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Chapter 7

HORI: A tool for computing Higher Order Residue Interactions in

structural domains and its application in protein structure analysis

Publications from this chapter:

• P. Sundaramurthy*, K. Shameer* S. Raashe, S. Gakkhar and R. Sowdhamini: A bioinformatics protocol for identification of spatial clusters and the calculation of higher order residue interactions in protein structures (2010) Nature Protocols Network, DOI: 10.1038/nprot.2010.91

• P. Sundaramurthy, K. Shameer, S. Raashe, S. Gakkhar and R. Sowdhamini: HORI: A web server to compute Higher Order Residue Interactions in protein structures (2010) (Presented at APBC 2010; BMC Bioinformatics18; 11 Suppl 1:S24)

* Equally contributed first authors

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7.1 Introduction

This chapter discusses about a novel structural bioinformatics tool developed for the

computation of higher order residue interactions in protein structural domains. Folding of a

protein into its three-dimensional structure is influenced by both local and global interactions

within a protein. Higher order residue interactions, like pairwise, triplet and quadruplet ones,

play a vital role in attaining the stable conformation of the protein structure. Derivation of a

three-dimensional structure of a protein from its primary sequence is controlled by a complex

and largely unknown set of principles called the folding code. Higher order residue

interactions also contribute to the potential energy landscape of proteins and hence it is

important to understand such interactions mediated in the level of active site residues to

whole structure [41, 43, 112, 543-546]. In the current era of high-throughput sequencing, due

to huge lacunae in the sequence to structure ratio, computational approaches are playing a

significant role in understanding the design principles and functional aspects of protein

structures [1, 20, 307, 547-551]. This chapter describe the details of a new tool called HORI

(Higher Order Residue Interactions in proteins) developed for the calculation of generic and

specific higher order residue interaction patterns in protein structure and its application in

protein structure analysis.

7.2 Materials and Methods

7.2.1 Concept of higher order residue interactions

Basic algorithm of HORI is designed based on the classical concept of four-body nearest-

neighbor propensities of amino acid residues. According to the earlier studies, each residue

can be approximately represented as a sphere centered on its location, it is possible for a

maximum of four closely packed spheres to make mutual contact, thus giving rise to pair

wise, triplet and quadruple interactions. Just as no more than four same-sized spheres can be

in mutual contact in 3D space, higher order interaction beyond quadruple interactions are

generally not considered [543, 552, 553]. Higher order residue interactions in HORI server

were computed using following equations:

7.2.1.1 Pairwise interactions

Interactions that involves two residues that are four residues apart for residue i.

Epairwise = (T(i),T(i+4))

i=1

j

! (7.1)

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where T(i) is the residue in the template sequence and j is the length of the sequence.

Pairwise interactions in the structure of crambin (PDB ID: 1CRN) within a distance cut-off

7$ is provided in Table 7.1.

7.2.1.1.1 Triplet interactions

Interactions that involves three residues that are four residues apart from residue i.

Etriplet (i,j,k) = Ep(i,j)! + Ep(j,k)! + Ep(k,i)! (7.2)

where i,j,k are each four residues apart. Triplet interactions in the structure of crambin (PDB

ID: 1CRN) within a distance cut-off 7$ is provided in Table 7.2.

7.2.1.2 Quadruple interactions

Interactions mediated by four residues that are four residues apart from residue i.

quadruple p p p p p pE (i,j,k,l) = E (i,j) + E (j,k) + E (k,l) + E (l,i) + E (i,k) + E (j,l)! ! ! ! ! ! (7.3)

Where i,j,k,l are each four residues apart and the cut-off distance is defined as 7$. Quadruple

interactions in the structure of Crambin (PDB ID: 1CRN) within a distance cut-off 7$ is

provided available from the following URL:

http://caps.ncbs.res.in/hori/hori_results/er_hori_SatDec13185917IST2008.html

Brief description and graphical representation of three different categories (pairwise, triplet

and quadruple interactions) of higher order residue interactions mapped on crambin (PDB ID:

1CRN) sequence and structure is provided in Figure 7.1.

Interactions between amino acids within a protein structure are considered as a measure of

the contact potential contributed by different residues. A pair potential cannot discriminate

between local and long-range interactions [554]. With two particles, the only distance that

can be considered is that between the two particles. Understanding multi-body interactions in

protein structures are important to analyze interaction patterns of protein structures. Previous

work on the geometric cooperativity and anti-cooperativity of three-body interactions in

native proteins showed the contribution of multi-body interactions in stabilizing protein

structures [555, 556]. The concept of higher order interactions has been introduced and

successfully employed in structure analysis and fold recognition by different groups [557,

558]. Earlier work also reported that the higher order interactions could be used to improve

accuracy of fold recognition and generic protein structure analysis [546, 559]. As HORI

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server can be used to compute higher order interactions in different levels from single residue

to whole structure, analysis of higher order interactions mediated by residues in the functional

or active sites will provide better insights to understand the structural interactions contributed

by these important residues. The availability of a server to compute higher order interactions

will enable the computation of higher order interactions in easy steps. This chapter explains

various features of HORI server along with different example scenarios where the general

application of the higher order interaction and the server has been useful.

7.3 HORI server implementation and features

HORI Server is designed as three distinct modules: HORI - Global, HORI - Lite, and HORI –

Cluster. These modules are provided with different options to compute generic and specific

interactions from protein structures. All the programs in these modules are available for

computation based on C! and C" atom types. Separate web interfaces are available for both

single chain and multi-chain based computations. HORI Server provides user-friendly,

interactive interfaces for the submission of files and visualization of results. A detailed flow

chart of different modules available in HORI server is provided in Figure 7.2. Web interface

of HORI server is developed using HTML and JavaScript. HORI programs are coded in C-

language and compiled using GNU Compiler (gcc version 4.2). Interactive visualization of

higher-order interactions on query structure is implemented using Jmol [394]. RasMol [360]

scripts are also provided for the interactive analysis of structures in local machine. Wrapper

scripts and automated e-mail programs are coded in Perl. HORI server is running on an

Apache web server powered by Athlon Quad-core processors.

7.4 HORI – Global module

HORI – Global module provides a set of programs for the computation of higher order

interactions at the complete structural level. Using options in HORI - Global module,

pairwise, triplet and quadruple interactions can be computed using residues in a given protein

structure. Here, the complete set of all possible interactions in each category will be

computed. The higher and lower-cut distance cut-off for identifying probable higher order

interactions are provided as a user-defined option. Preferred range for higher order residue

interactions are 1 – 7Å. Using efficient utilization of different parameters, like atom-type for

distance calculation, interaction type and distance cut-off, interesting information on higher

order interactions can be obtained from the protein structure of interest. HORI – Global is the

computationally intensive module available in the HORI server. This module is designed

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such that results will be sent as emails due to computation intensive nature of the back-end

programs.

7.5 HORI – Lite & HORI – Cluster modules

Hori – Lite, the second module available from HORI server offers a set of programs for the

computation of higher order interactions in a structure, based on specific residue numbers and

specific distance. The third module, HORI – Cluster, offers a set of programs for computation

of higher order interactions of different types of residues in a structure. Both HORI – Lite and

HORI - Cluster provide nine different programs under the category of three different classes

of higher order interactions. Programs are available in the class of pairwise interactions to

compute pairwise distances for any two residues in PDB file and pairwise distance around

any one residue. Triplet interaction class provides programs to compute triplet distance for

any three residues, triplet distance for around any two residues and triplet distance around

any one residue. Quadruplet interaction class provides options to compute quadruple distance

for any four, three, two or one residue in a PDB file.

7.6 HORI Server Input options

All the three modules of HORI server require three-dimensional co-ordinates of protein

structures in PDB format. Structures from PDB and modeled structure derived from program

like Modeller [284] or SWISSMODEL [560] can be provided as input for HORI server. For

NMR structures, higher order residues can be computed using data from specific chain. In the

current version, HORI server can be used to analyze both single chain and multi-chain

proteins. Due to computational-intensive nature of HORI-Global programs, currently, the

server allows only two chains in the multi-chain based higher order interaction calculations.

User of HORI - Global module should also submit a valid, non-commercial email address to

the server to receive the notification about the availability of results. HORI server will send

the result URL to the email address. In comparison to HORI – Global, both HORI – Lite and

HORI – Clusters offer specific and faster computation of the higher order interactions in a

protein structure. General parameters in different modules of HORI server are atom type,

interaction type and range of distance to calculate interactions. Apart from these general

parameters, user can also mention the range of residues (option available in HORI – Global),

exact residue numbers (available as a parameter in HORI – Lite) and amino acid type

(available as a parameter in HORI – Cluster). A collage of web-interfaces (screen shot) of

HORI-Global, HORI-Lite and HORI-Cluster is provided in Figure 7.3.

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7.7 HORI Server Output details

HORI server computes pairwise, triplet and quadruple interactions within a protein structure

based on different parameters provided by the user. Among the three modules available in

HORI server, HORI – Global provides more detailed output. User can generate customized

output based on pairwise, triplet and quadruple computations using HORI – Global. HORI –

Global module also provides a tab-delimited text file of the results for the customized

analysis of higher order interactions by the user. User can also visualize the interaction; either

using pre-installed RasMol [360] on local machines or on browser using Jmol plug-in [360].

All three modules in HORI server provide output in html, tab-delimited text files for parsing

and further analysis of higher-order interactions and visualization options to see individual

interactions.

A pictorial summary of HORI server input and output options are provided in Figure 7.4.

Briefly, the submission of protein structure to HORI server and computing higher order

residue interactions can be summarized in 5 steps:

1. User submit the protein structure to HORI server

2. User select various parameters and provide email address

3. Job scheduler running in the web server will submit the HORI process as a

background job

4. Once the output files are available; the ‘sendmail’ based program will send an

email to the user supplied email with a URL to access the results

5. User can visualize, analyze higher order interaction over the web interface or

after downloading the output files.

7.8 Results and Discussion

Bioinformatics tools are widely used in the study of protein structures to understand

structural, functional and interaction aspects of protein structures. Several tools are also

available for the calculation of interaction; interface, bonding patterns and disulphide

connectivity. In PDBWiki [561] various tools are listed to define or select interacting

residues. For example, Protein Interaction Calculator [146] can be used to calculate several

interaction parameters like intra-protein interactions, solvent accessibility, protein-protein

interactions and depth calculations. Other tools like SCOPPI [562] can be used for analysis of

protein-protein interface, LPC/CSU [563, 564] can be used for ligand-protein contacts &

contacts of structural units. Irrespective of such wide array of structure tools for protein

structure analysis, according to the best of my knowledge, HORI server is a primary attempt

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to provide a web server for the computation of higher order residue interactions in proteins in

a whole structure as well as at a residue-specific level.

7.9 Applications of HORI Server in protein structure analysis

Higher order interactions calculated using a set of computationally intensive algorithms

available in HORI server will be useful in fold prediction, protein modeling, protein-protein

interaction, active site identification and to understand higher order interaction characteristics

of active site residues within specified distance shells. Knowledge about the higher order

interactions will be of great importance in structural biology due to its wide range of

applications in fold recognition, structural analysis, protein engineering, protein-protein

interactions, active site identification and to understand mechanism of action of enzymes

[546, 557, 558]. In order to illustrate the usefulness of higher order interactions, four different

examples scenarios of protein structure analyses are discussed below to illustrate analysis of

higher order residue interactions from single chain and multi-chain crystal structures from

PDB. URLs to access the HORI output of the case studies discussed here is provided in Table

7.3. Output data from HORI server for various examples discussed in this thesis is available

from the online web server. HORI output provided six-column output for pairwise

interactions (See Table 7.1), nine-column output (See Table 7.2) for triplet interactions and

quadruple interactions output is provided in a 18 column output. Such large output files are

difficult to paginate in doc files and hence provided in the HORI server URLs provided in

Table 7.3.

7.9.1 Analysis of pair wise interactions in a protein involved in 3D domain swapping

Domain swapping is a structural phenomenon observed in proteins with diverse sequence,

structure and function [123, 126, 565]. Ruberythrin from Pyrococcus furiosus is reported to

be involved in 3D domain swapping [566]. The structure of Ruberythrin with PDB ID:

1NNQ is used to perform HORI-Global based computation and retrieved all pairwise

interactions. HORI server reported a total of 580 pairwise interactions with in the cut-off of

1Å to 7 Å. Out of these pairwise interactions, the residues between the two chains mediated

13% of interactions. Such detailed analysis on proteins with peculiar structural features will

give a better understanding of the role higher order interactions in orchestrating the structural

integrity.

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7.9.2 Analysis of Higher order interactions in structures from TIM fold and Rossmann

fold

Prior knowledge of spatial and higher order interactions will add more value to the

interpretation of HORI results and to discriminate between folds of roughly similar secondary

structural topology but dissimilar overall shape. To demonstrate where triplet and quadruplet

interactions might be crucial, the example of two !/" folds from SCOP database [176, 391]

with strikingly similar secondary structural topology but distinct overall spatial arrangements

were considered: triose phosphate isomerase (TIM) fold [567] and the doubly-wound

dehydrogenase or Rossmann-fold [568]. Despite the similar repeats of super secondary

structural elements ("!"), the TIM barrel is a closed structure with the first and eighth "–

strand in hydrogen bonding, whereas the doubly wound Rossmann folds have open structures

with two distinct domains holding the entire polypeptide fold. Due to the similar secondary

structural arrangements, most fold recognition methods have failed to distinguish the correct

fold. When the pairwise interactions between the residues in the first two " strands in 1WYI

(TIM fold) [569] and 1G5Q (Rossman fold) [570] are compared, they lie in the same range.

The pairwise interaction between the C" atoms of residues 8 and 39 that lie in adjacent

parallel strands in 1WYI (TIM fold) is found to be 5.17Å while that between residues 7 and

34 in 1G5Q (Rossman fold) is 4.49 Å. While the topology appears to be similar, the higher

order interactions exhibit distinct profiles for the two proteins. While the triplet interactions

between C" atoms of residues 7, 21, 34 in 1G5Q lie within 10 Å, it is not so in the case of the

triplet formed between C" atoms of residues 8, 28, and 39 in 1WYI. In the case of 4MDH

[571], which is a Rossman fold protein, there are no triplet interactions observed between

adjacent " strands and the joining ! helix. For example, the only triplet interaction observed

for C" atom of residue 8 is with residue 38 of another " strand and residue 76 of ! helix. On

the other hand, in 1WYI, residue 8 is seen forming triplet interactions with adjacent " strands

on either side, forming the triplet residues-8, 231, 244 and residues-8, 28, 39. Graphical view

of the triplet interactions in 1WYI, 1G5Q and 4MDH is provided in Figure 7.5. HORI server

can be used in the analysis of distance-based interactions within proteins. It proves to be an

effective means of distinguishing protein folds through its map of higher order interactions.

7.9.3 Intramolecular higher order interaction mediated by a cysteine residue in

Crambin

Crambin [572] is a member of Crambin-like superfamily plant seed protein with three

disulphide bonds. HORI Server was used to compute quadruple distances around 1 cysteine

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residue in Crambin structure (PDB ID: 1CRN) with in the distance range of 1-7 Angstroms.

This option provided an insight in to all intramolecular quadruple interactions mediated by

the six cysteine residues in crambin [573]. A detailed screenshot of HORI server with results

of HORI-Global runs on crambin (PDB ID: 1CRN) is provided in Figure 7.6. Screenshot of

output file from HORI server for crambin highlighted with various sections is provided in

Figure 7.7.

7.9.4 Higher order residue interactions mediated by active site residues in Barley 1,3-

1,4-beta-glucanase

The functionally important residues, including the one at the active site, may not be strictly

conserved for protein domains at the level of superfamily [574]. HORI server can be used to

search for alternate active site residues based on specific higher order residue interactions.

Barley 1,3-1,4-beta-glucanase [575] from Hordeum vulgare belongs to the TIM barrel

glycosyl hydrolases superfamily. This PDB entry (PDB ID: 1AQ0) belongs to the Glycosyl

hydrolases family 17 (Pfam ID: PF00332) in Pfam database [106, 173]. Three of the four

active site residues reported in Catalytic Site Atlas [180] were used in the option to compute

quadruple distances around any three residues in a PDB file and successfully identified the

fourth active site residues in a quadruple interaction.

7.9.5 Identification of alternate active site residues and suitability of residues for

mutational studies based on higher order residue interaction

Cutinase [576] is a serine esterase containing the classical Ser, His, Asp triad of serine

hydrolases [577]. Catalytic Site Atlas [180] reports five active site residues based on

homologous entries, found by PSI-BLAST [139] alignment to one of the PDB entries (PDB

ID: 1AGY). Two of these residues were used to identify potential third interacting residue

(using the compute triplet distances option) around any two residues in PDB file (PDB ID:

1CEX). Approaches like this can be used to identify potential alternate active site residues

and residues suitable for mutational studies, based on number of intermolecular interactions

contributed by residues in active site regions.

7.9.6 Analysis of higher order interaction in a multi-chain protein involved in 3D

domain swapping

CD2 is shown to have ability to fold in two ways as a monomer or as a swapped dimer [358,

381]. HORI-Global based computation of higher order residue interactions using the two

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chains of CD2 structure (PDB ID: 1A64) 1A64:A and 1A64:B were performed. The higher

order interactions within the cut-off of 0-8Å clearly indicate that the swapped structure is

stabilized by several higher order interactions between the residues in chains A and B [578].

7.10 Discussion

HORI server provides a landscape of all possible higher order residue interactions in protein

structures. The information provided by HORI server is important to understand the role of

higher order residue interaction in stability, to recognize alternate patches of functionally

important residues, structural integrity and folding properties of modeled and experimentally

solved protein structures. Availability of HORI server in the public domain will enable the

structural bioinformatics community to analyze and study higher order interaction patterns

from protein structure data in easier way and gain better insight about the structure. This will

also aid biochemists and biologists in designing of mutation experiments. By providing

various options in three different modules, HORI server offers a complete computing

platform online for higher order residue interactions and for the analysis of protein structures.

The tool has been used extensively for the calculation of higher order interactions reported in

protein involved in domain swapping to understand the role of higher order residue

interactions in mediating 3D domain swapping in proteins. HORI server will be a useful

resource for the structural bioinformatics community to perform analysis on protein

structures based on higher order residue interactions.

As biology is accelerating towards an integrated science, network approaches are gaining

importance. HORI can be leveraged as a tool to explore structure networks from the

perspective of network theory. Further biological networks can also be utilized for various

applications, for example Chapter 8 explores the usability of protein-protein interaction from

the unique concept of ‘interacting sequence space’. The concept is used to build a new tool,

which can identify peptide substrates and functional motifs from the local interactomes. The

approach defined as ‘PeptideMine’ utilized protein-protein interaction data, domain-domain

interaction data, GO annotations, domain architecture etc to find putative functional motifs

from interactomes.

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Table and Figures of Chapter 7:

Residue No. Interacting Residues Distance

1-34 THR-ILE 5.652

1-35 THR-ILE 5.446

1-36 THR-PRO 6.314

1-37 THR-GLY 6.508

1-38 THR-ALA 5.738

2-32 THR-CYS 6.742

2-33 THR-ILE 5.587

2-34 THR-ILE 4.597

2-35 THR-ILE 6.394

3-32 CYS-CYS 5.610

3-33 CYS-ILE 5.365

3-34 CYS-ILE 6.483

3-40 CYS-CYS 5.687

3-44 CYS-TYR 5.309

3-45 CYS-ALA 6.498

3-46 CYS-ASN 6.148

4-9 CYS-ALA 5.999

4-10 CYS-ARG 5.517

4-31 CYS-GLY 6.762

4-32 CYS-CYS 4.090

4-33 CYS-ILE 6.332

4-44 CYS-TYR 4.836

4-45 CYS-ALA 6.286

4-46 CYS-ASN 4.828

5-9 PRO-ALA 6.398

5-31 PRO-GLY 6.792

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5-32 PRO-CYS 6.140

5-43 PRO-ASP 6.165

5-44 PRO-TYR 4.176

5-45 PRO-ALA 6.155

5-46 PRO-ASN 5.858

6-10 SER-ARG 6.483

6-46 SER-ASN 5.403

7-11 ILE-SER 6.203

7-46 ILE-ASN 5.681

8-12 VAL-ASN 6.169

9-13 ALA-PHE 6.065

9-30 ALA-THR 5.407

9-31 ALA-GLY 6.627

9-32 ALA-CYS 6.609

10-14 ARG-ASN 6.238

10-46 ARG-ASN 6.489

11-15 SER-VAL 6.506

12-16 ASN-CYS 6.131

12-30 ASN-THR 6.470

13-17 PHE-ARG 6.269

13-26 PHE-CYS 5.787

13-30 PHE-THR 6.087

14-18 ASN-LEU 6.573

16-21 CYS-THR 6.402

16-26 CYS-CYS 5.798

17-21 ARG-THR 5.176

17-26 ARG-CYS 6.921

21-25 THR-ILE 6.827

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21-26 THR-CYS 6.778

22-26 PRO-CYS 6.505

23-27 GLU-ALA 5.974

24-28 ALA-THR 6.669

25-29 ILE-TYR 6.140

26-30 CYS-THR 6.087

27-31 ALA-GLY 5.572

27-32 ALA-CYS 4.627

27-33 ALA-ILE 5.364

28-32 THR-CYS 6.935

40-44 CYS-TYR 6.143

40-45 CYS-ALA 6.295

41-45 PRO-ALA 6.297

Table 7.1: Pairwise interactions in Crambin structure (PDB ID: 1CRN)

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RN IR Dis RN IR Dis RN IR Dis

3-40 CYS-CYS

5.687 40-44 CYS-TYR

6.143 44-3 TYR-CYS

5.309

3-40 CYS-CYS

5.687 40-45 CYS-ALA

6.295 45-3 ALA-CYS

6.498

4-9 CYS-ALA

5.999 9-31 ALA-GLY

6.627 31-4 GLY-CYS

6.762

4-9 CYS-ALA

5.999 9-32 ALA-CYS

6.609 32-4 CYS-CYS

4.090

4-10 CYS-ARG

5.517 10-46 ARG-ASN

6.489 46-4 ASN-CYS

4.828

5-9 PRO-ALA

6.398 9-31 ALA-GLY

6.627 31-5 GLY-PRO

6.792

5-9 PRO-ALA

6.398 9-32 ALA-CYS

6.609 32-5 CYS-PRO

6.140

6-10 SER-ARG

6.483 10-46 ARG-ASN

6.489 46-6 ASN-SER

5.403

9-13 ALA-PHE

6.065 13-30 PHE-THR

6.087 30-9 THR-ALA

5.407

13-17 PHE-ARG

6.269 17-26 ARG-CYS

6.921 26-13 CYS-PHE

5.787

13-26 PHE-CYS

5.787 26-30 CYS-THR

6.087 30-13 THR-PHE

6.087

16-21 CYS-THR

6.402 21-26 THR-CYS

6.778 26-16 CYS-CYS

5.798

17-21 ARG-THR

5.176 21-26 THR-CYS

6.778 26-17 CYS-ARG

6.921

Table 7.2: Triplet interactions in Crambin structure (PDB ID: 1CRN). RN = Residue

Number; IR = Interacting Residues; Dis=Distance

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PDB ID Molecule URL to access HORI server results / Example runs using PDB structures mentioned in this chapter

1CRN Crambin http://caps.ncbs.res.in/hori/hori_results/er_hori_SatDec13185917IST2008.html http://caps.ncbs.res.in/hori/hori_cluster_quadruple_sc_4_er.html

1NNQ Ruberythrin http://caps.ncbs.res.in/hori/HORI_1NNQ.html 1AQ0

Barley 1,3-1,4-beta-glucanase

http://caps.ncbs.res.in/hori/hori_lite_quadruple_sc_2_er.html

1CEX Cutinase http://caps.ncbs.res.in/hori/hori_lite_triplet_sc_1_er.html 1A64 CD2 http://caps.ncbs.res.in/hori/hori_results/er_hori_SatDec13182345IST

2008.html Table 7.3: Details about PDB structures mentioned in this chapter with URL to access the

HORI results

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Figure 7.1: Graphical representation of the approach used for the computation of pairwise,

triplet and quadruple interactions of Crambin molecule (PDB ID: 1CRN) (Reproduced with

permissions from BMC Bioinformatics 2010 11:1:S24 DOI: 10.1186/1471-2105-11-S1-S24)

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Figure 7.2: Flow chart of different modules available in HORI server (Reproduced with

permissions from BMC Bioinformatics 2010 11:1:S24 DOI: 10.1186/1471-2105-11-S1-S24)

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Figure 7.3: Screenshots of web-interfaces of HORI-Global, HORI-Lite and HORI-Cluster

(Reproduced with permissions from BMC Bioinformatics 2010 11:1:S24 DOI:

10.1186/1471-2105-11-S1-S24)

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Figure 7.4: HORI server workflow

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Figure 7.5: Graphical view of the triplet interactions identified using HORI server in PDB

IDs: 1WYI, 1G5Q and 4MDH (Reproduced with permissions from BMC Bioinformatics

2010 11:1:S24 DOI: 10.1186/1471-2105-11-S1-S24)

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Figure 7.6: A detailed screenshot of HORI server results from HORI-Global runs using

Crambin (PDB ID: 1CRN) structure as an input (Reproduced with permissions from BMC

Bioinformatics 2010 11:1:S24 DOI: 10.1186/1471-2105-11-S1-S24)

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Figure 7.7: Screenshot of output file from HORI server highlighted with various sections