VALUE ADDED COURSE MINI PROJECT

SWISS MODEL

PROJECT MEMBERS

Gunapiriya S(19BTBT010)
Jaya Barani (19BTBT016)
Kiruba Shree(19BTBT021)
Vishali S(19BTBT037)
Nilasha Maria W(19BTBT039)
Homology model using Swiss Model
Here, we provide a step-by-step example on how to build a homology model for the Superoxide
Dismutase [Cu-Zn] protein from Drosophila melanogaster (SOD, UniprotKB AC: P61851). This
protein destroys radicals which are normally produced within the cells and which are toxic to
biological systems.
In its native form, the protein is a homodimer with two metal-binding sites per subunit: one for Cu
and one for Zn.
Click the "Start Modelling" button or select the option "myWorkspace" from the navigation bar
(Modelling → myWorkspace) to start a new modelling project.
You can either paste the protein sequence or provide the UniprotKB AC (P61851) of your target
sequence in the input form.
Alternative input formats can be selected using the apposite drop-down menu. A project title is
automatically suggested from the information retrieved from SWISS-MODEL's own copy of the
UniprotKB annotation, if a UniProtKB AC is used.
To search for available template structures, click on the “Search for Templates” button.
Once the search is started, you can verify the status of the job, and optionally you can bookmark
the page using the provided link and come later to visualise the results. Otherwise you need to
wait until the search finishes.
The automated mode selects templates that maximise the expected quality of the model.
However, this does not garantee that the best template available is selected.
Depending on the intended application of a model, selecting a different template than the top-
ranked one might be necessary.
If the aim is to build a model of a protein in complex with a ligand rather than its apo form, a
template with a similar ligand or in a holo conformation should be preferred.

When the template search is finished, the output page includes a main table showing the list of
available templates ranked according to the expected quality of the resulting models For each
template, the following information is provided:

• A checkbox to select and visualise the template in the 3D panel

• The SMTL ID of the template and a link to the SWISS-MODEL Template Library page
associated to that SMTL entry
• The protein name of the template
• The coverage to the target sequence (darker shades of blue refer to higher sequence
identity)
• The GMQE (Global Model Quality Estimation)
• The QSQE (Quaternary Structure Quality Estimation)
• The target–template sequence identity
• The experimental method used to determine the structure (and resolution, if applicable)
• The oligomeric state of the SMTL biounit
 • The ligands present in the experimental structure (if any)
• A clickable icon to expand the box with the description of the template

As you can see from the table, there are multiple templates which cover the complete sequence
and share a considerable sequence identity with our target sequence. Let's analyse them in
more detail.

By clicking on the checkbox, different template structures can be visualised and compared to by
structure superposition in the 3D viewer.
Let's rank them according to their coverage to the target sequence, and let's select the four top-
ranking templates. We can observe that they all share a high sequence identity to the target
(Identity > 67%).
As expected, their structures also look very similar as they superimpose very well. Some local
differences are present at the level of loop regions.
What can be the possible reason of this?
Typically, due to their higher variability in terms of sequence and structure compared to the rest
of the protein, loops are the regions that are the most susceptible to modelling errors.
On the other hand, they are often involved in protein function, hence accurately modelling their
structure is of crucial importance.
Are these loops important for the function of the protein?
To get an overview of the similarity between the selected templates click on the "Sequence
Similarity" tab.
An interactive chart shows the relationship between the templates in the sequence similarity
space and your target protein is represented as a filled red circle.
Each template is displayed as a blue circle, where the thick blue arc indicates target coverage.
The distance between different templates is proportional to the pairwise sequence similarity, i.e.
evolutionarily closely related templates will be clustered together.
By clicking on a circle you can look at available information on the specific template. You can
also select and visualise a group of similar templates by hovering over the cluster.
The superposed structures of the selected templates will be instantaneously displayed in 3D,
where you can inspect their structural differences.
Would you expect large structural variations within a cluster?
By looking at the "Alignment" tab, you can notice that the sequences of the selected templates
are very similar overall.
Compared to the sequence of the target, they show some insertions and deletions as well as
some amino acid substitution.
By clicking on the cog icon, you can change the colouring scheme of the alignment by selecting
one of the many available options. Let's select the Clustal colouring scheme.
Based on the alignment you see, can you now explain the observed structural difference
between the templates?

As expected, the regions that show most of the structural differences correspond to positions in
the alignment where amino acid substitutitons and indels are present.
Even if these differences seem to have little or no impact on the overall structure of the protein,
they can still play an important role for the function of the protein, i.e. ligand- and/or substrate-
binding.
If we go back to the template table ("Templates" tab), we can see that not all of the selected
templates have been solved in presence of the natural ligands (Cu and Zn).
In particular, only the top-ranked template, after sorting by sequence identity, binds to the Zn ion,
while the second-best template structure contains both the Zn and the Cu ion.
Which template(s) would you select to build your model?
After you have selected the template(s), click the 'Build Model' button to run the modelling job.
For each model generated based on the selected templates, the following information is
provided:

• A file containing the model coordinates along with relevant information on the modelling
process
• The oligomeric state of the model
• The modelled ligands
• QMEAN model quality estimation results
• The target–template sequence alignment
• The template name (SMTL ID)
• The sequence identity to the target
• The target sequence coverage

Models can be displayed interactively using the 3D viewer. By default, models are coloured by
model quality estimates assigned by QMEAN to highlight regions of the model which are well- or
poorly modelled.
If several alternative models have been built for a target sequence, they can be interactively
superposed and visualised.
Local estimates of the model quality based on the QMEAN scoring function are shown as a per-
reside plot and as a global score in relation to a set of high-resolution PDB structures (Z-score).
In the first plot below, a score is associated to each residue of the model (reported on the x-axis),
reflecting the expected similarity to the native structure (y-axis). Typically, residues showing a
score below 0.6 are expected to be of low quality. Different chains are shown in different colours.
The second plot shows the quality of the model, as normalised QMEAN score (y-axis), in
comparison to the scores obtained for high-resolution crystal structures. Higher values indicate
that the model is of comparable quality to experimental structures of similar size.
Based on the results you obtained, can your homology model be considered a reliable model?
How to model the correct oligomeric state of a
protein using ornithine carbamoyltransferase
In this example we show how to infer the quaternary structure of Ornithine Carbamoyltransferase
from Thermus thermophilus (OTC, UniProtKB AC P96134). OTC is involved in arginine
biosynthesis, where it catalyses the formation of citrulline and phosphate from ornithine and
carbamoyl phosphate.
Please access the SWISS-MODEL website and click "Start modelling" to start a new modelling
project. Now, please provide your target protein. You can either paste the protein sequence or
provide the UniprotKB AC (P96134) of your target sequence in the input form. Alternative input
formats can be selected using the apposite drop-down menu. A project title is automatically
suggested if a UniProtKB AC is used.

To search for available template structures, click on the “Search for Templates” button. Once the
search is started, you can wait and verify the status of the job or you can bookmark the page
using the provided link and visualise the results later. When the template search is finished, the
output page includes a table showing the list of available templates ranked according to the
expected quality of the resulting models. For each template, the following information is provided:

• A clickable icon to expand the box with the description of the template
• A checkbox to select and visualise the template in the 3D panel
• The SMTL ID of the template and a link to the SWISS-MODEL Template Library page
associated to that SMTL entry
• The protein name of the template
• The coverage to the target sequence (darker shades of blue refer to higher sequence
identity)
• The GMQE (Global Model Quality Estimation)
• The QSQE (Quaternary Structure Quality Estimation)
• The target–template sequence identity
• The experimental method used to determine the structure (and resolution, if applicable)
• The oligomeric state of the SMTL biounit
• The ligands present in the experimental structure (if any)

As you can see from the table, the best template according to GMQE shares a sequence identity
of 99.34% and is the only template annotated as monomeric, followed by homo-12-mers, homo-
trimers and homo-hexamers with sequence identities ranging from 40 to 50%. It is hence very
difficult to decide which template with which quaternary structure should be selected, if we only
consider GMQE and sequence identity.
A more indicative view can be obtained by selecting the “Quaternary Structure” tab. Here, the
result of structural template clustering is reported as a decision tree.
Each leaf is a template labelled with the SMTL ID and a bar indicating sequence identity and
coverage.
The decision tree follows the levels of clustering based on oligomeric state, stoichiometry, the
topology of the complexes, and interface similarity.
The first clustering level shows two groups of templates, monomeric and homomeric
respectively. At the stoichiometry level, templates are grouped according to the number of
different subunits. At the topology level, templates are grouped according to the arrangement of
the different subunits. At the last level of clustering, templates are grouped according to their
interface similarity.
In this example, we see that interfaces are coherent between templates, as there is a single
cluster for each topology level group.
Different template structures can be visualised and compared to by structure superposition in the
3D viewer by clicking on their name. A check mark near the sequence identity and coverage bar
indicates the selected template.
According to the clustering level, which can be selected using the radio button provided at the top
of the page, a PPI Fingerprint curve is shown for the different groups of templates.
The latter curve offers an indication of how interface conservation (shown on the y-axis) varies as
a function of the evolutionary distance within a protein family (expressed as sequence identity on
the x-axis) and thus provides an additional criterion for quaternary structure prediction. More
specifically, the degree of conservation of an interface is expressed as the log-ratio of the
variability of interface residues over surface residues, as calculated from a MSA. Hence, values
below zero indicate that surface residues are less prone to mutate when compared to surface
residues.
The PPI Fingerprint curves follow a characteristic pattern: when only very similar sequences are
considered (80-90% sequence identity thresholds), the ratio is close to zero and there is no
difference among different oligomeric states. When more remote homologs are included, the
interface conservation becomes stronger and eventually reaches a minimum (sequence identity
below 40%) supporting the evidence that the trimeric form is the more conserved among the
other possible options.
This evidence, along with other features of the templates, is used to estimate the accuracy of the
predicted interchain contacts for a model given a specific template. This value of accuracy,
defined as quaternary structure quality estimation (QSQE) score, is a number between 0 and 1
and is used to sort templates in each interface similarity level cluster. The QSQE score
associated to each template can be visualised by mouse over. A value close to 1 indicates a high
confidence that this template is representing the correct quaternary structure of our model.
Templates showing a QSQE above 0.7, most likely leading to a model with the correct
quaternary structure (4nf2.1.A in our example), are highlighted with green lines.
Even in absence of direct structural evidence, the quaternary structure analysis indicates that the
trimeric interface is subject to greater evolutionary constraints than the rest of the protein surface,
exactly as would be expected of a true trimeric interface.
This is in contrast to the template with the highest sequence identity (~99%) and the highest
GMQE score (2ef0.1.A).