The document discusses protein 3D structure determination using computational modeling software. It describes different computational modeling methods like homology modeling, threading/fold recognition, and ab initio modeling. Homology modeling involves comparing the target sequence to known protein structures while threading/fold recognition compares the target to known structural templates. Ab initio modeling produces structures based only on the sequence. Popular software tools for each method are discussed like Modeller, SwissModel, I-TASSER, and Rosetta. The document also provides an overview of using nuclear magnetic resonance (NMR) spectroscopy to study protein structures experimentally.

1. Practical
Introduction to proteomics
Contents:
 Nuclear Magnetic Resonance
 Protein 3D model determination using
software
Biotechnology & Bioinformatics
Government College University Faisalabad

2. Practical No. 02
Protein 3-D model determination using software
Introduction:
Protein structure prediction is the biggest problem in the structural biology. Currently,
two major techniques are used for structure determination, which are X-ray Crystallography
and nuclear magnetic resonance. Determination of 3D structure by X-ray crystallography is
not always simple, usually takes as much as three to five years. NMR is another useful
technique and advantage of NMR over X-ray crystallography is that protein can be studied in
an aqueous environment that may resemble its actual physiological state more closely. The
main limitation of NMR is that it is only suitable for small proteins that have less than 150
amino acids. And gaps between known protein structure and sequence are increasing
exponentially, so there is need to develop computational based methods.
The large and huge sizes of search space and toughness of the fitness landscape make it
challenging. This is even quite difficult for computerized based hardware and state-of-the-art
optimization algorithms. And, these problem lead to discovery of many online software
which are used for 3d modeling of protein structure prediction and one of the most popular
software is the rosette which need to create a substantial number of protein models due to the
typically large number of local minima. Software offered for calculating and displaying the
3-D structure of oligosaccharides and proteins. With the two protein analysis sites the query
protein is compared with existing protein structures as revealed through homology analysis.

3. A model:
The three-dimensional re-presentation of a person or thing or of a proposed structure
is called as model.
Modeling – in the world of Proteins:
Computational protein structure prediction provides three-dimensional structures of proteins
that are predicted by in-silico techniques. Such protein modeling relies on principles from
known protein structures obtained via: X-Ray crystallography, NMR spectroscopy and
physical energy functions. Many structure-function model relationships can be inferred from
a reasonable model and these structures are then used for development of successful drug
design. Molecular docking can also help in structure determination. 3D structure of protein
can illustrate the how individual residues interact to form a functional entity.
Method of Modelling:
1- 2- 3-
1-Homolgy Modelling:
It is the prediction of 3d structure of a target protein from the homologous protein for which
an X-ray or NMR structure is available. These are steps in homology modelling.
Steps in homology modeling:
 Comparative modelling by comparing query sequence with the template sequence.
 Sequence Alignment (by BLAST and PDB)
 Backbone model building
 Loop modeling and side chain refinement
 Model refinement using energy function
 Model evaluation
Software usedfor Homologymodelling:
1- Modeller 2- Swiss Model
Modeller:
Step 1- (take query sequence)
Homology
Modelling
Threading Ab Initio
method

4. Step 02: (Run BLAST)
Step 03: (Run PDB)
Step 04: use software Modellerfor building model and 3d structure

5. Swiss Model:
Step 01: (query sequence) Step 02: Open Swiss model software
Step 03: SWISS Model:(Putting sequence and search for templates)
Step 04: (After getting template hits run build model)
Step 05: (Model building gives result of 3d protein with different information)

6. 2-Threading/fold recognition:
This method compares the target sequence against library of structural templates, producing a
list of scores.
Software:I-TASSER:
Step I: Open server and paste query sequence and run the model but this server
require pre-registration and then give ID and password of server.
Step 02: Information and link will be sent to through email just click on link
Step 03: Information about 2d and 2d structures were given. 5 models were given to
you select with high C-value.

7. 3-Ab- Initio Method:
As the name suggest it is the method to produce all protein models based on sequence
information alone without the aid of known protein structures.
Software:Rosetta:
Step I: Selectfragment consistentwith the localsequence preference and
open server.
Step II: Assemble fragments into models with native like globalproperties.
Step III: Identify the best model for the population of decoy.

8. Practicalno. 02
Nuclearmagnetic Resonance
NMR (nuclear magnetic resonance) spectroscopy is also called Magnetic resonance
spectroscopy (MRS). This technique is mostly used to study the magnetic properties around
the atomic nuclei. It is also used to study the chemical, physical and chemical properties of
matter. This method is commonly used by the biochemist to study the structure of protein. It
provides detail information about the structure, function, and reaction state. This technique is
actually based on the absorption of electromagnetic radiations. NMR is one of most precise
method to observe the monomolecular organic compounds.
Nuclear magnetic resonance (NMR) spectroscopy is an important analytical tool for living
chemicals. Research conducted on the biological board has been greatly improved with the
help of NMR. Not only does it provide details about molecular structure, it can also
determine the content and purity of the sample. Proton (1H) NMR is one of the most widely
used NMR methods for biochemistry. The protons present in this molecule will behave
differently depending on the chemical environment surrounding them, making it easier to
determine their structure.
Types of NMR:
There are two types of NMR that can be used in chemical analysis and medical field.
1. H-NMR
2. C-NMR
In this practical H-NMR will be used for the analysis of a compound. In H-NMR on the basis
of hydrogen atoms the analysis of a compound can be done.NMR spectroscopy determines
the physical and chemical properties of atoms or molecules.

9. Principle:
 Nuclear magnetic spectroscopy record the absorption of radio waves that is stimulated
through the changes in nuclear spin.
 It also measures the changes in the nuclear magnetism. NMR signal is generated when
the sample is ascended on the electric field this signal is produced due to the agitation
of the radio waves with the sample nuclei and detected through the sensitive radio
receivers.
 At different frequencies, different nuclei are absorbed.
 And produce the
detailed information
about the structure of
the protein or other
complex molecules.
Instrumentation:
 Sample holder
 Magnetic Coils
 Permanent Magnet
 Sweep the generator
 Radiofrequency
transmitter
 Radiofrequency
 RF Detector
 Recording
 Learning Program
Setup of experiment:
Sample preparation:
NMR tubes should be clean and solid samples should be dissolved in the lost solvent and
liquid samples can be processed or purified. The NMR sample tube itself is 175 mm long
with 5 mm O.D. The minimum filling rate is about 2 cm. Overcrowding can disrupt
uniformity and lead to lower resolution and tube is inserted into the probe.

10. Temperature adjustment
Chemical changes and the formation of signal lines are highly dependent on temperature.
In addition, protein composition can change with temperature.
Lock:
In the introduction it was stated that the current energy producing magnetic field continues
indefinitely. This is almost certainly true but actually decreases slightly over time and this
means that if this is not compensated, the magnetic field at the beginning and end of the study
may vary and have a lower quality effect.
Tuning:
As a radio transmitter and / or receiver of radiofrequency radiation, the investigation must
be well-organized in order to operate efficiently.
Shimming:
The magnetic field is strong enough for an effective NMR test, and it is also important
that the magnetic field be matched to the volume of the sample. If it were not so, the nuclei of
one part of the tube would receive a different field than the nuclei of the other part of the tube
with different resonance waves as a result.
Determination of water frequency:
When making protein NMR usually contains 1 mM protein dissolved in water. The filter
of water thus is 55 M and since there are two protons in water molecule the total proton is ~
100 M, so 100,000 is higher than the heart you are interested in.
Power range drive:
The highest NMR signal is obtained when the pulse width is exactly 90 °, which means that
the magnetization vector is rotated from the z-axis to the rotating plane. In addition, most

11. tests require that some lumps be 180 ° exactly. So we need to measure this too. Contrary to
perceptual perception, it is not so easy to detect a high signal and therefore set up a search for
a signal (or as low as possible) signal.
Procedure
 Placed the sample in a magnetic field.
 Excite the nuclei sample into nuclear magnetic resonance with the help of radio waves
to obtain NMR signals.
 NMR signals are detected with the help of sensitive radio receivers.
 The resonance frequency of an atom in a molecule is changed with intra-molecular
magnetic field surrounding it.
 This gives details of the functional group of the individual molecule and its electronic
structure.
 NMR spectroscopy is a very precise method for the identification of monomolecular
organic compounds.
 Provide the details of reaction state, structure, chemical environment and dynamics of
a molecule.

12. What we canlearn from NMR spectra?
 Chemical shift: Information about the composition of atomic groups within the
molecule.
 Spin-Spin coupling constant: Information about adjacent atoms.
 Relaxation time: Information on molecular dynamics.
 Signal intensity: Quantitative information, e.g. atomic ratios within a molecule that
can be helpful in determining the molecular structure, and proportions of different
compounds in a mixture.
 Observations and results:
This test gives us the basic concepts of nuclear magnetic resonance (NMR)
spectroscopy - spin, energy levels, radiation absorption, and multiple NMR viewing
parameters, and provides experience in identifying the unknown 1H (proton) NMR
spectra. A series of well-known samples will be used to present sample preparation
methods, the performance of NMR spectrometers, and the 1 H-NMR spectra where
students will measure chemical shifts, J-couplings and spectral energy. After that, it
will be able to record three unknown objects and will use these fields to find the
structure and chemical composition.