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Guidelines and Questions

The document provides instructions for completing an assignment involving analyzing a lysozyme protein structure using Coot and PyMOL. It describes downloading a lysozyme electron density map and mutated model from online sources. Using Coot, students are asked to identify 4 incorrectly inserted residues by examining how well side chains fit the electron density. They then use Coot tools to mutate residues to correct the sequence. Students export the corrected structure and open it in PyMOL to generate images highlighting individual corrected residues and the overall structure. The document also provides questions for students to answer about the assignment.

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84 views6 pages

Guidelines and Questions

The document provides instructions for completing an assignment involving analyzing a lysozyme protein structure using Coot and PyMOL. It describes downloading a lysozyme electron density map and mutated model from online sources. Using Coot, students are asked to identify 4 incorrectly inserted residues by examining how well side chains fit the electron density. They then use Coot tools to mutate residues to correct the sequence. Students export the corrected structure and open it in PyMOL to generate images highlighting individual corrected residues and the overall structure. The document also provides questions for students to answer about the assignment.

Uploaded by

d n
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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 Please submit the following assignment as a single pdf file with all the images and answers

 Name the pdf file as : “yourname_rollno._assign1”


 Upload the single file in the following google form. Upload this using your IITB mail
address.
https://docs.google.com/forms/d/14oJvE73zX2Jtbs4H04iwK6tiSvQyThujvwBzRYeAyHg/
There is an option to upload the file at the end of this form

PROCEDURE FOR THE ASSIGNMENT COOT:

1. First, download the electron density map of the lysozyme structure from
https://www.ebi.ac.uk/pdbe/entry/search
2. Enter PDB code 1VAU (Gallus gallus lysozyme structure)
3. Go to the Download section on the left, and click Maps.
4. Generate & download the map that will be opened in Coot. Use the following selections: Map
Format - CCP4
5. Start WinCoot (on Desktop).
6. File>Open Map”, select the map you just downloaded.

7. File>Open Coordinates>1VAU-MUTATED.pdb (found on our Course Site), this opens the


mutated lysozyme model.
8. Use the Crib Sheet at end of the handout to get familiar with the Mouse controls. (Zoom, Slab
etc)
9. In this structure there are 4 residues that have been incorrectly inserted. You should examine
each residue in the structure to find the incorrect ones. Since you have loaded the electron
density map for the wild-type structure, and a mutated model there will be differences in how
the map matches the model. To find these:
a) Draw> Go to Atom (Start at residue 1 and progress through all lysozyme residues, excluding
ligands, using the space bar)
b) Examine the electron density around the side chains- does is fit? (Is it too large too small,
wrong shape etc.)
c) When you think you have found an incorrect residue
i. Can you intuit the correct residue from the shape of the electron density?

ii. Once you have a guess for the correct residue go to the panel on the right (expand by clicking
on the last button and selecting “Icons and Text”) and click “Simple Mutate”. iii. Next,

click anywhere on the incorrect residue. A list of all the amino acids will pop up;

choose the one you wish to substitute. iv. On the right panel click “Real Space Refine”. Click the
button again, and then select your newly added residue. Coot will try to fit the residue into the
density. Does it fit well into the density? If not, try again with a different residue.
10. When you believe you have the entire correct sequence, save your coordinate (pdb) file on the
desktop: File> Save Coordinates. Make sure to give it a unique name.

Exercise
1. Start PyMOL. (Two windows will appear if running on a PC vs one on a Mac).
2. File >Open> yourlysozymefile.pdb
3. Change visualization such as ribbon diagram vs. sticks etc. c. H- Hide d. L- Label- can add
atom labels etc. e. C- Color – changes colors of selected structure

a. Overall structure:

1. First some practice changing views:


a. In all: S>Ribbon. H>Lines
b. Now show as Sticks. Hide Ribbon. Show as Cartoon. Hide Sticks.
c. Hide waters. Rotate and zoom in.
d. Change your background to white. Display> Background> White
2. Choose the view (cartoon, stick, ribbon, colors etc.) you prefer. Make sure you can see the
entire structure.
3. Find the command line: PyMOL>.
4. Into the command line box type: set use_shaders. Click Enter/Return
5. Next, type: png image.png, 4000, 3000. Click Enter/Return.
6. DO NOT click on your structure. File >Save Image As…> PNG.
Give the file a name.
b. Individual corrected amino acids:
1. In the lower right hand corner, there is another button “S”. When clicked, this displays the
amino acid sequence on the top of the viewer window showing your structure. You should now
see the entire aa sequence show up at the top of your black window. You can choose residues in
the sequence by clicking on them here, and they will be highlighted in the viewer window.
2. Using the panel at the top of your screen, select one of the amino acids you corrected prior in
Coot. “sele” should now show up in your right panel and the amino acids should be highlighted on
your structure.
3. Rename this new selection (e.g. Ala78Trp [indicating you changed residue 78 from Ala to Trp
to correct the overall sequence]).
4. In all: S>cartoon (hide other views)
5. Show your selection as Sticks (S> sticks) and color by element (C > by element)
6. Zoom in so you can clearly see your aa side chain and some of the overall structure.
7. Setting > Cartoon > Side Chain Helper 8. Setting >Transparency > Cartoon > 80% (optional)
9. Now type the command png image.png, 4000, 3000 again to save your image as instructed
previously.
10. File> Save Image As > PNG (Save your file with a unique name) REPEAT FOR ALL
FOUR AMINO ACIDS CORRECTED.

Example of a finished amino acid figure generated in Educational PYMOL1 :


You should have now have 5 figures
Make sure to give them a descriptive caption.
• Remember to note what the incorrect amino acid was, as well which amino acid you mutated it
to.

Questions Set A:
1. What is the source organism of the 1vau protein? (Answer from the PDBe site)
2. What are the mutations that existed in the crystal structures?
3. What are the wild type amino acids you added?
4. What option in coot was used to change the mutations back to the wild type?
5. What was the significance of the real space refine option in coot?
6. Name any other options you could find to refine the structure in coot after changing the amino
acids?

LYSOZYME STRUCTURE
1. In the PyMOL command line type: fetch 3pbl. This downloads the structure directly from the
PDB into PyMOL. The dopamine structure should appear.
2. All> Color > by chain. You will see there are two different colored chains. These are two
(identical) copies of the protein present. We will select just one to work with.
3. In the command line, type select Dopamine1, /3pbl/A/A .
A tab called "Dopamine1" will show up in the right hand column of the viewer window.
4. All > Hide > Everything
5. Dopamine1 >Show > cartoon. Now the cartoon ribbon diagram of just chain A should appear
on your screen. To make manipulating this chain easier, in the “Chain A” menu click on A (for
actions), then center and zoom in.
6. Look at the structure. Can you identify the two individual proteins dopamine receptor and
lysozyme? Scrutinize the AA sequence by using the “S” button in the lower right hand corner
again to show the AA sequence. Is something distinct about the numbering?
7. Once you have identified the residue range for the lysozyme, Into the command line type select
lysozyme, resi 1-10 (note replace 1-10 with the correct residue range). This will select all the
residues in the lysozyme only & a tab “lysozyme” will appear. Make lysozyme a different
color from the dopamine receptor. Show lysozyme as spheres (or other different representation
than cartoon).
8. Create an image with white background. Submit the image.

Questions Set B:
1. Navigate to www.rcsb.org. Find the structure with PDB ID: 3PBL.
2. Explore all the tabs (Summary, Sequence etc.) to answer the following questions about 3PBL,
the dopamine receptor:
a. What is the organism is the dopamine receptor from (scientific name)?
b. How many ligands are bound to the receptor? And what are their names?
c. What is the resolution of the crystal structure?
d. What method was used for crystallization?
e. What temperature was it crystallized at?
f. Was the data collected at a synchrotron?
g. What software was used to complete refinement?

Questions Set C:
Consider the following 2 PDB structures 2MHR and 1HEW. Try to identify at least 2 different
possible non- covalent interactions in these systems (eg: H-bond and hydrophobic interactions).
Do not show 2 H-bonds alone, interactions must be different. Submit the PyMOL images of these
2 interactions – (Refer the procedure of first Tutorial). Make sure to properly label the residues
and write the name of the interaction.

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