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Investigating Connectivity in Secondary Structure Matching
PDBeFold searches and matches protein structures by considering the three-dimensional (3D)
arrangement of secondary structure elements (SSEs) - β-strands and α-helices. It can do this in
two different ways. PDBeFold can take into account the order that these elements occur in the
protein sequence, their connectivity. Alternatively, it can ignore this information, relying solely on
the position and orientation of SSEs in space. Ignoring connectivity, motifs 1 and 2 in Figure 1
below are equivalent, but when taking connectivity into account they are different.
Figure 1. Whilst the relative positions and
orientations of the helix and strands in the
two motifs are the same, the order in which
they are connected differs between the two
motifs.
In the Quips episode about the ultraviolet-sensing protein UVR8, we saw that UVR8 has the
same tertiary structure as the DNA-binding protein RCC1, but the two proteins’ SSEs are
connected differently: they are circularly permuted relative to each other. From the diagram
below, you can see that the highlighted β-sheet in UVR8 is made of four strands from the
N-terminus, but in RCC1, the equivalent sheet is formed from strands of both termini of the
protein.
Figure 2. UVR8 is circularly
permuted
relative
to
RCC1.
Compare the connectivity of the βstrands (arrows) in the two proteins.
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You can use PDBeFold to analyse the similarity of the two protein structures with and without
taking their connectivity into account. PDBeFold allows you to search for a fold within the whole
PDB or to compare folds of specific proteins or chains within a single structure. In this example
we are going to align two specific PDB entries.There are several ways to do this, but let’s launch
PDBeFold by going to pdbe.org/fold.
This takes you to the PDBeFold homepage; press the ‘Launch PDBeFold’ button (screenshot
1)
Screenshot 1.
Launch PDBeFold from
the Structure similarity
page.
You are now on the PDBeFold submission form (screenshot 2, below). You will need to fill it
in to align two specific proteins only.
Choose PDB entry 4d9s (UVR8) as the Query structure (left-hand side) by typing ‘4d9s’ in the
PDB code box on the left. For the Target (right-hand side), select ‘PDB entry’ from the pull
down menu. An input box will then appear, similar to that on the left. Choose to align 1a12
(RCC1) by typing ‘1a12’ in the new input box.
Both PDB entries contain several copies of the protein of interest, and we only need to align
one of each. Pressing the ‘Find chains’ button will fill in all the chains present in an entry; you
can then delete the extra ones to leave ‘A’ remaining. Alternatively, type ’A’ into the boxes
labeled ‘Chains:’ (NB: make sure it’s a capital A).
Your form should now look like screenshot 2.
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Screenshot 2.
Setting up the search.
Fill in the PDBeFold form
as shown. Make sure to
note whether the ‘match
connectivity’ box (arrowed) is
ticked.
In screenshot 2, the ‘match connectivity’ box (red arrow) is ticked. This means that structures
are aligned taking into account the sequence order of the SSEs (so motif 1 is not the same as
motif 2 in Figure 1 above).
When you have your form set up, press the ‘Submit your query’ button and wait for the
program to run.
Once PDBeFold has completed its task, you will be taken to a page that shows the alignment
results (screenshot 3).
Screenshot 3.
Structure alignment results
from PDBeFold.
You can see more details of the alignment by clicking on the ‘1’ in the leftmot column of the
results table (highlighted in screenshot 4). This will take you to an in-depth analysis of which
SSEs align between the two structures and which residues align within these SSEs.
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Screenshot 4.
Detailed analysis of the
alignment.
Press ‘View superposed’
(highlighted) with the Jmol
viewer selected (arrowed)
to obtain a new window
showing the two protein
structures overlaid.
The superposed structures can be displayed with the 3D viewer Jmol by clicking the ‘view
superposed’ button (highlighted in screenshot 5). (Make sure that Jmol is the selected viewer.
Alternatively, use the download buttons to download the molecules separately. They will overlay
when both are loaded into a graphics viewer such as Pymol.
Now repeat the alignment by opening PDBeFold again in a different tab, window or even
web browser. Keep everything the same, except for the ‘Match connectivity’ box (arrowed in
screenshot 2). Make sure it’s NOT ticked for this analysis, so that connectivity will not be taken
into account, and motifs 1 and 2 in Figure 1 above are considered to be equivalent.
When you compare the statistics of the two different alignments (with and without matched
connectivity), you will immediately see that they differ. Notably, the number of aligned residues
(Nalign), arrowed in screenshot 3, increases from 318 to 347 when connectivity matching is
turned off.
In screenshot 6 you can see that the structures have two β-strands in common that are
coloured grey (arrowed), indicating that they do not occur in the same sequence order in the
two proteins. This is because this alignment was done with connectivity matching on, and
the circular permutation between UVR8 and RCC1 is such that these strands differ in their
sequential position (see Figure 2).
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Screenshot 5.
3D
views
of
overlaid proteins.
the
The
alignment
with
connectivity
matching
on is to the left whilst
that without connectivity
matching is to the right.
The two strands which
are in different parts of
the sequence due to the
circular permutation are
arrowed.
By performing the two analyses and carefully comparing the results, in particular those on the
page shown in screenshot 4, the nature of the circular permutation can be readily identified.
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