V.V.

VANNIAPERUMAL COLLEGE FOR WOMEN

BIOINFORMATICS (Belonging to Virudhunagar Hindu Nadars)
An Autonomous Institution Affiliated to Madurai Kamaraj University
Re-accredited with ‘A’ Grade (3rd cycle) by NAAC
LABORATORY MANUAL VIRUDHUNAGAR – 626 001 (TAMIL NADU)

DBT STAR COLLEGE SCHEME

Department of Biotechnology, Ministry of Science and Technology
Government of India, New Delhi
Chairman & Principal : Dr.(Tmty.) S.M. MEENA RANI, M.Sc.,M.Phil.,P.G.D.C.A.,Ph.,
K.Sudha Rameshwari Contact number: 9498088703
e-mail:[email protected]
Coordinator & Member Secretary : Dr.(Tmty.) M.TAMILSELVI, M.Sc.,M.Phil.,Ph.D.,
Contact Number: 9894883106
e-mail:[email protected]

C April,2023@authors FOREWORD
This Lab Manual on “BIOINFORMATICS” is prepared in accordance with
All rights reserved. No part of this publication may be reproduced or
the updated syllabus under DBT Star College Scheme sponsored by the Department
transmitted any form or by any means without permission of the author. of Biotechnology, Ministry of Science and Technology, MHRD, New Delhi to
Any person who does any unauthorised act in relation to criminal fulfill the needs of students.
prosecution and civil claims for damage.
This manual could enable the students to retrieve protein, nucleic acid sequence
using different tools and to interpret the results. The protocols included in this
manual elaborate the step by step procedure with URL link and illustrations.

We thank the Department of Biotechnology, Ministry of Science and

Technology, MHRD, New Delhi for providing a good opportunity under Star
College Scheme (No.HRD11011/163/2020-HRD-DBT Dt.24.8.2020).

We hope this manual will definitely meet out the student’s needs to perform the
experiments that enhance their research skills in the drug development.

Member Secretary/Coordinator Chairman/Principal

 FOREWORD PREFACE

Bioinformatics is an evergreen, emerging interdisciplinary field in life The field of bioinformatics, which applies both information technology
science. It provides a medium for the exchange of information in the fields of and biological science make to prediction about biological processes using
computational molecular biology and the post-genome era, with an emphasis bioinformatics tools. Moreover bioinformatics is an essential part of biological
on the documentation of large data sets and databases that allow the progress of research and has applications in different practical fields.
biomedical research in a significant manner.
This bioinformatics manual helps the beginners to learn step-by-step
This lab manual presents a collection of twenty practical exercises, aimed at
procedure that will make the learners to follow the experiments easily. This
providing standard protocols to access nucleic acid and protein sequence
manual covers the curriculum for the Biotechnology, Microbiology and
databases, perform sequence alignments, predict secondary structure, and
Biochemistry students at the undergraduate level.
visualize proteins. This manual introduces the theory and provides a systematic
procedure to facilitate students to carry out the practical exercises in an easy I would like to express my heartfelt gratitude to the reviewer,
manner. Overall understanding NCBI resources, accessing biological Mrs.P.Sugapriya Menaga, Assistant Professor, Department of Biotechnology,
sequences from GenBank, performing sequence alignments, and using protein ANJAC, Sivakasi for her useful suggestions to improve the quality of this
visualization tools will become easy if the students and researchers use this manual.
manual effectively. I assure that this will become a handy tool and motivation
K.Sudha Rameshwari
factor for the basic analysis of sequences. Assistant Professor,
Department of Biochemistry,
When the readers finish practicing the exercises in this manual, they will
V.V.Vanniaperumal College for Women,
possess the knowledge to use the various online tools available for processing Virudhunagar-626001,
the biological data and will have a platform to develop their skills in handling Tamilandu, India.
bioinformatics techniques in the future.

P. Sugapriya Menaga
Reviewer
Assistant Professor of Biotechnology,
ANJAC, Sivakasi.
 BIOINFORMATICS
LABORATORYMANUAL
LIST OF PRACTICALS
S.No. TITLE Page No.
1. Introduction to Bioinformatics 1
2. Retrieval of Nucleotide sequence from GenBank 4
3. Retrieval of Protein sequence from GenBank 8
4 Sequence Similarity Search using BLASTN 14
5. Sequence Similarity Search using BLASTP 22
6. Accessing Structural Database and Download the 28
Protein Structure
7. Working with Ensembl 32
8. Multiple Sequence Alignment 36
9 Predicting Physiochemical properties of protein 42
sequence
10 Predicting Peptide mass of protein sequence 48
11 Predicting cleavage site of protein sequence 54
12 Predicting secondary structure using SOPMA tool 58
13 Predicting secondary structure using CFSSP tool 62
14 Predicting transmembrane region of Protein sequence 66
15 Predicting hydrophilic region in the protein sequence 70
16 Detecting alignment of repeats in protein sequences 74
17 Predicting the peptide structure for the given protein 76
sequence
18 Conversion of nucleotide sequence into Protein 78
sequence
19 Molecular Visualization using RasMol 82
20 Measurement of bond length in protein structure 94
using RasMol
21 Measurement of bond angle in protein structure using 99
RasMol
22 Web links 101
 INTRODUCTION TO BIOINFORMATICS personalized medicine depends on the application of
bioinformatics approaches.
Bioinformatics is a newly emerging scientific discipline for
This course focuses on employing existing bioinformatics
the computational analysis and storage of biological data.
resources - mainly web-based programs and databases - to access
Bioinformatics is the field in which biology, computer science and
the wealth of data to answer questions relevant to the average
information technology merges into a single discipline for managing
biologist, and is highly hands-on. Different types of career
and analyzing biological data using advanced computing techniques.
opportunities are available for the students of bioinformatics like
OBJECTIVES Scientific Curator, Gene Analyst, Protein Analyst, Phylogenitist,

 To organize data, to access existing information and to Research Scientist / Associate, Data Base programmer,

submit new entries as they are produced. Bioinformatics software developer, Computational biologist,

 To develop tools and resources that aid in the analysis of data Network Administrator / Analyst, Structural Analyst, Molecular
Modeler, Biostatistician, Biomechanics, Database programmer,
 To analyze the data and interpret the results in a biologically
Cheminformatician, Pharmacogenetician, Pharmacogenomics etc.
meaningful manner

SCOPE OF BIOINFORMATICS

Biological and medical labs use methods that produce

extremely large data sets, which cannot be analyzed for instance
sequencing human genomes. Thus, modern biological and
medical research and development cannot be carried out without
bioinformatics. In addition, bioinformatics plays an important role
in biomedical research. Research in the area of genetic diseases
and medical genomics is rapidly developing and the future of

1 2
 RETRIEVAL OF NUCLEOTIDE SEQUENCE FROM
GENBANK

AIM
To retrieve the gene from Genbank and to save the sequence in
FASTA format
Click
INTRODUCTION
Entries of nucleotide sequence are stored in Genbank nucleotide
database. Each entry contains complete information about the particular
gene. Genbank website is accessible for anyone who is interested in
Figure 1: Open the web browser getting the entry for research.
PROCEDURE
 Type NCBI in the web browser and click search, click National
Center for Biotechnology Information, it directs to the URL :
https://www.ncbi.nlm.nih.gov/ (Figure 1) OR
 Type the URL https://www.ncbi.nlm.nih.gov/ directly in the
space for address in the address bar and press the enter key.
 NCBI homepage will appear.
 Click the All Databases drop –up menu and drag the bar and
Click select nucleotide (Figure 2)
 Search list will be displayed, click the suitable accession number
or any gene of interest (figure 4)

Figure 2: NCBI Home page

3 4
 Enter the Gene name in the search
area  Click the gene of interest/accession number (Figure 4)
Click  Type Collagen in the search area and click (Figure 3)
 A new window will appear and shows the entry of the collagen
gene in detail (Figure 5)
 Click the FASTA, FASTA sequence appears in the new window
(Figure 6)
Figure 3: Home page of NCBI the gene enter in the search area
 Copy the FASTA sequence and paste it in note pad
 Save it for further investigation.
[[ RESULT
The collagen gene was retrieved and saved as FASTA format in
notepad.
OUTCOME

Students acquire the knowledge to retrieve any gene from

Figure 4: search list page Genbank. From the retrieved sequence flat file, details of sequences,
Click
submitter’s details, biological significance, and the scientific name
and taxonomy of the organism is understood. A feature table shows
CLICK characteristics that indicate coding regions, transcription units,
FAST
mutation locations, etc. Retrieved gene sequence is useful for gene
Figure 5: Genebank entry format
analysis. It can be compared with other sequences to determine which
gene is mutant. It enlightens the diagnosis of hereditary disease.

Figure 6: FASTA sequence in new window

5 6
 RETRIEVAL OF PROTEIN SEQUENCE FROM GENBANK

Click AIM
To retrieve the protein from Genbank and to save the sequence in
FASTA format
INTRODUCTION
Entries of protein sequence are stored in Genbank database.
Each entry contains complete information about a particular protein.
Genbank website is accessible for anyone who is interested in getting
the entry for research.
PROCEDURE
Figure 7: Home page of NCBI, Selection of Protein  Type NCBI in the web browser and click search, it shows
National Center for Biotechnology Information, click National
Center for Biotechnology Information, it directs to the url :
https://www.ncbi.nlm.nih.gov/ or home page of NCBI (Figure 7)
Or
 Type the url: https://www.ncbi.nlm.nih.gov/ web/Genbank/
directly in the address bar and press the enter key
 NCBI homepage will appear
Click  Click the All Databases drop –up menu, and drag the bar to
select protein (Figure 8)

Figure 8: Search list page of protein

7 8
  Type collagen in the search area and press enter key or click
search
 Search list will be displayed (Figure 8)
 Select the suitable accession number that describes collagen
 Click the accession number of collagen
 A new window will appear and shows the entry of the collagen
K gene (Figure 9)
 In display format, in place of summary choose “FASTA”
 Only sequences will be shown (Figure 10)
 Copy the sequence (Figure 11) and paste it on note pad (Figure
12) and save it for the further investigation
RESULT
Figure 9: Collgen gene Genbank entry page
The Protein sequence was retrieved and saved as FASTA
format in notepad.
OUTCOME
Students master the technique of sequence retrieval which is the
basic step in bioinformatics. From the retrieved sequence flat file,
details of sequences, submitter’s details, biological significance, and
the scientific name and taxonomy of the organism are understood.
Sequence retrieval is essential for the analysis of primary, secondary
and tertiary structure of any protein sequence.

Figure 10: Fasta format Page

9 10
 Figure 11: Copy the Fasta Sequence

Figure 12: Paste the Fasta Sequence in the note pad

11 12
 SIMILARITY SEQUENCE SEARCH USING BLASTN
AIM
To find the similarity of sequence for the given nucleotide or
Click
protein sequence
INTRODUCTION
BLAST is Basic Local Alignment Search Tool. It is a
technique for finding homology and similarity. It is a tool for
Figure 13: Search list of BLAST searching of sequences that are similar to one another in databases. By
matching the novel sequence with previously defined genes, it
compares novel gene sequences with nucleotide databases. This tool
focuses on identifying areas of sequence similarity. It will provide
information on the structure and function of the novel sequence.
Instead of using the best alignments, it looks for areas of sequence
Click
Nucleotide similarity. It produces ungapped alignments. It reports multiple local
BLAST for
similarity alignments between the query and database. It is based on an explicit
search of
nucleotide
statistical theory.
PROCEDURE
 Type BLAST in the web browser and click search
 Click the first link, BLAST (Figure 13)
 Go to Blast Home page (Figure 14)

Click Protein BLAST  Click nucleotide blast BLASTN (Figure 15)

for similarity
Figure 14: BLAST home page & Select Protein blast search of Protein
sequence

13 14
Check blastn
is selected Depending on the type of sequence, the programs in BLAST
differs. They are
 Blastp compares an aminoacid query sequence against a
protein sequence database
 Blastn compares an nucleotide query sequence against a
Paste the Fasta nucleotide nucleotide sequence database
sequence in the search box
 Blastx compares a nucleotide query sequence translated in
all reading frames against a protein sequence database.
 tblastn compares a protein query sequence against a
nucleotide sequence database dynamically translated in all
reading frames.
 tblastx compares the six frame translation of a nucleotide

Figure 15: BlastN Page query sequence against the six frame translation of a
Click Tick the box nucleotide sequence database.
 Paste the sequence (FASTA format) in the given box of
BLASTN or paste the accession number (Figure 15)
 Tick the box “results in new window” in the last line of
BLASTN Home page (Figure 15)
 Click BLAST (Figure 15)
 In the new window, format request display is shown, wait for
few seconds, it leads to result page

15 16
  In the result page, the blast results for sequence similarity
search appear as graphically and as text (descriptions,
alignment)
 Click each text and find the similarity between the two
sequences
 Click graphic summary (Figure 16)
 View the graphical representation of sequence alignment
 Lines in pink, red colors represent the sequence in a set of
score values.
 Length of the line indicates the length of the local
alignment with the query sequences. (Figure 17)
RESULT
The most significant and similar sequences fetched by the

Click
blast are Homo sapiens collagen type II alpha 1 chain (COL2A1),
graphical transcript variant 2, mRNA,
summary OUTCOME
By learning BLASTN, students obtain the skill to compare the
new gene sequence with the nucleotide database by aligning the
novel sequence with previously characterized gene/protein. From
the comparison, students gather the functional and evolutionary
clues about the structure and function of the novel sequence.

Figure 16: Result window of BLASTN

17 18
Figure 17: Result page of Graphical summary

19 20
 SIMILARITY SEQUENCE SEARCH USING BLASTP

AIM
To find the similarity of sequence for the given protein sequence
INTRODUCTION
BLAST is Basic Local Alignment Search Tool. It is a technique
for finding homology and similarity of protein sequences. It is made
for the Windows platform and is employed to carry out DNA or protein
similarity searches. The source is NCBI. It is a tool for searching for
sequences that are similar to one another in databases. This tool
focuses on identifying areas of sequence similarity. It is used to
Figure 18: Select Protein blast Click
compare a novel protein sequence against protein database by aligning
the novel sequence with previously characterized protein. The
emphasis of this tool is to find regions of sequence similarity. It will
yield functional and evolutionary clues about the structure and function
of the novel sequence. It finds out patches of sequences similarity
rather than best alignments. It produces un gapped alignments. It
reports multiple local alignments between the query and database. It is
based on an explicit statistical theory.
PROCEDURE
 Go to NCBI Blast page
 Chose the Blast program for proteins BLASTP (Figure 18)
Figure 19: BlastP Page
Click Tick
21 22
  Copy the sequence from the notepad. Avoid the first FASTA line.
Paste the protein sequence in the search window of BLASTP.
(Figure 19)
 Tick the box “results in new window”, in the last line of blast
Home page
 Click BLAST
 In the new window, format request display is shown, wait for few
seconds, it leads to result page (Figure 19a)
 In the result page, the blast results for sequence similarity search
appear as graphically and as text
 Click each text and find the similarity between sequence
 Click graphic summary
 View the graphical representation of sequence alignment
(Figure 19b)
 Lines in pink, red colors represent the sequence in a set of score
values.
 Length of the line indicates the length of the local alignment with
the query sequences.
RESULT
The most significant and similar sequences fetched by the
blastp are hemoglobin [Pseudoterranovadecipiens],CAA77743.1
Figure 19a: Result page of Blastp
23 24
 OUTCOME
By learning BLASTP, students gain the knowledge to compare
the new protein sequence with the protein database by aligning the
novel sequence with previously characterized protein. From the
comparison, students can obtain functional and evolutionary clues
about the structure and function of the novel sequence.

Figure 19b: Graphical summary page

25 26
 ACCESSING STRUCTURAL DATABASE AND
DOWNLOADING THE PROTEIN STRUCTURE

AIM
To access the PDB (Protein Data Bank) structural database and
Click to download the protein structure.
INTRODUCTION
Figure 20 : Web browser page The three dimensional structure of biomolecules plays an
important role in the functions and maintenance of the structural

Click features of an organism. These structures were deciphered by research

Enter the scientists and deposited in the databases, specifically designed for
interested Click Protein BLAST
protein
structural submission, for worldwide use. Research Collaborator for
name or ID Structural Bioinformatics (RCSB), manages of the PDB. It provides
free resources to assist the fields of biology and bioinformatics. It
provides detailed information about sequence, atomic coordinates,

Figure 21: PDB Home page

structure factors, and crystallization conditions etc.
PROCEDURE
 Type PDB in the web browser search field (Figure 20)
 Click the RCSB page (Figure 20)
 Go to the WWW.rcsb.org (Figure 21)
 Enter the protein name of interest in the search box
 Click Go
Click

Figure 22: Result summary page

27 28
  Choose the protein name of interest (Figure 22)
 Click the ID
 Click the download file (Figure 23)
 Click PDB format (Figure 23)
Click
 Download file as PDB format (Figure 24)
 Save file
Click
 View the protein structure in the molecular visualization
tool like RASmol, Swisspdbv (Figure 25)
Figure 23: Download the protein structure in PDB format
RESULT
The protein structure was downloaded and their descriptions were
Protein structure
downloaded in observed.
pdb format
OUTCOME
Students get trained to retrieve any protein structure from
structural protein database and can understand the characteristics of
Figure 24: Protein structure was downloaded
the protein structure.

Figure 25: Downloaded protein structure is visualized in Molecular visualization

tool (Rasmol and Swisspdbv)

29 30
 WORKING WITH ENSEMBL

Type AIM
ensembl
To retrieve the vertebrate genomic information
INTRODUCTION
Ensembl is one of the several well-known genome browsers for

Click the retrieval of genomic information. It is considered to be the

universal information source for the human genome. Data available in
Ensembl include genes, SNPs, repeats and homologies. Genes may
Figure 26: Google search page either be known experimentally or deducted from the sequence;
because the experimental support for annotation of the human genome
is so variable. It presents the evidence for identification of every gene.
Extensive linking to other databases containing related information
such as OMIM or expression databases is also available. Ensembl tools
include BLAST, BLAT, BioMart and the Variant Effect Predictor
(VEP) for all supported species.
PROCEDURE
 Type Ensembl in the web browser search field
Select Type your interested  Click Ensembl genome browser 109 (Figure 26)
species
gene or disease  Go to the home page, First choose the species of interest
and type gene of interest or disease name (Figure 27 & 28)
Figure 27: Ensembl home page and click go

31 32
  List of genes were displayed (Figure 29)
 Select one and click
 Details of diabetes were shown (Figure 30)
 Click hyperlink one by one; the details of variant, genomic
Diabetes
Select location, reported genes, phenotype/genotype trait, annotation
Human
source, submitter, and external reference were known. (Figure
30)
Figure 28: Ensembl home page
RESULT
The diabetic gene annotations were retrieved from the Ensembl
genome browser.

Click OUTCOME
Students can retrieve any vertebrate gene and its details from the
Ensembl genome browser.
Figure 29: List of diabetes gene

Figure 30: Details of diabetes were listed

33 34
 MULTIPLE SEQUENCE ALIGNMENT
AIM
To study the closely related genes or proteins
Click INTRODUCTION
ClustalW tool is used for aligning multiple nucleotide or
protein sequences. It uses progressive alignment methods, which align
the most similar sequences first and work their way down to the least
Figure 31: Google Search similar sequences until a global alignment is created. A multiple
sequence alignment tools called Clustal Omega creates alignments
between three or more sequences by using HMM profile-profile
algorithms and seeded guide trees. Multiple sequence alignment is a
tool used to study closely related genes or proteins in order to find the
evolutionary relationships between genes and to identify shared
patterns among functionally or structurally related genes.

PROCEDURE
Figure 32: ClustalW Home page
 Type multiple sequence alignment tool in the web browser
search field (Figure 31)
 Click Clustal Omega < Multiple Sequence Alignment <
EMBL-EBI Or
 Click Multiple Sequence Alignment-CLUSTALW –
Genome Net ((Figure 31)

Figure 33: sequences in the notepad

35 36
  Go to home page (Figure 32)
 Copy the sequences from the notepad (Figure 33)
 Paste the more than two sequences in input sequences box
(Figure 34)

Choose protein  Choose the sequence as protein or DNA (Figure 34)

or DNA  Click submit or execute multiple sequence alignment
(Figure 34)
Paste more than
 Result page is shown (Figure 35)
two sequences in
the box  Choose fast tree and click
 Phylogenetic tree was displayed (Figure 36)

Click execute RESULT

multiple sequence
alignment  The close relationship for this NP_001275068.1 (any one
Id which sequences are pasted in input sequence box) is
Figure 34: Paste the sequences in the box
NP_001104966.1.
 The far relationship for this NP_001275068.1 (any one Id
Choose tree which sequences are pasted in input sequence box) is
and click
AAA29796.1.
OUTCOME
Using this multiple sequence tool, students attain the ability to
infer the evolutionary relationships between the sequences under
study.
Figure 35: Result page

37 38
Figure 36: Phylogenetic tree

39 40
 PREDICTING PHYSIOCHEMICAL PROPERTIES OF
PROTEIN SEQUENCE

AIM
Click To predict the physiochemical properties of protein sequence
using ExPASy resources.
Figure 37: search bar INTRODUCTION
With the help of the tool ProtParam, users may compute a
number of physical and chemical parameters for a specific protein that
is contained in Swiss-Prot or TrEMBL, as well as for a user-entered
protein sequence. Molecular weight, theoretical pI (Isoelectric point),
Click amino acid composition, atomic composition, extinction coefficient,
assumed half-life, instability index, aliphatic index, and grand average
of hydropathicity (GRAVY) are among the calculated characteristics.
Figure 38: Home page of ExPASy
PROCEDURE
 Type ExPASy in the web browser search box (Figure 37)
 Click ExPASy
 Go to ExPASy page, enter specific proteomic tool in search
 box (Figure 38) Or
 Enter the url: https://web.ExPASy.org/ProtParam/
 For predicting physiochemical properties, type ProtParam
Click and enter

Figure 39: Search Results Page

41 42
  Select protoparm tool (Figure 39)
 Click “browse the resource website”, protoparm tool page is
opened in new window (Figure 40)
 Paste the protein sequence / accession number in the given box
(Figure 41)
Click  Click compute parameters (Figure 41)

Figure 40: Resource page RESULT

Using ProtParam tool, the physicochemical properties of protein
sequence were predicted as follows (Figure 42):
 Number of aminoacids:718
 Molecular weight: 72106.72

Paste the sequence in the box  Theoretical pI: 7.31

 Total number of negatively charged residues (Asp + Glu): 77
 Total number of positively charged residues (Arg + Lys): 77

OUTCOME
Students learnt the art of analysing the physical and chemical
properties for a specific protein.

Click

Figure 41: sequence pasted in the ProtParam tool box

43 44
Figure 42: Result page of Protoparam tool

45 46
 PREDICTING PEPTIDE MASS OF PROTEIN SEQUENCE

AIM
To predict the peptide mass of protein sequence using ExPASy
resource
INTRODUCTION
In the analytical method of protein identification known as
peptide mass fingerprinting (PMF), the unknown protein of interest is
first broken up into smaller peptides whose absolute masses may be
Click
precisely determined with a mass spectrometer like the MALDI-TOF
or ESI-TOF.
Figure 43: ExPASy Proteomic tool search site PROCEDURE
 Type ExPASy in the web browser search field
Tool opens in new window
 Click ExPASy
 Go to ExPASy page, Enter Peptide Mass in the search field
(Figure 43) Or
 Type url : https://web.ExPASy.org/peptide_mass/
 Click peptide mass tool
 It leads to peptide mass resource page
Click  Click the “Browse the resource website”, Peptide mass tools
site opens in new window (Figure 44)
 Paste the protein sequence of interest
Figure 44 : Peptide Mass resources site

47 48
  Move the cursor to choose which type of enzyme to cleave the
interested protein sequence (Figure 45)
 Select "trypsin" from the drop-down box under "Select enzyme:
in the tool," if we want other enzyme use drop menu and select
the desired enzyme (Figure 45)
 Click perform (Figure 46)
 Result page shown (Figure 47)
RESULT
Choose any
enzyme
 The high molecular weight peptide mass sequence is
TGPPGKPGPPGPPGPPGIQGIHQTLGGYYNK, position 309-
339and its molecular mass is 3036.5689
Figure 45: Peptide Mass tool site showing different enzymes for selecting to cleave
protein sequence
 The low molecular weight peptide mass sequence is QELK,
position 174-177 and its molecular weight is 517.2980.

OUTCOME
Students can identify the molecular weight of peptide after
cleavage of protein using different enzymes or chemicals.

Click

Figure 46: PeptideMass tool site with pasted sequence and click to perform

49 50
Figure 47: Result page of peptide mass

51 52
 PREDICTING CLEAVAGE SITE OF PROTEIN SEQUENCE

AIM
To predict the cleavage site of protein sequence using ExPASy

Click resource (Peptide cutter).

Figure 48: Google search list with peptide cutter
INTRODUCTION
Peptide Cutter explores for protease cleavage sites in a protein
sequence given by the user, a protein sequence from the SWISS-PROT
Paste the protein sequence and/or TrEMBL databases, or both. Users can use a protease, a group of
proteases, or the entire list of proteases and compounds. Different
If you want to know the cleavage
forms of output of the results are available: Tables of cleavage sites that
sites of all enzymes and chemicals,
select this option are either ordered alphabetically by the names of the enzymes or
sequentially by the number of amino acids are available. A map of the
cleavage locations is a third possible result. The user can choose the
block size to print out the sequence and the cleavage sites that have
Figure 49: Peptide cutter Home page been mapped onto it.
PROCEDURE
 Type Peptide cutter in the web browser search field and press
If you want to know the cleavage sites of
any one or two of enzymes and chemicals, enter
select the option of particular enzymes or  Click Peptide cutter tool (Figure 48) Or Type url :
chemicals
https://web.ExPASy.org/peptide_cutter/
Click

Figure 50: Paste the sequence and click to perform

53 54
  Go to the peptide cutter tool home page (Figure 49)
 Paste the protein sequence of interest in the given box
 Move the cursor to choose the enzyme type to cleave the
sequence of interest or select all available enzymes and
chemicals; Click perform, (Figure 50)
 Result page is displayed (Figure 51)

RESULT
Proteinase K enzyme (Specific enzyme choose) cleaves 328 sites
in a given protein sequence.

OUTCOME
Students can predict the potential cleavage sites cleaved by
proteases or chemicals in a given protein sequence. Peptide Cutter
returns the query sequence with the possible cleavage sites mapped on
it and /or a table of cleavage site positions.

Figure 51: Result page

55 56
 PREDICTING SECONDARY STRUCTURE OF PROTEIN
SEQUENCE USING SOPMA TOOL

AIM
Click To predict the secondary structure of protein sequence using
ExPASy resources
INTRODUCTION
Secondary structure prediction is a group of techniques
in bioinformatics that aim to calculate the secondary structures of
proteins and nucleic acid sequences based on the information from
Figure 52: Google search list with secondary structure prediction tool
their basic structures (primary structures). Through base pairing and
base stacking interactions, it predicts the formation of nucleic acid
structures like helixes and stem-loop structures while predicting the
formation of protein structures like alpha helices and beta strands for
proteins. The Self-Optimized Prediction Method with Alignment
(SOPMA) is a tool to predict the secondary structure of a protein.
Based on the query (primary sequence of a protein), SOPMA will
predict its secondary structure. Protein secondary structure prediction
offers insight into the activity, interactions, and functions of proteins
as well as serving as an important initial step toward tertiary structure
prediction. The polypeptide backbone of the local conformation
Figure 53: Home page of SOPMA tool proteins is referred to as the protein's secondary structure.

57 58
 PROCEDURE

 Type SOPMA tool in the web browser search field and press
 enter
 Click the NPS@SOPMA secondary structure prediction
(Figure 52)
 Open the SOPMA secondary structure prediction tool home
page (Figure 53)
Click
 Paste a protein sequence in the given box (Figure 54)
Figure 54: sequence pasted in box  Click submit (Figure 54)
 Result page (Figure 55) is displayed.

RESULT
 The secondary structure location present in the protein
sequence is predicted.
 The secondary structures present in the given sequence are
alpha helix, extended strand, beta turn and Random coil.
 Random coil is the most prominent secondary structure
(70.89%) in the given sequence.
 The lowest percentage of secondary structure in the given
sequence is Beta turn.
OUTCOME
Students learn how to predict the regions of different forms of
Figure 55: Result page of SOPMA secondary structure from the protein sequence.

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 PREDICTING SECONDARY STRUCTURE OF PROTEIN
SEQUENCE USING CFSSP TOOL

AIM
To predict the secondary structure of the given protein sequence
through CFSSP tool.
INTRODUCTION
CFSSP (Chou and Fasman Secondary Structure Prediction
Click Server) is an online protein secondary structure prediction server. The
Figure 56: Search list of Secondary structure prediction tool output predicts regions of secondary structure from the protein
sequence such as alpha helix, beta sheet, and turns from the amino
acid sequence. The method implemented in CFSSP is Chou and
Fasman algorithm, which is based on analyses of the relative
frequencies of each amino acid in alpha helices, beta sheets, and turns
based on known protein structures solved with X-ray crystallography.
CFSSP is freely accessible via ExPASy server or directly from
BioGem tools at http://www.biogem.org/tool/chou-fasman.
PROCEDURE
 In web browser, type Chou Fasman secondary structure
prediction tool, search list will be displayed and click CFFSSP
(Figure 56)
 Click CFSSP or http://www.biogem.org › tool › chou-
Figure 57: CFSSP Home Page fasman

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  Open the CFSSP secondary structure prediction tool home
page (Figure 57)
 Paste a protein sequence in the given box (Figure 58)
 Click predict (Figure 58)
 Result page (Figure 59) is displayed.
RESULT
Click  The secondary structures present in the given sequence are
alpha helix, beta sheet and betas turn.
Figure 58: Sequence pasted in the box
 Alpha helix is the most prominent secondary structure
(32.9%) in the given sequence.
 The lowest percentage of secondary structure (16.7) in the
given sequence is β turn.

OUTCOME
Students understand how to predict the regions of different
forms of secondary structure from the protein sequence.

Figure 59: Result page

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 PREDICTING TRANSMEMBRANE REGION OF
PROTEIN SEQUENCE

AIM
To predict the transmembrane region in the given protein
Click sequence/ID.
INTRODUCTION
The orientation of membrane-spanning sections is predicted
by the TMpred software. The technique is based on a statistical
investigation of the transmembrane protein database known as
TMbase. A combination of various weight matrices for scoring are
Figure 60: ExPASy home page
used to make the prediction.
PROCEDURE
 Go to ExPASy page, enter TMpred in search box (Figure 60)
 Click TMpred
 Go to the home page of TMpred tool (Figure 61)
 Paste the protein sequence in the search field
 Click Run TMpred (Figure 61)
 Results page is displayed (Figure 62)

Click

Figure 61: Home page of Mpred

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 RESULT

 Outside to inside helices : 1 found

from to score center
565 (568) 587 (587) 42 577
 outside->inside
"++" symbol indicates a strong preference of orientation
(565- 587 (23) 42 ++)

OUTCOME
Students comprehend the technique to predict the membrane-
spanning regions and their orientation. They also understand that the
transmembrane proteins act as gateways for transporting specific
substances across the membrane.

Figure 62: Result page of TMpred

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 PREDICTING HYDROPHILICITYREGION IN THE
PROTEIN SEQUENCE

AIM

Click To predict the hydrophilic region in the given protein

sequence
INTRODUCTION
The hydrophobicity or hydrophilicity of the amino acids in a
protein is statistically analysed using a hydrophilicity plot. It is used
Figure 63: Search list of Hydrophilicity plot to analyse or identify a protein's potential structure or domains. If the
protein fragment is sufficiently hydrophobic to interact with or
remain in a membrane can be predicted from the plot. The plot's x-
axis represents the amino acid sequence of a protein, while the y-
axis represents the degree of hydrophobicity and hydrophilicity. The
degree of interaction between certain amino acids and polar solvents
like water can be determined using a variety of techniques. For
instance, the Hopp-Woods scale assesses hydrophilic residues while
the Kyte-Doolittle scale identifies hydrophobic amino acids.
Understanding the plot's shape reveals the details of the protein's
partial structure. For instance, if a group of roughly 20 amino acids
exhibits positive hydrophobicity, then it is possible that these amino
acids are a portion of an alpha-helix that spans a lipid bilayer made
up of hydrophobic fatty acids.
Figure 64: Hydrophilicity home page

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 On the other hand, amino acids with high hydrophilicity show
that these residues are in touch with a solvent, such as water, and are
therefore likely to be found on the protein's outer surface. First, a
hydrophobicity rating between 4.6 and -4.6 is provided to each
amino acid. The highest hydrophobic value is 4.6, and the least
hydrophilic score is -4.6.

PROCEDURE
 Type hydrophilicity plot in the web browser and press enter
 Search list will be displayed, Click protein hydrophilic city
Figure 65: Home page plot –novoprolabs (Figure 63)
 Go to the home page (Figure 64)
 Paste the protein sequence in the given field
 Click submit (Figure 65)
 Results will be displayed (Figure 66)
RESULT
The hydrophilic residues in given Protein sequence was
predicted.
OUTCOME
Students learn to predict whether or not the protein segment
has enough hydrophilicity to either interact with or reside in a
membrane.
Figure 66: Hydrophilic regions in the protein sequence

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 DETECTING ALIGNMENT OF REPEATS IN A PROTEIN
SEQUENCES
Type tool name
AIM
To detect the alignment of repeats in a given protein sequence.
INTRODUCTION
Click
RADAR stands for Rapid Automatic Detection and
Alignment of Repeats in protein sequences. RADAR identifies
Figure 67: Google search page gapped approximate repeats and complex repeat architectures
involving different types of repeats.
PROCEDURE
 Type Radar protein tool in the web browser and press enter
 Click the RADAR tool (ebi.uk)in the search list (Figure 67)
Figure 68: RADAR tool home page
 Go to the RADAR home page (Figure 68)
 Paste the fasta protein sequence in the input box
 Click submit
 Result page was shown (Figure 69)
RESULT
The given sequence contains 2alignment repeats.
OUTCOME
Students can able to predict the number of alignment repeats
in the proteins sequences.

Figure 69: Result Page

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 PREDICTING THE PEPTIDE STRUCTURE FOR THE
GIVEN PROTEIN SEQUENCE
AIM
Click To predict the peptide structure of given protein sequence
using Pepdraw tool
INTRODUCTION
Figure 70: Google search list Pepdraw tool is used to draw primary peptide and also
calculate the physico chemical properties.
PROCEDURE
 Type Pepdraw tool in the web browser and press enter
 Click the Pepdraw in the search list (Figure 70)
Figure 71: Pepdraw home page  Go to the Pepdraw home page (Figure 71)
 Paste the fasta protein sequence in the input box
 Click draw peptide
 Result page will be displayed (Figure 72)
RESULT
The protein sequence was converted into peptide chain.

OUTCOME
Students acquire the skill to draw the primary chemical
structure of an amino acid sequence and to predict chemical
properties for any protein sequences.

Figure 72: Result page

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 CONVERSION OF NUCLEOTIDE SEQUENCES INTO
PROTEIN SEQUENCES

AIM
To convert the nucleotide sequences into protein sequences and to
identify the correct reading frame.
INTRODUCTION
Select compact Translate is a tool which allows the translation of a nucleotide
(DNA/RNA) sequence into a protein sequence. Translate accepts a
DNA sequence and converts it into a protein in the reading frame as
specified. Translate supports the entire IUPAC alphabet and several
genetic codes. A raw sequence or one or more FASTA sequences is
pasted in the text area. Input limit is 200,000,000 characters.
Determining is a complex process if a nucleic acid sequence actually
codes for a protein. Because, generally it is not known which strand is
the coding strand or which is the correct reading frame. Both these
questions are resolved by translating both strands in all three reading
frames and looking for the one that gives the longest amino acid
sequence before a stop codon is encountered. A stop codon is expected
to appear on average once for every 20 amino acids when reading a
sequence in the incorrect frame. It is possible for an out of frame

Figure 73: translate Homepage

translation to extend over 100 amino acids before a stop codon is
reached.

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 PROCEDURE
 Select the nucleotide sequence, copy it and then paste it into the
translate sequence window in the ExPASy translate tool
 Under Output format, select "Compact" or nucleotide sequence
without space
 Click on Translate Sequence
Select nucleotide

 Result page is displayed (Figure 73 and 74).

sequence with no
spaces

RESULT
 Output compact is selected; it gives the amino acid sequence as
one letter code with stop codons indicated by a hyphen with
different frames.
 Output nucleotide sequence without space is selected; it gives the
nucleotide sequence with one letter code aminoacid with different
frames.
 Red colour indicates the open reading frame.

OUTCOME
Students gain knowledge to convert any unknown nucleotide
sequence into protein sequence and to identify the most correct reading
frame. They also acquire knowledge about the exons, pseudogenes, non-
coding region of DNA and regulatory functions.

Figure 74: Result page of translate with nucleotide sequence with aminoacid

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Click file Click open
MOLECULAR VISUALIZATION USING RASMOL

AIM
To visualize the tertiary structure of protein molecule in
graphic view and command line

INTRODUCTION
RasMol is free software for molecular visualization created by
Roger Sayle. It is a molecular graphics programme intended for the
visualization of proteins, nucleic acids and small molecules. The
programme aims at display, teaching and generation of publication
with quality images. The program reads in a molecule coordinate file
Figure 75 : Open the PDB file in RASMOL tool and interactively displays the molecular screen in variety of colour
schemes and molecular representations.
PDB co-ordinate file
REQUIREMENT
RASMOL software, PDB molecule
PROCEDURE
 Open a molecular visualization tool (Figure 75)
 From the file menu open a PDB atom co-ordinate file
(Figure 76)
 Rotate the molecule

Figure 76: Protein structure open in molecular visualization tab

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  Try various options (Figure 77,78,79)
 Try different commands in command line and visualize the
changes in structure (Figure 80, 81)
 Save the required structural view
 Exit the application

COMMANDS
 Select
 Colour
 Zoom on
 Zoom off
 Label on
 Label off
 Spacefill
 Star on
 Background pink
 Stereo on
 Stereo off
 Pick angle
 Pick distance
 Label 250
 Star

Figure 77:Visualise the different forms of protein structure

 Rotate

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  Hbonds
 Wireframe
 Cartoon
 dots
 Quit

Other command reference:

Backbone Background Bond Bulgarian Cartoon Centre Chinese Clipboard

Colour ColourMode Connect CPK CPKnew Defer Define Depth

Dots Echo English Execute Exit French HBonds Help

Italian Japanese Label Load Map Molecule Monitor NoToggle

Pause Play Print Quit Record Refresh Renumber Reset

Restrict Ribbons Rotate Save Script Select Set Show

Slab Source Spacefill Spanish SSBonds Star Stereo Strands

Structure Surface Trace Translate UnBond Wireframe Write Zap

Zoom

RESULT
The tertiary structure of protein molecule is visualized in graphic
view and command line
Figure 78: Visualise the different forms of protein structure

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 OUTCOME
The students are imparted the ability to evaluate and interpret
molecular models. Students can interpret the complicated molecule
structure, properties, and interactions with the use of molecular
visualisation tools. These resources aid their study in the fields of
chemistry, pharmacology, biology, and bioinformatics.

Figure 79: Visualise the different forms of protein structure

87 88
Figure 80: Visualise the different forms of protein structure

89 90
 Select carbon
Colour green

Figure 81: Various commands used in command line and visualize the
changes in structure

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 MEASUREMENT OF BOND LENGTH IN PROTEIN
STRUCTURE USING RASMOL

AIM
To measure the bond length of atoms in the protein structure
and to visualize it in graphic view and command line.
Label on INTRODUCTION
RasMol is a free software for molecular visualization created
by Roger Sayle. It is a molecular graphics programme intended for
the visualization of proteins, nucleic acids and small molecules. The
program aims at display, teaching and generation of publication with
quality images. The program reads in a molecule coordinate file and
interactively displays the molecular screen in variety of colour
Stereo on
schemes and molecular representations.
PROCEDURE
 Open RasMol and import a file of Pdb atom coordinates
 Use the various menu options and get a composition of the
molecule
 Set the display style to “ball and stick” (Preferable, but works
with other displaying style as well)

Background green  Use Shift+ mouse down to zoom in on the molecule to see the
bonds more clearly.
Figure 82: Various commands used in command line and visualize the  Open the command line window
changes in structure
 Type set picking distance and press enter key
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  Open the display Window and select the two atoms
participating in the bond formation by clicking on them
successively.
 The command line window displays the bond length.
 Record the results
 Alternatively, to show bond and to measure the bond length
between two atoms, type set picking monitor in the command
line window.
 Now click on the two atoms again.
 A bond line appears (The bond is removed when a click on
the atoms is made more than once.)
 Note the results from the command line window.

RESULT

The distance between two atoms is 90.0Å.

OUTCOME
Students learn how to find out the distance between two atoms
using command line and directly picking atoms in structure.

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 MEASUREMENT OF BOND ANGLE IN PROTEIN
STRUCTURE USING RASMOL

AIM
To measure the bond angle between the atoms in the protein
structure and to visualize it in graphic view and command line.

INTRODUCTION
RasMol is a free and most popularly used software for
molecular visualization created by Roger Sayle. It is a molecular
graphics programme intended for the visualization of proteins, nucleic
acids and small molecules. The program aims at display, teaching and
generation of publication with quality images. The program reads in a
molecule coordinate file and interactively displays the molecular
screen in variety of colour schemes and molecular representations.
PROCEDURE
 Open RasMol and load a file of Pdb atom coordinates
 Use the various menu options and get a feel of the molecule
 Set the display style to “ball and stick”(Preferable, but works
with other displaying style as well)
 Use Shift + mouse down to zoom in on the molecule to see the
bonds more clearly.
 Go to command line window.
 Type set picking angle and press enter key

97 98
  Go to display Window and select the three atoms forming the
bond angle by clicking on them successively.
 The command line window displays the bond angle.
 Note the results.

RESULT

The bond angle between atoms is 25.0°

OUTCOME
Students acquire the skills necessary to find out the bond angle
between atoms using command line and directly picking atoms in
structure.

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 V.V.VANNIAPERUMAL COLLEGE FOR WOMEN
(Belonging to Virudhunagar Hindu Nadars)
An Autonomous Institution Affiliated to Madurai Kamaraj University
Re–accredited with ‘A’ Grade (3rd cycle) by NAAC
VIRUDHUNAGAR.
WEBLINKS:

 https://www.careerindia.com/courses/unique- courses/what-is- About the Author

bioinformatics-scope- career-opportunities-012034.html
 https://www.ncbi.nlm.nih.gov/ Sudha Rameshwari is a Biochemist and her native is Kingmaker Kamaraj birth
 https://www.ebi.ac.uk/Tools/sss/ncbiblast/ town Virudhunagar, Tamilnadu, India. She did her B.Sc. and M.Sc. (Biochemistry)
from V.V.Vanniaperumal College for Women, Virudhunagar, Tamilnadu, India.
 https://en.wikipedia.org/wiki/Clustal She obtained her M.Phil degree in Life Science (2003) from Manonmaniam
 https://www.ebi.ac.uk/Tools/msa/clustalo/ Sundaranar University, Tirunelveli. She has completed her Post graduate diploma
in Bioinformatics (2005) in Bharathiyar University, Coimbatore. She has completed
 https://www.bing.com/ck/a?! 24 years’ of teaching experience in Biochemistry and her Research interests include
 https://web.ExPASy.org/ Microbiology, Pharmacology, Green nanotechnology and Bioinformatics. She
submitted one sequence to GEN BANK on April 2018. She regularly teaches
 https://web.ExPASy.org/ProtParam/ Techniques, Enzymology, Clinical biochemistry, Microbial biochemistry and
 https://web.ExPASy.org/peptide_mass/ Bioinformatics. She is well trained in Microbiology and Molecular Biology
Techniques. She has published more than 30 research articles in well reputed
 https://web.ExPASy.org/peptide_cutter/ International journals which are Scopus indexed (8), Web of Science (4) and UGC
approved. She is interested in participating and to organize workshops. She guided
 https://www.academia.edu/3112992/CFSSP_Chou_and_
2 M.Phil students and 25 PG students. She has filed and published one patent
Fasman_Secondary_Structure_Prediction_Server/ (Indian) Publication. She is a reviewer in 15 reputed International journals. She also
published 3 chapters in edited books. She received grants from TNSCST-DBT,
 http://www.openrasmol.org/ autonomy fund and MRP grant from VVVCMB-MRP Scheme sponsored by
 https://www.novoprolabs.com/tools/protein-hydropathy VVVCollege Manage Board. She has also been awarded as Honorary Doctorate
(D.Litt.(hc)) from University of Central America, CV Raman Prize 2022 from
 https://www.ebi.ac.uk/Tools/pfa/radar/ Institute of Researchers, Wayanad, Kerala and Bharat Excellence award 2022,
 https://pepdraw.com/ Leading educationist of India Award 2022 from Friendship forum , New Delhi,
Global Personalities of Asia 2022 from Global brotherhood forum, New Delhi and
 https://ase.tufts.edu/biology/bioinformatics/exercise3.asp Outstanding Researcher in Microbial Biochemistry from VIHA, 2018. She is a life
member (LM052202) in Institute of Researchers, Waynad and also SAS eminent
Fellow Membership (SAS/SEFM/077/2021) in Scholars Academic and Scientific
society. She has scored 8.7/10 in VIDWAN expert database and National
Researchers network.
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