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General instructions
Save your answers into a separate Word file for your own reference.
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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/
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
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
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 ----------------------------------------------KYYS 24
uniprot|Q86M91|Q86M91_ANOST ----------------------------------------------AKTF 24
uniprot|Q9VSA5|Q9VSA5_DROME TVYNGHCRQKTRADVANCFDGKDLPAEVAKPSKGNELVKKTSPTPKAKIY 598
uniprot|P00698|LYSC_CHICK ---------------------------------------------LGKVF 21
uniprot|Q95V66|Q95V66_PENVA ---------------------------------------------DAKVF 21
uniprot|P00713|LALBA_CAVPO ----------------------------------------------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
:* : : : *:* * .* : .
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
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.
2. Select enzyme inhibitor (NAG) from the control panel (you can select several residues by
holding the Ctrl-button while selecting).
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
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:
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.
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?
Question:
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