Tutorials

A murder mystery with molecular structures

Sat, 24 Jul 2010 09:35:55 -0700

Three people were found dead at the scene of a crime. It’s your job to find out how they died.

In the first part of this work, you will need to investigate the structure of the normal protein and find the active site so that you can recognize it later when you example samples from the victims. In the second part, you will look find the active sites in the structures from the crime scene samples and determine if the samples contain a poison.

Before you begin, you will need to download and install Cn3D from the NCBI.

Part I. Investigating the active site of cytochrome c oxidase
1. Download the structure (1OCC.cn3 or the gzipped version 1OCC.cn3_zip, gzipped versions download more quickly).
2. Open the structure in Cn3D.
3. Cytochrome C oxidase is found in the inner membrane of the mitochondria. Turn the structure around and identify the part of the protein that’s normally inside the membrane by looking for the green alpha helices.
4. Find the parts of the molecule that are located outside the membrane by looking for the tan beta sheets.
5. Open the Style menu, choose Coloring Shortcuts, and Molecule.
Each of the different protein chains and all the other molecules in the structure will shown in a different color. The amino acid sequences of the protein chains are shown in the Sequence/Alignment Viewer window.
A. How many protein chains are in this structure?
B. The protein in this structure also happens to be a dimer. How many protein chains are in each monomer?
1. Notice the letters that appear in the structure. These letters represent different elements.
2. Zoom in and find a copper ion located close to an iron. The iron is part of a structure called heme. Hold the shift key down and select both the heme group and the copper ion by clicking them with your mouse.
3. Open the Show/Hide menu and choose Select by Distance > Other molecules. Set the distance at 3 angstroms and click OK. You should see some of the residues become highlighted in one of the protein chains in the Sequence/Alignment Viewer window.
4. Scroll through the Sequence/Alignment viewer to see which residues became highlighted.
A. Which chain contains the highlighted residues?
B. Which residues were highlighted?
1. Open the Show/Hide menu and choose Show Selected Residues. You will see the heme, the copper, and the amino acid residues from the protein chain that binds to these substances.
Heme is a prosthetic group and copper is a cofactor. Both of these are very important for the enzymatic activity of cytochrome C oxidase.
1. Open the Style menu and choose Rendering Shortcuts, then Ball and Stick.
2. Click your mouse inside the Sequence/Alignment Viewer to deselect the highlighted residues.
You will see ionic bonds (white) between the copper, and the iron, and some of the amino acids.
1. Select the bound residues with your mouse and look for the highlighted amino acids in the Sequence/Alignment Viewer to identify the names and position numbers for those amino acids.
A. Write down the names and PDB position numbers for any amino acids that are bound to copper.
B. Write down the names and PDB position numbers for any amino acids that are bound to iron.
1. Open the Style menu and change the Coloring Shortcut to Element.
A. Which element forms an ionic bond to copper?
B. Is this the same element for all the bound amino acids?

Part II. What killed the victims?
The copper ion and the iron that you looked at in part I are both important for cytochrome c oxidase activity. During oxidative phosphorylation, molecular oxygen (O2) binds to the iron, and is reduced, leading to the production of two molecules of water. If other substances are bound to the iron, they can block binding by the oxygen and inhibit the activity of the enzyme. Your job is to determine if there might be other substances bound to the active site in the structures from the crime scene.

1. Download one of the structures from a victim (See below. Note - the gzipped files will download faster. If you’re using a Mac, you can unzip these by double-clicking them. If you’re using a Windows-based computer, you may need to use a program like WinZip to unzip them. Start out with the “harder” versions of the structures, use the easier versions if you’re having difficulty with selecting the proper molecules.)
2. Open the structure in Cn3D.
These structures contain alignments between the protein chain that was investigated in part I and structures from the crime scene.
1. Use your mouse to click the heme and copper from both structures and the other substance (if there is one).
2. Open the Show/Hide menu and choose “Show Selected Residues.”
3. Use your arrow keys to toggle back and forth between the normal version of the protein chain and the version from the crime scene.
4. If there is another substance besides the heme and copper in the victims’ structure, use the color of the substance to identify the elements. Then, go to Wikipedia (www.wikipedia.com) and search Wikipedia with the name of the substance to find out more about it and whether or not it could be used as a poison.
5. If the substance is poisonous, read about the properties of that substance and propose a theory to explain how it could have been administered to the victim.

Funding for this project was provided by the National Science Foundation, grant DRL-0833779, as part of a collaboration between Digital World Biology, the Northwest Association for Biomedical Research, and the EdLab Group.

DNA barcoding

Tue, 6 Jul 2010 17:24:33 -0700

DNA barcoding has become an important technique for identifying many kinds of animals, insects, and plants. In this technique, PCR is used to amplify a short 650 base region of the MT-COI gene from mitochondrial DNA. The DNA sequence is then determined from the PCR product. If this sequence has been found before, it can be used to identify the type of organism that contributed the DNA. If a barcode sequence has not been found before, one can still identify related species, by comparing the new sequence to other sequences in a database.

The bioinformatics steps in DNA barcoding involve identifying high quality regions of the trace files generated from DNA sequencing instruments, extracting the DNA sequences from those files, assembling the sequences, and identifying the most likely source by blasting the GenBank nucleotide database.

The trace files supplied here were obtained from the NCBI. These data come from actual sequencing experiments that were submitted to this public database.

Use DNA barcoding to identify unknown samples

Before you begin, download FinchTV from Geospiza, Inc. and install it on your desktop computer. FinchTV is a free program that will show base calls and quality values (if available).

A. Get the data
1. Download the trace files for an unknown organism by clicking one of the sample links in the data set.
2. Unzip the archive to obtain the individual files.

B. Review the data quality
1. Open the file in FinchTV
2. Select the high quality region with your mouse.
High quality bases have well-resolved peaks, with quality values that fall above the dotted line on the histogram plot (these values are 20 or higher).
1. Export the FASTA formatted DNA sequence to a text file as shown below.

C. Assemble the FASTA sequences into a contig
1. When you have obtained all of your sequences in a FASTA format, go to the CAP3 assembly site.
1. Open each sequence file in a text editor.
2. Copy and paste each sequence in the box on the assembly page. Be sure to include the part that begins with “>”.
3. Click Submit.
4. When the assembly is complete, select the Contigs link.
5. Copy the contig sequence and paste it into a text file.
A “contig” is a sequence that is produced by putting shorter sequences together. Each letter in the sequence represents a base in a strand of DNA. Assembling sequences into a contig is another way to check the quality of the DNA sequences and see if all the sequences are alike. If there are differences between sequences you may wish to review the trace file and determine if the base was misidentified.

D. Identify the most like source of the DNA
Now, you’ll be a DNA detective. You’ll use a program called blast to compare your contig sequence to a database of sequences (the nr database) at the NCBI. You may be able to identify your organism, and determine which types of organisms are most closely related to yours, by looking for the database sequence that matches the best. In the case of barcoding, look for the DNA sequence (or sequences) that matches most of your contig and has the highest percentage of identical bases.

1. Go to the blastn page at the NCBI.
2. Paste your contig sequence in the text box on the blast page.
3. Choose the nucleotide collection database as shown below.

1. Click BLAST.
2. Use the BLAST results to identify the potential source of the DNA in your sample.
Multiple sequences will probably be similar to your entry. The organisms that are most closely related to yours, are the ones whose DNA sequences are most similar to your contig. The best match will the sequence that matches over the longest region of DNA, with the greatest number of identical bases, and the lowest E value.

E. Learn more about your organism
1. Select the accession number for the best matching sequence to see where the sequence came from and to get the scientific name of the organism.

Some entries will have the entire scientific name (genus and species). Some entries might only have the name of a taxonomic group (i.e the order, family, or genus).

1. Use the scientific name to search the Encyclopedia of Life.
2. Use the scientific name to search Google.
3. What can you learn about this creature and where it lives?

DATA SETS
July 2010 data set

Funding for this project was provided by the National Science Foundation, grant DRL-0833779, as part of a collaboration between Digital World Biology, the Northwest Association for Biomedical Research, and the EdLab Group.

Exploring with microarrays: how do plants respond to drought?

Thu, 3 Jun 2010 11:43:49 -0700

Microarray and Next Generation DNA sequencing technologies are giving us new abilities to understand how living things respond to environmental change. One of the ways we can use this new information is to study gene expression under different conditions. In this activity, we will explore microarray data from plants as they experience drought.

1. View information for the data set.
2. View the instructions and background material.
3. Instructors may obtain a set of questions for students to answer and an updated answer key (available soon).
4. Microarray data have been pre-loaded and partially analyzed using GeneSifter Analysis Edition (Geospiza, Inc.)

An introduction to Entrez

Tue, 10 Feb 2009 20:24:23 -0800

This six and half minute video shows how to find out the kind of information that’s stored in each of the NCBI databases.

Learn how to:
• find the database fields
• use the database index
• obtain metadata for a certain type of information, for example, the number of human proteins in GenBank

An introduction to Cn3D

Tue, 10 Feb 2009 19:05:38 -0800

This five and half minute video introduces basic skills for using Cn3D. You will learn how to:
• make structures larger or smaller
• move structures across the screen
• change the color
• change the drawing style
• view selected parts of a structure
• annotate parts of structures