Swiss PDB Viewer exercises & answers

General instructions
Save your answers into a separate Word file for your own reference.

To import images from Swiss PDB Viewer to Word you can take screenshots and paste them into
Word. Activate the desired window in Swiss PDB Viewer by clicking it and press Alt + Print Scrn
to take a screen shot of that window. In Word, place the cursor and press Ctrl + v to paste the image.

1. Checking PDB file against its Uniprot entry

Cutting parts of the protein is often required for obtaining protein crystals needed in tertiary structure
determination. Due to this, the sequence numbering in PDB files may be different from the
corresponding Uniprot sequences.

Find out what is the sequence difference between chicken egg-white lysozyme from Uniprot
(LYSC_CHICK) and one of its corresponding sequences in PDB file: 1hew.

Instructions:

1. Obtain the sequences for LYSC_CHICK and 1hew in fasta format from the PDB
database:

http://www.rcsb.org/pdb/

Fetch the entry for 1hew, and Download sequences in the entry in FASTA format.
Alternatively you can use the European PDB mirror at:

http://www.ebi.ac.uk/msd-srv/msdlite/

2. Obtain the sequence of LYSC_CHICK from Uniprot.

3. Compute a global alignment of the two sequences with EBI’s EMBOSS Pairwise
Alignment tool:

http://www.ebi.ac.uk/Tools/emboss/align/index.html
########################################
# Program: needle
# Rundate: Wed Jan 14 12:06:27 2009
# Align_format: srspair
# Report_file: /ebi/extserv/old-work/needle-20090114-12062619149154.output
########################################

#=======================================
#
# Aligned_sequences: 2
# 1: LYSC_CHICK
# 2: SEQUENCE
# Matrix: EBLOSUM62
# Gap_penalty: 10.0
# Extend_penalty: 0.5
#
# Length: 147
# Identity: 129/147 (87.8%)
# Similarity: 129/147 (87.8%)
# Gaps: 18/147 (12.2%)
# Score: 720.0
#
#
#=======================================

LYSC_CHICK 1 MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA 50
||||||||||||||||||||||||||||||||
SEQUENCE 1 KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA 32

LYSC_CHICK 51 KFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSA 100

||||||||||||||||||||||||||||||||||||||||||||||||||
SEQUENCE 33 KFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSA 82

LYSC_CHICK 101 LLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL 147

|||||||||||||||||||||||||||||||||||||||||||||||
SEQUENCE 83 LLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL 129

Which part of the LYSC_CHICK sequence is missing from the PDB entry?
How many residues are missing from 1hew.pdb?
MRSLLILVLCFLPLAALG
18 N-terminal residues

DeepView exercises

2.Molecular surface and active site analysis

For PDB file 1hew (lysozyme in complex with inhibitor) create a view that shows the molecular
surface of the protein and the bound enzyme inhibitor in wireframe.

Colour conserved residues for the LYSC_CHICK protein listed below in green and the rest of the
surface grey. The image should look similar to figure 1.
Figure 1 Example showing molecular surface for Myoglobin (1mbn), prosthetic heme group is shown in
wireframe.

uniprot|P50717|LYS_HYPCU
uniprot|Q86M91|Q86M91_ANOST
uniprot|Q9VSA5|Q9VSA5_DROME
uniprot|P00698|LYSC_CHICK
uniprot|Q95V66|Q95V66_PENVA
uniprot|P00713|LALBA_CAVPO
----------------------------------------------KYYS 24
----------------------------------------------AKTF 24
TVYNGHCRQKTRADVANCFDGKDLPAEVAKPSKGNELVKKTSPTPKAKIY 598
---------------------------------------------LGKVF 21
---------------------------------------------DAKVF 21
----------------------------------------------AKQL 22

uniprot|P50717|LYS_HYPCU TRCDLVRELRK----QGFPENQMGDWVCLVENESGRKTDKVGPVNKNGSK 70
uniprot|Q86M91|Q86M91_ANOST SKCDLAKALAN----NGIAKASLPDWICLVQHESAFSTSATN-KNKNGSK 69
uniprot|Q9VSA5|Q9VSA5_DROME NRCELAKELYHR---HKFPMREIPTWVCIAEHESSFNTAAVGKLNADGSE 645
uniprot|P00698|LYSC_CHICK GRCELAAAMKRHGLDNYRGYS-LGNWVCAAKFESNFNTQATN-RNTDGST 69
uniprot|Q95V66|Q95V66_PENVA GKCEFAELLKR---DYYLSNDDIKNWVCIAEFESSFNTAAIN-RNRNRST 67
uniprot|P00713|LALBA_CAVPO TKCALSHELND---LAGYRDITLPEWLCIIFHISGYDTQAIV--KNSDHK 67
:* : : : *:* * .* : .

uniprot|P50717|LYS_HYPCU DYGLFQINDKYWC--SNTRTPGKDCNVTCADLLLDDITKASTCAKKIFKR 118

uniprot|Q86M91|Q86M91_ANOST DYGIFQINNKFWC--DSSYG-ANDCKMACSNLLNDDITDDIKCAKMIFKR 116
uniprot|Q9VSA5|Q9VSA5_DROME DHGLFQISDIYWC--THDQTSGKACHIECDRLLDSDISDDVQCIRTIHEE 693
uniprot|P00698|LYSC_CHICK DYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSD 119
uniprot|Q95V66|Q95V66_PENVA DYGIFQINNKYWC--GSDYG-KNVCKIPCSDLMSDDITEALRCAETIRRD 114
uniprot|P00713|LALBA_CAVPO EYGLFQINDKDFCESSTTVQSRNICDISCDKLLDDDLTDDIMCVKKILDI 117
::*::**.. :* : *.: * *: .*:: * . *

uniprot|P50717|LYS_HYPCU HN--------FRAWYGWRNHCDGKTLPD-TSNC----------------- 142

uniprot|Q86M91|Q86M91_ANOST
uniprot|Q9VSA5|Q9VSA5_DROME
uniprot|P00698|LYSC_CHICK
uniprot|Q95V66|Q95V66_PENVA
uniprot|P00713|LALBA_CAVPO
HG--------FNAWYGWKDHCKGKTLPS-VSACF---------------- 141
HTRLS--GDGFNAWTVYNGHCRNQNLAK-LSDCFDGNEISEADKTSHYAP 740
-------GNGMNAWVAWRNRCKGTDVQAWIRGCRL--------------- 147
TERFRGRGKGYSAWVAYNSKCKNRDLDQYMAECWSHGSNSVFPF------ 158
KG--------IDYWLAHKPLCSDK-LEQWYCEAQ---------------- 142

There are 17 highly conserved residues marked by * in the above alignment of lysozyme family
sequences, out of these 7 are cysteines. Two cysteines can form a disulfide bridge that helps to
stabilize the fold of the protein; these residues are typically buried inside the structure can be ignored
here.

The position of the conserved residues (other than cysteine) for LYC_CHICK (P00698) obtained
from the alignment:
 W S T G Q I D I W

LYSC_CHICK 46 54 58 72 75 76 105 116 126

Convert LYSC_CHICK residue numbering to correspond to 1hew residue numbering by subtracting
18.

  W S T G Q I D I W
LYSC_CHICK 46 54 58 72 75 76 105 116 126
1hew 28 36 40 54 57 58 87 98 108

Instructions:

1. Change the setting for molecular surface calculations, so as to allow you to exclude
selected residues from the surface.

Prefs > Surface > Ignore Selected Residues

Prefs > Surface > General appearance > Filled triangles

2. Select enzyme inhibitor (NAG) from the control panel (you can select several residues by
holding the Ctrl-button while selecting).

3. Show the selected inhibitor residues in wireframe.

4. Next, compute the surface of the protein. You need the NAG residues selected so as to
exclude them from the surface rendering.
Tools > Compute Molecular Surface

5. You should also turn on OpenGL rendering see a solid surface.

Display > Render in solid 3D

6. Colour the protein surface grey, use the control panel for this.
7. Select conserved residues (see list below) and colour them green
You have to change LYSC_CHICK sequence numbering to correspond to
1hew’s numbering!!!

Tips:
To discard molecular surface:

File > Discard > Discard Surface

Questions:

2.1 Where are the conserved residues located?

The conserved residues are located mainly in a groove on the protein’s surface, where the inhibitor is
also bound. This is the active site of the enzyme
2.2 Where is the inhibitor molecule bound?
Also in the active site groove.

3. Ramachandran plot
Ramachandran plots can be used to check that the given/assumed 3D structure of a model is of
reasonable quality. For a given protein, the torsion angles (or conformational angles): phi and psi of
the polypeptide backbone may be plotted. For example, in the plot below, each dot is the phi/psi
angle of one residue. In Swiss PDB Viewer, the Ramachandran plots can be used to modify torsion
angle by dragging points on the plot – try it and see the effect on the structure!
 Beta sheet
region

Left
handed
alpha helix
region

Right
handed
alpha helix
region

Questions.

3.1 For protein 1hxw (HIV protease dimer), make a Ramachandran plot and identify the
residues which are outside the expected (yellow) regions of the plot. They are almost exclusively one
kind of residue – which? Why do you think this residue type is more likely to have greater freedom
in backbone torsion angles than other residues?

Answer:
The residues which are mostly outside the expected regions are glycines – this is because they have
no side chain and are therefore the smallest residues, with the least constraints on backbone
conformation or position.

4. Analysis of a Protein-DNA complex

Obtain the full PDB entry for protein: 1flo, and load the protein into Swiss PDB Viewer. Try and
obtain a view like the one below, but with all four hetero atoms. Note that your image should include
the following:

 Only DNA and hetero atoms are shown

 The DNA backbone is displayed in ribbon form, coloured by secondary structure
 the heterogeneous atoms are highlighted and solid,
 the heterogeneous atoms are labelled

Hint: Select GroupKind:HETATM will identify the heterogeneous atoms. Once you have selected
this, you can colour them directly by right-clicking directly on the word: ‘col’ in the Control panel,
and picking a strong colour. Then scroll down through the control panel, to find where the
HETATM’s are.

Create an image, like the one below, but which includes all four hetero atoms and DNA chains in the
PDB file.
Question
4. 1 What does this structural model represent?

The model represents DNA recombination, a so called Holliday junction structure.

5. Analyzing interacting surfaces

Obtain the PDB file 1dkf. It is a heterodimer complex of the ligand binding domains for RAR
(retinoic acid receptor) and RXR (retinoid x receptor-alpha) nuclear receptors.

Question:

5.1 Which chain corresponds to RAR and which to RXR?

Hint: Look at the PDB file in text format

Chain A corresponds to RXR and chain B to RAR.

5.2 Which residues are responsible for the interaction between the two monomers? Start by
finding intermolecular hydrogen bonds. Mark the residues that are hydrogen bonded to each other.

Hint: Colour the backbones of the chains with different colours. Select one of the chains. Then
Select: Groups Close to another Chain …Select groups that are within 5 Å of another chain. This
amounts to selecting the residues within 5 angstroms of the subunit interface.
H-bonds:
A-chain B-chain
arg426 asp338
glu406 arg364
arg431 ser386