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EXPERIMENT – 08

PROTEIN STRUCTURE ANALYSIS AND MODELING

EB3233: BIOINFORMATICS LABORATORY

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ABSRACTS

The prediction of protein structure is the assumption from its amino acid sequence of the
three-dimensional structure of a protein, namely the prediction of its folding as well as its
secondary and tertiary structure from its main structure. The prediction of structure is
essentially distinct from the opposite issue of protein structure. A variety of species’ full
genomes has been sequenced and several more are under way. In order to promote the
analysis of their functions and modes of action, structural biology now faces the arduous task
of characterizing the shapes and dynamics of the encoded proteins. The prediction of protein
structures is one of the most important tasks sought by bioinformatics and theoretical
chemistry; it is of considerable significance in medicine such as drug design and
biotechnology for example, in the design of novel enzymes. An organization called the
Worldwide Protein Data Bank, wwPDB, manages the PDB. In structural biology fields, such
as structural genomics, the PDB is a crucial resource. A crystallographic server for the three-
dimensional structural data of large biological molecules. For example and nucleic acids, is
the Protein Data Bank (PDB). So, through this practical we used Protein Database website or
PDB, Jmol software, and SWISS-MODEL.

Therefore, this experiment was used to understand the information contained in the PDB
database as well as to visualize and present biological structures using Jmol or molecular
graphic tools.

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INTRODUCTION

The three-dimensional configuration of atoms in an amino acid-chain structure is the

structure of proteins. Proteins are polymers derived from amino acid sequences, the
monomers of the polymer, specifically polypeptides. A residue suggesting a repeated unit of
a polymer can also be considered a single amino acid monomer. Proteins are formed by
amino acids that are exposed to condensation reactions wherein the amino acids remove one
molecule of water per reaction to bind to each other with a peptide bond. Standard, rather
than a protein sometimes know a chain under 30 amino acids known as a peptide (wwPDB:
Worldwide Protein Data Bank, n.d.).

. Proteins fold into one or more complex spatial conformations guided by a variety of non-
covalent hydrogen bonds bonding, ionic interactions, Van der Waals forces, and hydrophobic
packing, to be able to fulfill their biological function. Determining their three-dimensional
structure is also important to understand the roles of proteins at the molecular level. This is
the subject of structural biology in the scientific world that uses techniques, such as X-ray
crystallography, NMR spectroscopy, cryo electron microscopy, and dual polarization
interferometry to evaluate protein structure.

A crystallographic server for the three-dimensional structural data of large biological

molecules. For example and nucleic acids, is the Protein Data Bank (PDB). Data usually
obtained from X-ray crystallography, NMR spectroscopy or, gradually, cryo-electron
microscopy and sent by biologists and biochemists from everywhere in the world are widely
available on the Internet through the websites of member organisations such as PDBe, PDBj
and RCSB. An organization called the Worldwide Protein Data Bank, wwPDB, manages the
PDB. In structural biology fields, such as structural genomics, the PDB is a crucial resource.
Scientists are now needed by most major scientific publications, and some funding
organizations, to send their structure data to the PDB (wwPDB: Worldwide Protein Data
Bank, n.d.). Protein structures recorded in the PDB are used for numerous other databases.
This is located at https://www.rcsb.org/

Jmol is a 3D open-source Java molecular modeling platform for chemical structures. Jmol
returns a 3D image of a molecule that can be used for example, in chemistry and
biochemistry, as a teaching instrument or for analysis. It is software that is free and open

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source, written in Java and runs on Windows, Mac OS X, Linux and UNIX systems. There
seems to be a standalone framework that can be merged into other Java systems, such as
Bioclipse and Taverna, and a development tool kit.

SWISS-MODEL is a browser of structural bioinformatics devoted to 3D protein structure

homology modeling. Homology modeling is actually the most accurate way of producing
consistent three-dimensional models of protein structure and is widely used in many
functional applications. Methods of homology modeling use experimental protein structures
to construct models for evolutionary proteins (Swiss-model - Wikipedia, n.d.). This is located
at httD://swissmodeI.exDasy.org/.

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OBJECTIVES

1. To understand the information provided in PDB database

2. To visualize and present biological structures using Jmol (molecular graphic tools)

MATERIALS

1. Computer
2. Internet connection
3. Protein Database website
4. Jmol software

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METHODS AND RESULTS
1. Understand the information provided in PDB database

1. The PDB database homepage was accessed by visiting https://www.rcsb.org/

Figure 1-The PDB database homepage

2. The PDB ID ‘1qys’ was copied and pasted into the box and the search button was
clicked.
3. A typical output is shown in the figure below.

Figure 2-A typical output

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 This one-page Structure Summary form presents the essential information on this
protein structure including a small graphic, a bibliographic reference, its functions,
and its organism of origin. Additional details on various aspects of this protein can be
obtained by clicking the various tags at the top of the form.

Figure 3-A typical output

4. The FASTA sequence of this protein was obtained by clicking on the 'Display File'
link in the left corner of the page for PDB access 1qys and then FASTA sequence was
obtained.

Figure 4-A typical output

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 If you want to download the FASTA sequence and the PDB file, click the ‘Download
Files’. Click to download the ‘PDB text’ file and the ’FASTA sequence file’.

Figure 5-FASTA sequence file

Figure 6-‘PDB text’

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2. To visualize and present biological structures using Jsmol

1. In the PDB 1qys search, the 3D view was clicked as indicated by the arrow.

Figure 7- A typical output

2. 1qys appeared in the NGL program. The viewer was changed to Jsmol as shown by
the arrow in the image below.

Figure 8-3D view

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3. The 1qys structure appeared in the Jsmol program. Once launched, Jsmol will be
displayed as an image below, also known as the display window.

Figure 9-The 1qys structure-Jsmol

4. The windowpane menu is accessed by right clicking anywhere in the pane, A new
window named Console Window opens with the click of a "Console" button.

Figure 10-The windowpane menu

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 Figure 11-Console Window

5. To browse all display formats, right-click on the display window, click 'Style', then
click 'Suggestion Scheme', and then select the desired display format.

Figure 12-Console Window

6. To emphasize specific protein structure or active site region ball and stick scheme is
preferable for more clear view.

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7. The water molecule in the protein structure was highlighted as a different color, for
which the following was done.
a) After selecting everything from the console window, press enter.
b) Yellow (or any color you like) was typed and inserted.
c) Then select water was typed and inserted.
d) After that, it was typed in blue (see image below). All water molecules were
influenced by the command.

Figure 13-Result 01

Figure 14-Result 02
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Figure 15-Result 03

Figure 16-Result 04

Figure 17-Result 05

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 Figure 18-Result 06

Figure 19-Result 07
8. After that, it was typed in color blue. All water molecules were influenced by the
command.

Figure 20-Result 08
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 Figure 21-Result 09

9. The beta-pleasant sheet of the protein structure was continuously highlighted.

10. The cartoon scheme did the following:
a) Type the selection sheet in the console window and press enter.
b) When the cartoon was typed.

Figure 22-Result 10

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Figure 23-Result 11

Figure 24-Result 12

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Figure 25-Result 13
3. Homology modelling using Swiss-Model

1. The Q9ERI2.1 FASTA file was retrieved from the NCBI database in protein
sequence.

Figure 26-The Q9ERI2.1 FASTA file

2. The swissmodeI database homepage was accessed by visiting

httD://swissmodeI.exDasy.org/.

Figure 27-The swissmodeI database homepage

3. The initial format was clicked, then the sequence was pasted in the query box and
the build model was clicked.

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Figure 28-The swissmodeI database homepage
 Figure 29-The swissmodeI database homepage

4. Two different protein models were obtained as shown in the picture below.

Figure 30-Two different protein models

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DISCCUSION
The structures of proteins vary in size from tens to several thousand amino acids. Proteins are
known as nanoparticles by physical dimension, from 1-100 nm. From protein subunits, very
large aggregates may be created. For starters, several thousands of molecules of actin are
assembled into a microfilament. In performing its biological function, a protein can undergo
reversible structural changes. Various conformational isomers are referred to as the alternate
structures of the same protein, or simply, conformations, and transitions between them are
considered conformational changes (Protein Data Bank, n.d.).

There are four levels of protein structure.

1. Primary structure - Simplest level. The linear amino acid residues in a polypeptide
chain. This sequence will determine how a protein will fold and therefore determine how it
will function.

2. Secondary structure - The next level of arrangement of the primary structure. There are
two types of structures.

 α helix - Most common. Stabilized by hydrogen bonds between residues

 β sheets - Stabilized by hydrogen bonds between adjacent segments. Antiparallel or
parallel orientation.

3. Tertiary structure - 3D arrangement of secondary structures packed together into a

globular structure. Classes;

 Purely αhelixes
 Purely βsheets
 Mix

4. Quaternary structure - Arrangement of two or more tertiary structures into one

functional molecule with several subunits.

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Figure 31-Levels of protein structure
An organization called the Worldwide Protein Data Bank, wwPDB, manages the PDB. In
structural biology fields, such as structural genomics, the PDB is a crucial resource. Scientists
are now needed by most major scientific publications, and some funding organizations, to
send their structure data to the PDB. Protein structures recorded in the PDB are used for
numerous other databases. This one-page Structure Summary form presents the essential
information on this protein structure including a small graphic, a bibliographic reference, its
functions, and its organism of origin. Additional details on various aspects of this protein can
be obtained by clicking the various tags at the top of the form.

SWISS-MODEL is a browser of structural bioinformatics devoted to 3D protein structure

homology modeling. Homology modeling is actually the most accurate way of producing
consistent three-dimensional models of protein structure and is widely used in many
functional applications (Swiss-model - Wikipedia, n.d.).

SWISS-MODEL consists of 3 closely interconnected components:

 The SWISS-MODEL pipeline, a suite of automatic protein structure modeling

software tools and databases
 The SWISS-MODEL Workspace, a web-based graphical user workbench
 The SWISS-MODEL Repository, a continually modified homology model database
for a range of high biomedical interest model organism proteomes

The four key steps involved in constructing a homology include the SWISS-MODEL
pipeline. A provided protein structure model:

I. Structural template recognition (s). First, BLAST is used to fetch homologous

sequences, and HHsearch is used to detect remotely similar sequences if no
appropriate templates are detected. The templates are contained in the PDB-derived
SWISS-MODEL Template Library (SMTL).
II. Target sequence and template structure alignment II. Target sequence and template
structure alignment.
III. Building of models and minimization of energy. For simulation, SWISS-MODEL
implements a static fragment assembly approach.
IV. Assessment of the consistency of the model using three independent techniques such
as QMEAN, ANOLEA, and GROMOS.

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The first model from the figure 30 is considered the best protein model for the protein
sequence. The scores around QMEAN Z-zero indicate good agreement between experimental
structures of similar size to the model structure. Scores of -4.0 or lower indicate lower quality
models. This is also highlighted by changing the “thumbs up” symbol to a “Thumbs down”
symbol adjacent to the score level. And also, residues displaying a score below 0.6 are
usually expected to be of poor quality. For this practical, Residues displaying a score greater
than 0.6 was obtained. So it was expected to be in good quality (Figure 32).

Figure 32-Errors Vs residue plot

In contrast to the scores obtained for high-resolution crystal structures, the plot indicates the
model quality as a normalized QMEAN score. Higher values indicate that the model is
qualitatively comparable to experimental structures of similar size. If the star is in or near the
black band, as shown in the figure below, it indicates a quality relative to the experimental
structures.

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 Figure 33-Comparison with Non-redundant set of PDB structures

Positive values mean that on average, the model scores better than experimental systems.
Negative numbers suggest that on average, the model scores worse than experimental
systems. The black bar in the figure below is more towards the blue side, so this model was
shown to have a higher value than the experimental structures in general.

Figure 34-Global quality estimate

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REFERENCES

1. n.d. RCSB PDB: Homepage. [online] Available at: <https://www.rcsb.org/> [Accessed 2020].
2. n.d. Wwpdb: Worldwide Protein Data Bank. [online] Available at:
<https://www.wwpdb.org/> [Accessed 2020].
3. n.d. Protein Modelling Using Swiss Model [ Expasy ] - Youtube. [online] Available at:
<https://www.youtube.com/watch?v=XNqtBkpQCTQ> [Accessed 2020].
4. n.d. RCSB PDB: Homepage. [online] Available at: <https://www.rcsb.org/> [Accessed 2020].
5. n.d. SWISS-MODEL. [online] Available at: <https://swissmodel.expasy.org/> [Accessed 2020].
6. n.d. SWISS-MODEL - SIB Swiss Institute Of Bioinformatics | Expasy. [online] Available at:
<https://www.expasy.org/resources/swiss-model> [Accessed 2020].
7. n.d. Swiss-Model - Wikipedia. [online] Available at: <https://en.wikipedia.org/wiki/SWISS-
MODEL> [Accessed 2020].

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POST-LAB QUESTIONS

1. Access the PDB with ID ‘2at9’ and answer the following questions based on the
page presented for the PDB entry 2at9 :
a) What is the function of the protein encoded by the PDB ID 2at9?

 transporter activity
 ion channel activity
 ion transmembrane transporter activity
 channel activity
 inorganic molecular entity transmembrane transporter activity
 passive transmembrane transporter activity
 transmembrane transporter activity
 photoreceptor activity
 signaling receptor activity
 molecular transducer activity

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b) What organism is it from?

 Halobacterium salinarum

c) Where and when was this structure published and by whom?

 Deposited: 1998-12-17 Released: 1999-04-27

 Deposition Author(s): Mitsuoka, K., Hirai, T., Murata, K., Miyazawa, A.,
Kidera, A., Kimura, Y., Fujiyoshi, Y.

d) How was this 3-D structure determined?

 Structure of bacteriorhodopsin at 3.0 angstrom by electron

crystallography

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 e) In which part ofa cell do you think it is found?

 Plasma membrane

2. Built protein model of the protein sequence obtain from NP_001317683.1 and
answer the following questions:
a) How many protein model(s) does Swiss Model able to build?

 Three models

b) Which of the protein model is consider the best protein model for the
protein sequences? Briefly explain and give evident showing that the
protein model that you choose is the best model of protein sequence
NP_00131 7683.1

 The Model 1 is consider as the best protein model for that protein
sequences. Because, the scores around QMEAN Z-zero indicate good
agreement between experimental structures of similar size to the model
structure. Scores of -4.0 or lower indicate lower quality models. This is
also highlighted by changing the “thumbs up” symbol to a “Thumbs
down” symbol adjacent to the score level. Therefore, above model one
has -3.85.

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