references for the tools used. 

See the authors' detailed response to the review by Sunando Datta
See the authors' detailed response to the review by Bernard Offmann and Stéphane Téletchéa

Introduction

Currently (as of April 3, 2014)1 there exist more than 72000 experimentally determined protein structures complexed with small molecule ligands, providing an extensive data resource on protein binding sites. These binding sites vary in size ranging from six to thirty residues depending upon the size and the nature of the ligand. In most cases, the contribution of the individual amino acids towards the binding of a given ligand is not well understood. A well-established method of demonstrating the importance of a residue at the site is to create point mutants through site-directed mutagenesis2. Efforts towards characterization of entire functional site include tools such as alanine scanning mutagenesis (ASM)3 where each residue is mutated to an alanine and its effect on the function is evaluated. ASM is indeed a well-used technique in experimental biology and has been successfully applied to the problems of protein folding and stability4, protein-protein5,6, and protein-ligand7 interactions. The experimental success of this technique has resulted in further developments, including high-throughput and low-cost variants8, greatly expanding its reach. Yet, given the time, cost and effort required for carrying out experimental biochemistry, a large majority of proteins are yet to be studied through this method.

Due to availability of a variety of structural bioinformatics tools, it is now feasible to carry out alanine scanning mutagenesis computationally9. Spurred by the successes and widespread adoption of the ASM technique, various computational resources now exist for in-silico alanine scanning. Prominent examples include Modeller10 and the Rosetta software suite11. However, most packages are command-line oriented and are out of reach for researchers. Alanine scanning webservers with intuitive user interfaces such as Robetta webserver12, the Rosetta Design web-server13, ROSIE14, FOLDX15, BeATMuSiC16, DrugScorePPI 17 exist for the problems of protein folding, protein stability and protein-protein interactions. Although, there are workflows to evaluate ligand-binding energetics which require significant computational time and setup through free-energy calculations involving Molecular Mechanics/Generalized Born Surface Area method (MM-GBSA)1820, there is however, no intuitive web-tool available for analyzing alanine-scanning mutations of small-molecule binding site residues in real time. A common requirement for an experimental biochemist is to identify which amino acids to mutate in the protein to generate loss-of-function mutants. A web-tool to cater to that specific need will therefore be highly useful. The analysis will also provide deep insights into critical residues for interaction, residue pairs or sets that when mutated will abolish ligand binding and provide analytical insights for lead refinement in the process of drug discovery, as well as understand drug resistance due to mutations.

We present a computational workflow and webserver, Alanine Binding Site-Scan (ABS-Scan), for automated alanine-scanning mutagenesis of protein-ligand interface residues. The workflow combines the libraries of widely used software packages including Modeller10 for site-specific alanine mutagenesis and Autodock21 for energetic evaluation of protein-ligand complexes.

Workflow

This workflow allows a user to submit a protein-ligand complex of their interest (Figure 1). The user is provided with an option of selecting a distance cut-off to define the binding site around a specific ligand for which, in-silico alanine scanning mutagenesis is carried out. Once the input parameters are obtained, the Modeller library is used to perform site-specific mutagenesis on all selected residues, coupled with steps of energy minimization22. This consists of initial steps of conjugate gradient (200 iterations with minimum atom shift of 0.001Å), followed by 200 steps of molecular dynamics simulation with steepest descent carried out at different temperatures. The initial restraints for the mutated model are derived from the wild-type protein structure. The analysis and results derived from alanine scanning mutagenesis relies on two assumptions: (a) The introduced point mutation does not drastically change the structure of the protein and (b) the mode of ligand interaction in point mutant is the same in comparison to wild-type complex. Care is taken to ensure that there are no steric clashes between the protein/ligand atoms during the process of minimization. The quality of the protein structures generated is estimated through Discrete Optimized Protein Energy (DOPE) score23, a statistical potential score that is calculated for each of the mutant. This scoring scheme is based on the improved reference consisting of non-interacting atom pairs in a homogenous sphere with radius dependent on sample native structure. The score therefore reflects the feasibility of interactions and the compactness of the modeled structure.

533bfb85-face-4680-8158-8efdeb2856b8_figure1.gif

Figure 1. ABS-Scan workflow.

Flowchart depicting various steps involved in ABS-Scan.

Each mutated structure, will then be scored by using Autodock 4.1 force field21, to calculate the energetics of a protein-ligand complex. The force-field is used here only to score the pose of protein-ligand interaction and no docking is performed. By default, ‘check_hydrogens’ flag is kept ‘on’ while preparing the receptor and Gasteiger charges are used for proteins and ligand. The contribution from a protein residue is determined by difference in interaction score of mutant and wild-type protein (∆∆G value). These results are graphically presented to the user, along with a ranked list of residues in the given site that could be experimentally explored for site-directed mutagenesis. A Jmol applet displays protein-ligand interactions with residues colored according to the computed extents of contribution towards interaction, while a table simultaneously displays inter-molecular energy scores. We also provide a help-section explaining the results along with selected examples.

Validation and case studies

We evaluate the significance of ∆∆G score used to assess the contribution of individual residues at the binding site by systematically analyzing two different datasets. The first dataset was derived from CSAR Community Structure-Activity Resource (CSAR - www.csardock.org/). Decoys in this dataset contain artificial docked complexes of protein with ligands having similar chemical properties to native ligands, but known not to interact with the protein. The protocol could be successfully applied on 288 of 343 protein-ligand native and decoy complexes. The distribution of average ∆∆G scores obtained through ABS-Scan analysis for residues in the binding site for decoy dataset is seen to be different from the native protein-ligand complexes (Figure 2A & B). An average ∆∆G score of 0.395 was obtained for the native protein-ligand complexes. The second dataset we used to obtain an estimate of ∆∆G score is derived from PDBbind database24 and comprises 195 protein-ligand complexes (PDBbind core dataset). Around 135 of these protein-ligand complexes could be successfully processed using ABS-Scan workflow. In this case, an average ∆∆G score of 0.387 was observed for each mutated residue at the binding site. Hence, to determine the sensitivity of ABS-Scan, a cut-off of 0.5, which is a more stringent value, is chosen. ABS-Scan is seen to effectively discriminate between the decoy and the native complexes of CSAR dataset (p-value ~0.004 calculated with Student’s t-test) in ~67% of the cases (∆∆G ≥ 0.5). This clearly indicates that residues important for ligand interaction can be identified through this protocol (Figure 2C). The detailed results of ∆∆G scores obtained for each of the mutation produced at the binding site for both these datasets can be accessed from the web-resource - http://proline.biochem.iisc.ernet.in/abscan/validation.

533bfb85-face-4680-8158-8efdeb2856b8_figure2.gif

Figure 2. ABS-Scan Sensitivity.

(A) The average ∆∆G score per residue distribution from the cognate and decoy protein-ligand complexes of CSAR dataset. (B) The scatter plot displaying the average ∆∆G score for native and the corresponding decoy complexes from the CSAR dataset. (C) Boxplot showing the difference in the % of the residues in the binding site of cognate and decoy complexes having a predicted ∆∆G score ≥ 0.5.

A suitable dataset for validation would be one that reports binding affinities for both wild-type and mutant proteins with same ligand, performed in a uniform experimental environment, for large number of proteins. Although such a dataset exists for protein-protein alanine scanning mutagenesis12,25, there are none reported for protein-ligand interactions. In order to compare the predictions of ABS-Scan with the experimentally reported alanine-scanning mutations, a methodical search was carried out to mine all the experimental results available in literature on alanine-scanning mutagenesis of residues at the binding site. Advanced search option in PDB was used for this purpose. All the PUBMED extracts were scanned for the term - "alanine scanning". The above search criteria mentioned yielded 126 structure hits with 56 citations. The list of entries obtained, was further pruned to remove biologically irrelevant ligands, metal ions and modified residues. The list of 79 entities/binding sites that we finally obtained can be accessed at http://proline.biochem.iisc.ernet.in/abscan/validation. Alanine scanning could be successfully undertaken for 54 of these structures. On an average, atleast two residues per binding site were predicted to have ∆∆G score ≥ 0.5. The details of the dataset and the ranked lists of residues in the order of their contribution to ligand binding identified for all the complexes is made available to the community - http://proline.biochem.iisc.ernet.in/abscan/validation.

Each of the above experiments involving alanine-scanning mutagenesis reports different mutant evaluation scores. The measures reported to test the fitness of the mutants include various attributes such as Kd, Ka, kcat/KM (for enzymes), specific substrate/product assays etc. These measures cannot be normalized to derive values having uniform units for direct comparison. We describe three such examples here, each with different experimentally reported mutant evaluation scores and the predicted ∆∆G values for the same as case studies to highlight the heterogeneity associated with the data.

A study on testosterone binding site of rat 3-alphahydroxysteroid dehydrogenase (PDBID: 1AFS) by Heredia et al.26 reports that binding site residue in direct contact with the ligand influences the rate determining step of the enzymatic reaction. In this case, the alanine scanning experiments performed on the residues in the binding site that recognize progesterone and testosterone reports the Kd values. The ABS-Scan analysis performed on 3-alpha hydroxysteroid dehydrogenase in complex with both testosterone and progesterone also predicted the residues W227 (∆∆G score = 1.43; Kd = 10.7±1.2), Y310 (∆∆G score = 1.31; Kd = 9.20±0.94), L54(∆∆G score = 0.5696; Kd = 7.24±0.79) to be important for ligand recognition. A good correlation was observed (0.829 for testosterone and 0.704 for progesterone) between the reported Kd value of the mutants and the corresponding predicted ∆∆G score.

A two-dimensional alanine scanning mutations were performed to understand the structure-function relationship between vitamin-D receptor (PDBID: 1IE9) and vitamin-D analogs by Shimizu et al.27. Since there was no structural information available for the analogs complexed to vitamin-D receptor, four of the vitamin-D analogs were docked on the receptor at the vitamin-D native binding site using Rosetta 3.4 docking protocol28. All the poses obtained were analyzed using ABS-Scan to determine the residues crucial for interaction of particular ligands. Since this is a nuclear receptor protein, a transcriptional activity assay was used in original study to evaluate the effect of mutants generated. The effect of each vitamin-D receptor mutant was measured by the downstream transactivation assay that quantifies luciferase activity under the influence of VDR (Vitamin-D Receptor promoter) promoter sequence. In this case, if the mutation affects the binding of ligand, correspondingly the expression of luciferase would reduce by a factor that can be quantified. A good negative correlation was also observed with all the four analogs complexed to vitamin D-receptor and atleast four residues - L233, W286, R274 and H397, important for interaction with all the analogs had ∆∆G score > 0.5. L233 and W286, present in H3 (helix 3) and β sheet are reported to have hydrophobic interactions with B and C rings of the ligand whereas R274 present in H4 (helix 4) is observed to have hydrogen bond interaction with 1α-OH group of the ligand.

A similar study was carried out on human trimethyl-guanosine synthase enzyme (Tgs1) that converts m7G caps (7-methyl guanosine caps) to 2,2,7-trimethylguanosine (TMG) caps. In the original study29 around 37 point mutations were introduced into human Tgs1 (PDBID: 3GDH) to study the interaction profile with mGTP (7-methyl guanosine tri-phosphate) and AdoMet (S-adenosyl methionine). The fitness of mutants generated in this case was evaluated by using the methyltransferase assay that determines the percentage of methylation by quantifying the levels of m7GDP to m2,7GDP. The residues - R807 and K646, reported to be the most affected mutants, are also predicted by ABS-Scan to be essential, with the highest predicted ∆∆G score of 3.63 and 3.39 respectively. These positively charged residues (R807 and K646) are observed to interact with α and β phosphate groups of m7GTP. The π -cation stacking observed between W766 and the m7G was also predicted to be crucial (∆∆G score of 2.66) and correspondingly no methylated products were detected for this mutant through methyl-transferase assay.

The details of the case-studies described above along with the results of the analysis can be accessed on the example section of the web-tool - http://proline.biochem.iisc.ernet.in/abscan/examples.

Implementation

The web-server was implemented using hypertext preprocessor (PHP). Autodock, Modeller and Pymol libraries have been used for modeling the mutation and evaluating the energetics. Integration of these back-end libraries for presentation as a functional and intuitive user interface is accomplished using Shell, Python, Java, HTML and PHP scripts. The web-server is platform independent and will run on any machine having internet access with browser installed. For the advanced users, a command-line interface in the form of a single python script can be accessed from github repository (https://github.com/praveeniisc/ABS-Scan). The script has been tested on Intel 2.83 GHz quad-core system running 32 bit linux OS(Ubuntu 12.04) with Modeller10, MGL AutodockTools30 & Pymol (http://pymol.org) installed. For the web-server d3.js library has been used for displaying the plots. Jmol Applet has been used to visualize the protein-ligand interaction.

Input

The input required for the server is the structure of a protein-ligand complex in PDB format. Users can either provide the four-letter PDBID or upload the PDB structure file of the complex. An option is provided to define the cut-off distance and select the ligand to obtain binding site residues which would be mutated to alanine for evaluating the interaction energetics. A default distance cut-off of 4.5 Å is set to select all the residues whose atoms lie within this distance from any ligand atom. In some the cases, metal ions31 and water molecules are observed to play a crucial role in stabilizing the interactions32. A major problem involved in incorporating the ligand metal ion in ABS-Scan worflow is fixing the charge parameter as metal atoms can have different ionic states (Ex. Fe2+, Fe3+ etc.) which is important for evaluating energetics. Enumerating all important structural water molecules involved in the ligand interaction is also highly dependent on the resolution of the crystal structure. Hence, an advanced option is provided to the user for uploading the PDBQT format of the ligand, to account for cases where the ligand contains unusual atom types, metal ions or uses bridge-water molecules for interaction. For practical purposes, the bridge water molecules can be considered to be the part of ligand and these can be incorporated into the pdbqt file of the ligand. As an example, ABS-Scan analysis was carried out on protein lysine methyltransferase (PDB: 3S7B) complexed with S-adenosyl methionine33 through four bridge water molecules. These four bridge-water molecules can be incorporated into the ligand pdbqt file and uploaded with the help of an advanced option provided on the server. The protocol correctly identified GLU135 and ASN182 as significant contributors to ligand binding through formation of water bridges. The output can be accessed through the example section of the web-server.

Output

All the results produced by ABScan can be visualized interactively on the web-server. Jmol Applet is used to visualize the contribution of residues towards ligand interaction (Figure 3).

533bfb85-face-4680-8158-8efdeb2856b8_figure3.gif

Figure 3. ABS-Scan interactive display.

Snapshot explaining the Jmol applet output on the ABScan server. The individual residues are colored in red to blue gradient depending upon the contribution towards the ligand interaction as predicted by ABScan ∆∆G score. Options to visualize the different kinds of interaction - polar, hbonds etc. is also provided.

d3.js library has been utilized to plot the predicted ∆∆G values and subcomponents of the energetic scores reported by Autodock4 (Figure 4). An option is provided to download publication quality images in SVG/PDF/PNG formats. Twitter bootstrap java library is used for framework development on the webserver. An option is also provided to download the raw files containing individual mutants in PDB format, ∆∆G scores in the raw CSV format along with autodock energy scores.

533bfb85-face-4680-8158-8efdeb2856b8_figure4.gif

Figure 4. ABS-Scan energy plots.

(A) ∆∆G values reported for each of the alanine mutation performed for the residues present at the binding site. The residues are ordered according to their contribution/∆∆G values. (B) The different energy component of autodock interaction score plotted for each of the alanine mutant produced at the binding site.

Conclusions

ABS-Scan webserver can provide valuable insights on molecular recognition involving protein-ligand interactions. Experimentally determined protein-ligand structures can be studied to understand individual residue contributions towards ligand binding. Modeled complexes can also be submitted to infer the feasibility of the interaction. We believe that ABS-Scan would add one more dimension to the analysis of binding sites in proteins, comparison of various ligand interactions and be of importance to researchers performing ASM studies.

Software availability

Source code as at the time of publication

https://github.com/F1000Research/ABS-Scan/releases/tag/V1.0

Archived source code as at the time of publication

http://dx.doi.org/10.5281/zenodo.1280634

Software license

ABS-Scan is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

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Version 2
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Anand P, Nagarajan D, Mukherjee S and Chandra N. ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.12688/f1000research.5165.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
VERSION 2
PUBLISHED 01 Dec 2014
Revised
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Reviewer Report 19 Jan 2016
Bernard Offmann, Unité Fonctionnalité et Ingénierie des Protéines, Université de Nantes, Nantes, France 
Approved
VIEWS 25
No more comments. This version of the manuscript has been improved on several ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Offmann B. Reviewer Report For: ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.5256/f1000research.6207.r11974)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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32
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Reviewer Report 04 Feb 2015
Srikrishna Subramanian, Protein Science and Engineering, CSIR - Institute of Microbial Technology, Chandigarh, India 
Gurmeet Kaur, Institute of Microbial Technology, Chandigarh, India 
Approved
VIEWS 32
In their manuscript entitled " ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex", the authors have discussed the development of an in silico workflow for the prediction of important residues for ligand recognition in a ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Subramanian S and Kaur G. Reviewer Report For: ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.5256/f1000research.6207.r7324)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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30
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Reviewer Report 22 Dec 2014
Sunando Datta, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal, India 
Approved
VIEWS 30
The authors have now  incorporated all the suggestions I made in my 1st review. In fact the revised web application is able to include the solvent molecules. I think that the revised version is now ready to be indexed in ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Datta S. Reviewer Report For: ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.5256/f1000research.6207.r7122)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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PUBLISHED 09 Sep 2014
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Reviewer Report 17 Oct 2014
Bernard Offmann, Unité Fonctionnalité et Ingénierie des Protéines, Université de Nantes, Nantes, France 
Stéphane Téletchéa, Unité Fonctionnalité et Ingénierie des Protéines, Université de Nantes, Nantes, France 
Not Approved
VIEWS 123
This paper reports an attempt to develop an original tool that simulates alanine scanning mutagenesis to probe residues involved in the process of ligand recognition in proteins.

More precisely, the work describes the development of a work flow that implements known ... Continue reading
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CITE
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Offmann B and Téletchéa S. Reviewer Report For: ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.5256/f1000research.5509.r6276)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 18 Nov 2014
    Nagasuma Chandra, Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
    18 Nov 2014
    Author Response
    We thank the reviewers for their time and effort. There were some useful suggestions, which we have incorporated but do not agree with all the points raised. A detailed point-by-point ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 18 Nov 2014
    Nagasuma Chandra, Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
    18 Nov 2014
    Author Response
    We thank the reviewers for their time and effort. There were some useful suggestions, which we have incorporated but do not agree with all the points raised. A detailed point-by-point ... Continue reading
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59
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Reviewer Report 30 Sep 2014
Sunando Datta, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal, India 
Approved with Reservations
VIEWS 59
The manuscript 'ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex' reports development of a web server for performing in silico alanine scanning mutations for studying protein-small molecule interactions. It further validates the tool by taking ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Datta S. Reviewer Report For: ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex [version 2; peer review: 3 approved]. F1000Research 2014, 3:214 (https://doi.org/10.5256/f1000research.5509.r6262)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 18 Nov 2014
    Nagasuma Chandra, Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
    18 Nov 2014
    Author Response
    We thank the reviewer for going through our manuscript and finding the work useful. These are indeed valid points and have been addressed in the revised version. 
    Bridge water molecules do ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 18 Nov 2014
    Nagasuma Chandra, Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India
    18 Nov 2014
    Author Response
    We thank the reviewer for going through our manuscript and finding the work useful. These are indeed valid points and have been addressed in the revised version. 
    Bridge water molecules do ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 09 Sep 2014
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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