Structure Analysis Tutorial Fall 2004

Tutorial written by: Beth Stadtmueller

The goal of this tutorial is for you to analyze several structures using some of the programs that I
discussed in my talk. Specifically you will perform the following:

1. Calculate electrostatics
2. Map conservation among species onto you’re your structure
3. Calculate surface areas
4. Investigate a dimer interface.
5. Investigate conformational changes
6. Investigate structure homology/similarity
7. Create a topology cartoon

Getting Started:

About half of this tutorial can be run on Windows. The ddmatrix program (Analyzing
conformational changes), Naccess and Dimplot must be run on Linux.

As this tutorial follows the structure analysis talk, it may be helpful to quickly run through
the presentation to get an idea of what you are looking for.

¾ cd tutorial
¾ mkdir struct_anal
¾ cp /home/tutorial/structure_analysis/* struct_anal/.
¾ cd struct_anal
¾ ls
1QGO.pdb 1TKL.pdb 1TLB_A.pdb copox.txt
1TKL_A.pdb 1TLB_AD.pdb aln_input.doc 1ID3.pdb

Calculating electrostatics:

1. Open Pymol
2. Open 1ID3.pdb (A histone octomer core of a nucleosome)
3. show: surface
4. actions: generate: vacuum electrostatics (relative)
5. You should now see the protein colored by electrostatics. Blue should equal ~ +70 and
Red should equal ~-70. To enhance the contrast, we will change these values to ~150
and ~-150. To do this hold down control and click on the Red/Blue bar with the middle
mouse button, you should then be able to move the values.
6. What is the electrostatic charge on the outer surface of the octamer? How is this charge
related to the function of the nucleosome?
7. To run a more rigorous calculation of electrostatics you can download an APBS plug-in
and run it under Plug-in APBS tools. This will take some time and we are still in debate
about how good this calculation is. If you are really motivated, generate electrostatics in
GRASP and compare results!

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Alternative methods for calculating electrostatics

1. GRASP
2. STING (http://mirrors.rcsb.org/SMS/index_m_mirror.html) which uses GRASP via a
server.

Does the active site have any particular electrostatic charge? The substrate of COPOX is a
tetrapyrrole containing both methyl, vinyl and carboxyl side chains around its periphery.

Mapping conservation onto your structure:

Here we will map sequence conservation among species onto a structure so that you can
visualize areas of conservation

1. First we have to create a sequence alignment file in aln format.

2. Open your preferred internet browser and go to Clustalw (www.ebi.ac.uk/clustalw)
3. To input a sequence we must use a specific format which I have already created for you
using several Cop Ox sequences. It can be found as a Word document aln_input.doc
4. copy these sequences into the “sequence box” in Clustalw

For Future reference the following format (FASTA) MUST be used as an input:
>yeast (name followed by any text and carriage return)
acgtacgtagc (sequence with or without carriage returns.)
>human (next sequence name – must have unique first words)
ataagtac
....
5. submit (you can choose to get results emailed back to you or you will be allowed to open
a window containing you results)
6. Save the aln file in a place you wont forget
7. open ESPrint 2.2 http://prodes.toulouse.inra.fr/ESPript/ESPript/
i. Click on execute
ii. Select expert (top EXP)
iii. input your aln sequence file
iv. Two boxes will be available for you to input a pdb file
v. Input copox.pdb ( this is simply 1TKL.pdb renamed to match our aln
input-remember these must have the same name)
vi. In addition you can also input the same pdb file (Copox.pdb) into the
bottom of the two boxes. This box maps secondary structure onto the aa
sequence. If you do not input a pdb, it will map a structure prediction
which can be quite useful when analyzing a sequence f an unsolved
structure, however, in this case we have a structure.
8. save output pdb file (if you look at the text file, it will have conservation mapped in the
B-factor column)
9. open output pdb in pymol (again-must be most recent version- 0.97)
10. show surface
11. color by B-factor

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a. Results are displayed from blue (no conservation) to red (complete conservation)

Can you identify the location of the active site?

Calculating molecular interfaces using Naccess

Here we are going to calculate total accessible surface area and calculate the surface area of a
molecular interface. A complex of the homodimer COPOX is in file 1TKL.pdb. We can separate
out the different monomers (A and B) and remove the water to generate three files. 1TKL_a,
1TKL_b and 1TKL_ab.

1. ssh sundance (must run NACCESS on Sundance)

2. change into your tutorial directory and run
a. > naccess 1TKL-a.pdb
3. Spits out three files
a. asa files where B-factor has been replaced by ??
b. log file nothing interesting
c. rsd file- this is what you want.
i. Provides a table that basically shows accessibility of each residue .
ii. Bottom line gives a total accessible surface area for the molecule (record
this number)
4. Now we can calculate the surface area of the dimer interface
5. Run Naccess on tkl-b.pdb and 1TKL-ab.pdb and record total accessible surface area.
6. Add accessible surface of 1TKL-a.pdb and 1TKL-b.pdb and then subtract the accessible
surface area of tkl-ab.pdb. Now divide by two (This is two molecules coming together
so if we don’t divide by two we have 2X the interface surface and of course two is not a
perfect number to use)
7. In this case tkl-ab.pdb = 28696.7 A2
tkl-a.pdb = 15601.7
tkl-b.pdb = 15744.2

Molecular interface = 1324.6 A2

What percent of the surface area is occupied by the dimer interface? Can you expect and
interface to be higher than say %20-30? What percent surface area would you expect two perfect
semi-spheres to share?

Investigating Molecular Interfaces

Dimplot/H-bond allows us to map all H-bond and Van der Waal interactions at a molecular
interface.

1. You have a file hemk.pdb

2. run ligplot
a. > source /home/local/IRIX/setup/ligplot
b. > ligplot hemk.pdb 300 L 300 L

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3. runs hbadd, etc

4. spits out
a. ligplot.hhb (no waters H-bonds)
b. ligplot.nnn (no waters van der waal interactions)
5. Also creates a postscript file (image)

¾ logout (to logout of sundance)

¾ ghostview ligplot.ps

What ligand is bound to hemk? Are all the atoms which can form hydrogen bonds forming
hydrogen bonds? S-adenosyl-homocystine is actually the product of the HemK
methyltransferase, but the substrate, S-adenosyl-methionine, differs only by a single methyl
group on the SD atom. At that point the SD atom becomes charged and is made more unstable by
the interaction with the nearby hydrophobic phenylalanine.

Determining the differences between two structures

Now we are going to analyze the conformational change associated with our two CopOx
structures.

1. Use 1TKL_a.pdb and 1TLB_A.pdb These have been adjusted to compare residues 4
through 328 in chain A of both structures

Difference distance matrix plots were produce using the DDMP program from the Center for
Structural Biology at Yale University, New Haven, CT.

Features of ddmp include:

• the dot matrix plot where the dots may be two shades of gray or colors to reflect ranges
of distance differences
• a variable window of residue numbers may be plotted
• an ASCII output of the difference distance matrix
• up to 1000 residue comparison

To use DDMP do the following:

• Obtain two PDB coordinate files for comparison (Equal number of residues)
• In your working directory (where you have write permission), type:

>ddmp

The program will prompt you for various file names and options.

Note: The title to be displayed MUST be 40 characters or less.

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Note: The range of residues to be displayed MUST be a multiple of 25.

After running DDMP you may look at the ASCII file output or you may look at the postscript
grapical output similar to the figure above. To look at the postscript file you may type the
following:

>ghostview ddmatrix.ps &

structure homology/similarity
Now we are going to compare our structure to other structures in the pdb using DALI. As Copox
doesn’t see to bring up anything interesting we are going to use another structure that I discussed
in the talk: Anaerobic Cobalamine Biosynthetic Cobalt Chelatase; Chain: A (1QGO) .

Note that if you want to compare your structure that is not in the pdb you will use a slightly
different method where you will upload your pdb into DALI.

1. Once again using your favorite web browser go to

http://ekhidna.biocenter.helsinki.fi/dali/start
2. This is the FSSP database access within the program DALI
3. enter pdb # 1QGO
4. you will get “Dali database query: 1QGO”
Browseing the results you can see that the first domain (1-114) of the cobalt chelatase is quite
similar to Ferrochelatase structures, despite only 11% sequence similarity. Interesting, lower
scoring Uroporphyinogen-III Synthase also contains a chelatase component
No Chain raw-score Z-score %id lali rmsd Description
1 1qgoA 4195.8 46.0 100 257 0.0 ANAEROBIC COBALAMINE BIOSYNTHETIC COBALT
2 1dozA 1925.3 18.8 11 234 3.1 FERROCHELATASE
3 1hrkA 1776.0 16.1 9 234 3.4 FERROCHELATASE
4 1lbqA 1709.8 15.9 9 232 3.5 FERROCHELATASE
5 1tjnA 640.7 9.8 10 113 3.5 SIROHYDROCHLORIN COBALTOCHELATASE
6 1dp4C 767.7 6.9 8 178 7.2 ATRIAL NATRIURETIC PEPTIDE RECEPTOR A
7 1jftA 702.7 6.9 8 165 4.8 PURINE NUCLEOTIDE SYNTHESIS REPRESSOR
8 1efaB 736.6 6.8 7 158 3.9 LAC REPRESSOR
9 1jr2A 442.7 6.8 10 99 3.1 UROPORPHYRINOGEN-III SYNTHASE
10 1bykA 759.5 6.7 8 182 5.2 TREHALOSE OPERON REPRESSOR
11 1o8oB 496.3 6.6 10 95 2.7 MOLYBDOPTERIN BIOSYNTHESIS CNX1 PROTEIN
12 1di0A 479.1 6.4 9 108 3.6 LUMAZINE SYNTHASE
13 1mkzB 484.8 6.3 13 97 3.0 MOLYBDENUM COFACTOR BIOSYNTHESIS PROTEIN B

Note that the uroporphyinogen-III Synthase is a heme biosynthetic enzyme, and though related to
a cobalamin biosynthetic enzyme, the structural similarity is not followed up with functional
relevance and hence defines a Z-score level which is not biologically significant.

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Creating Topology Cartoons

Now we will create a TOPS cartoon of 1QGO. This will show us a simple 2D rendering of
secondary structure.

1. Go to http://www.tops.leeds.ac.uk/
2. click on “cartoon generation”
3. enter your email and pdb 1QGO (or enter any protein you want!!)
4. you may also upload a pdb
5. submit and your cartoon will be emailed to you!
6. Information on interpreting your cartoon is available at this website and in TOPS papers
included in the “Literature Folder”

Other recommendations:

STING: STING is a web-based server designed for easy analysis of protein structures. Check it
out at: http://mirrors.rcsb.org/SMS/index_m_mirror.html

You will need Chime and Java plug-in to run STING

The original paper on STING can be found in the Literature Folder.

CATH Structure classification database

http://www.biochem.ucl.ac.uk/bsm/cath/