Download S11. Computational Molecular Modeling- Week 5. 3

S11-Analysis of three-dimensional (3-D) protein models
INSTRUCTOR PRE-LABORATORY PREPARATION
The students will need a computer with internet access and ICM-Browser
(www.molsoft.com/icm_browser.html) installed to complete these week 5 activities. In my classes I
ask that at least one student in the group downloads the ICM-Browser software on to their own
personal laptop computer. Only on occasion do I have a group where no member has a personal
laptop. I am fortunate that the teaching laboratories at my institution have a few laptops that the
students can borrow. If the instructor’s institution does not have laptops for students to borrow
perhaps the university will install ICM-Browser software, which is free, on the computers in a
computer laboratory.
Prior to the laboratory meeting, the students and instructor should review the Top 10 General ICM
Tips (Supporting File S12) so they understand the basic ICM-Browser commands required to
analyze the 3-D structure of proteins. In addition, users should explore the “Help” feature of ICMBrowser which contains basic help guides and tutorials. The instructor should be aware that there
are also several ICM Help videos available on YouTube
(https://www.youtube.com/playlist?list=PLF9DCF2F2C8938F73).
For the week 5 activities, I have provided two different student protocol instructions. The “Easy 3-D
Protein Modeling Instructions,” require the instructor and students to open and manipulate ICMBrowser files provided as part of these activities (Supporting files S17 and S18). The advantage of
using the easy instructions is that the instructions precisely match the 3-D structure and protein
alignment in the provided files (Supporting files S17 and S18). I strongly recommended that an
instructor performs the easy activity to familiarize themselves with the ICM-Browser software,
even if they plan to use the “Advanced 3-D Protein Modeling Instructions,” instructions in class. If the
instructor chooses to use the easy instructions in class, the students will need access to the AS2
normal_mutant and AS1 normal_mutant files (Supporting file 17 and 18). These files can be emailed
to students or uploaded to a course management system. It should be noted that in the file names
and within the ICM-Browser program, the term normal is used to describe the wild type proteins.
The advanced instructions require the students to load the 3-D protein models that the instructor
submitted to I-TASSER in the week 3 and 4 activities (Supporting files S7 and S8). These files are
downloaded from I-TASSER as .pdb files. The instructor will need to give students access to the .pdb
files for the normal and mutant AS1 and AS2 proteins. The .pdb files can be emailed or uploaded to
a course management system. In the advanced instructions, students generate protein sequence
alignments and upload them into ICM-Browser. Once uploaded into ICM-Browser, the instructor
and students determine what regions of the 3-D protein models to analyze, based on the sequence
alignment.
Compared to the easy instructions, the advanced instructions are more challenging to write because
they use the students’ own 3-D models generated from I-TASSER. These models will vary as the ITASSER program undergoes updates. Because the models may vary with each submission, I cannot
write specific instructions to guide the user through an analysis of the proteins. Therefore,
successful completion of the advanced instructions require the instructor to be familiar with the
ICM-Browser program and be comfortable helping students make informed choices about what
regions of the proteins should be analyzed.
I recommend that the instructor performs an internet background search on how protein 3-D
modeling works so they understand the various types of resources that are available and how
modeling software can be incorporated into biology research. The review article written by
Schwede (2013) will help instructors become familiar with the current state and uses of molecular
modeling (1). An instructor may wish to present their background research on molecular
modeling to students during the in-laboratory discussion.
STUDENT PRE-LABORATORY PREPARATION
Instructions to be given to students: At least one person in your group will need to have ICM-Browser
installed on their computers. Please download and install ICM-Browser
(www.molsoft.com/icm_browser.html) to your computer prior to the laboratory meeting. Depending on
your internet connection, downloading and installing ICM-Browser may take up to 45 minutes. If you
have problems downloading and installing the software, or no one in your group has a laptop computer,
please contact the instructor immediately.
This week’s activities require the use of the 3-D molecular modeling software ICM-Browser. To
familiarize you with the ICM-Browser software commands the instructor will provide you with a
document entitled the Top 10 General ICM-Browser 3.8-3 Tips. Please review this document prior to the
laboratory session. Please also explore the Help menu in ICM-Browser so that you know what resources
are available to help you navigate the software.
The two proteins that will be analyzed during this laboratory are ASYMMETRIC LEAVES1 (AS1) and
ASYMMETRIC LEAVES2 (AS2). AS1 is classified as a myb domain transcription factor and the AS2
protein contains a leucine zipper region. To help you understand the basic structure and significance of
these proteins, please perform an internet background search of myb domains and leucine zippers and
write a brief summary of your findings.
IN-LABORATORY DISCUSSION
At the beginning of the laboratory I have the students share their summaries of the myb domain and
leucine zipper internet research with a student from another group. After 5-10 minutes of student
discussion, I ask the students to volunteer their information on myb domain and leucine zippers proteins
and lead a whole class discussion to ensure that the students understand these topics.
I then spend about 10 minutes doing a brief demonstration of the ICM-Browser software. I simply open
the AS2_normal_mutant file (Supporting file S17) and show the students how to zoom in and out on the
protein, rotate the protein, change the color of the protein, and change the protein from a ribbon to a space
filling model. These instructions are written in the Top 10 General ICM-Browser 3.8-3 Tips (Supporting
file S11) but I find a brief demonstration helps the students feel more comfortable navigating the
software.
Once the demonstration is complete, I ask the students to volunteer how they believe this software could
be used in a research setting. I build on the student responses and lead a class discussion that briefly
explains that I-TASSER makes 3-D protein models based on similarities to known protein structures
found in databases (2). I also discuss how pharmaceutical companies use molecular modeling software to
develop new drugs (3).
1.
Schwede T. 2013. Protein Modeling: What Happened to the “Protein Structure Gap”?
Structure 21:1531–1540.
2.
Roy A, Kucukural A, Zhang Y. 2010. I-TASSER: a unified platform for automated protein
structure and function prediction. Nat Protoc 5:725–738.
3.
Schmidt T, Bergner A, Schwede T. 2014. Modelling three-dimensional protein structures
for applications in drug design. Drug Discov Today 19:890–897.
SAMPLE STUDENT HANDOUT: 3-D PROTEIN MODELING (EASY INSTRUCTIONS)
Introduction: The goal of today’s laboratory is to compare the three-dimensional (3-D) structures of the
normal (wild type) and mutant AS1 and AS2 proteins. During these activities, you will use computational
molecular modeling software (ICM-Browser) to view and compare the 3-D models of the AS1 and AS2
proteins. Successful completion of these activities will require careful reading of the instructions. The
instructor has provided you with a document entitled Top 10 General ICM-Browser 3.8-3 Tips. It is
highly recommended that you review this document prior to attending the laboratory session because it
has detailed instructions for completing various tasks (selecting/deselecting proteins, changing the color,
taking images, etc.).
Before you begin this laboratory, download and save this student handout to a computer. Complete the
laboratory by following the instructions provided. When you are finished with the laboratory, upload your
completed handout to the course management system. Also, please have the Top 10 General ICMBrowser 3.8-3 Tips open as you work through the activities. The “Help” feature of the ICM-Browser
program is also a useful resource.
Task 1-Analysis of AS2 proteins
Step 1: Your instructor will provide you with files entitled AS2 normal_mutant and AS1 normal_mutant.
Please note that in this week’s activities, the term “normal” in the file names and within the ICMBrowser program is used to describe the wild type proteins. To open these files, open ICM-Browser
and go to File>Open, then find the AS2 normal_mutant file on your computer. Simply clicking on the
downloaded AS2 normal_mutant file will not open it.
Step 2: Once the file is open, deselect the AS2 mutant protein to visualize only the AS2 normal protein in
the 3-D workspace. Color your AS2 normal protein green. If no secondary structure is visible, turn on the
secondary structure by clicking and holding the “ribbon representation” button in the display tab and
selecting “assign sec. structure” (first make sure “ribbon” is selected under the “ribbon representation”
button). Rotate, zoom in or out until the AS2 normal protein is arranged in the workspace so that an
image representing the protein’s structure can be taken. Take an image (see Top 10 General ICM-Browser
3.8-3 Tips). The image should look something like those shown in figure 1 (except it should be green).
A)
B)
F IGURE 1 A) P ROTEIN WORM VIEW OF THE NORMAL AS2 PROTEIN . B) RIBBON STRUCTURE
VIEW OF NORMAL AS2 PROTEIN WITH SECONDARY STRUCTURE ASSIGNED.
Paste an image of your AS2 protein here:
Step 3: View the AS2 normal protein as a space filling model, recolor your protein green and take an
image. The image should look something like the protein in figure 2 except the protein should be green.
Figure 2 Space filling model of normal AS2 protein
Paste an image of your space filling model of AS2 here:
Step 4: Toggle off the space filling view; zoom in and closely view the “zipper” region of the protein.
Turn on the stick and ball view. Notice how the amino acid side chains are positioned in this region
(figure 3). Structurally, what would you predict is the role of amino acid side chain interaction in this
region?
F IGURE 3 P ROTEIN BACKBONE AND BALL AND STICK VIEW OF NORMAL AS2.
Step 5: Deselect the ball and stick representation. Highlight and label amino acids E72 and R96. With
these amino acids still highlighted, turn on the ball and stick representation. You can highlight two amino
acids simultaneously by highlighting the first one and then clicking Ctrl before highlighting the second
one. Deselect the graphical selection; this will remove the green Xs from the display. Look closely at how
the position of the E72 and R96 amino acids (figure 4). These two amino acids form a salt bridge. Do a
quick internet search and briefly summarize what a salt bridge is and describe the role they play in protein
structure.
F IGURE 4 CLOSE UP OF THE BALL AND STICK VIEW OF THE SALT BRIDGE BETWEEN E72 AND
R96.
Step 6: Measure the distance between the atoms of E72 and R96 (figure 5). Click the distance between
two atoms button on the top tool bar. Then click on an oxygen of the E72 and a nitrogen of the R96.
Repeat for the other E72 oxygen and R96 nitrogen. What is the distance between these atoms? What
would you predict would happen to the structure of the AS2 protein if these amino acids were mutated?
Distance between atoms.
F IGURE 5 S CREENSHOT SHOWING THE DISTANCE BETWEEN ATOMS BUTTON AND DISPLAY .
Step 7: Turn off the distance between atoms (three buttons to the right of the distance between atoms
button). Turn off the labels by clicking the A5 button and turn off the ball and stick view by clicking the
ball and stick button. This should return you to the protein worm view (figure 6).
F IGURE 6 P ROTEIN WORM VIEW OF THE NORMAL AS2 PROTEIN ZIPPER REGION.
Step 8: Now deselect AS2 normal from the 3D workspace and view just the AS2 mutant protein. Color
the mutant protein orange. Assign secondary structure, rotate, zoom in and out until the AS2 normal
protein is arranged in the workspace in such a way that an image representing the structure of the protein
can be taken. Take an image of the mutant AS2 protein and paste it here:
Highlight and label the E72 and R96 in the mutant protein and turn on the ball and stick view for E72 and
R96. Deselect the graphical selection; this will remove the green Xs from the display. Take an image
and paste it here:
In the mutant AS2 protein, are E72 and R96 able to form a salt bridge? Why or why not?
Step 9: Look at the sequence alignment of the AS2 normal and mutant protein. If the sequence alignment
does not appear on your screen go to menu bar at the top of the screen and choose Windows>alignments.
Analyze the sequence alignment. Do the E72 and R96 align in the normal and mutant protein? Overall,
are the AS2 normal and mutant protein similar or dissimilar in sequence? Where is the most dissimilarity
in these proteins, N-terminal or C-terminal? Propose an explanation as to why the E72 and R96 are
separated from each other in the mutant AS2 protein and cannot form a salt bridge like they do in the
normal AS2 protein.
Step 10: Turn off the amino acid labels by clicking the A5 button and turn off the ball and stick view.
This should return you to the protein worm view of the AS2 mutant protein.
Step 11: View both the AS2 normal and mutant protein simultaneously in the 3-D workspace. Double
click on the AS2 normal object title and then Ctrl click the AS2 mutant object title. Both proteins should
have all of their amino acids selected (green Xs) in the 3D workspace. Now click the superimpose button.
Make the AS2 normal the static object when the box appears asking for this information. If necessary,
recolor the normal AS2 protein green and the mutant AS2 protein orange.
Step 12: On both the AS2 normal and mutant proteins, label the following amino acids, M1, E72, R96
and Q149. Highlight and label the E72 and R96 in the mutant protein and turn on the ball and stick view
for E72 and R96. Deselect the graphical selection; this will remove the green Xs from the display. Of the
amino acids you labeled (M1, E72, R96 and Q149), which are located in the same region when the 3-D
images of the mutant and normal proteins are superimposed and which are located in different regions?
Take an image of the superimposition and insert it here:
Now, without rotating or zooming the objects in the 3D window, turn off the AS2 mutant and leave the
AS2 normal on. Take an image and insert it here:
Again, without rotating or zooming the objects in the 3-D window, turn off the AS2 normal and turn on
the AS2 mutant. Take an image and insert it here:
Task 2-Analysis of AS1 protein
Step 1: Open ICM-Browser and open the file entitled AS1 normal_mutant. Again, you will need to go to
File>Open within ICM-Browser.
Step 2a: Deselect the AS1 mutant protein and visualize only the AS1 normal protein in the 3-D
workspace. Color your AS1 normal protein green. Assign secondary structure by clicking and holding the
“ribbon representation” button in the display tab and selecting “assign sec. structure.” How many alpha
helices does the normal AS1 protein have? How many beta-pleated sheets?
Step 2b: Rotate, zoom in and out until the AS1 normal protein is arranged in the workspace in such a way
that an image representing the structure of the protein can be taken. Take an image and insert the image
here (your image should look something like the one in figure 7 except is should be green):
F IGURE 7 RIBBON VIEW OF THE AS1 NORMAL PROTEIN .
Step 3: View the protein as a space-filling model, recolor the protein green and take an image. Insert the
image here:
Step 4: Select and label the M1, K112 and P223 amino acids. Deselect the graphical selection; this will
remove the green Xs from the display. Take an image and insert it here:
Step 5a: Deselect the AS1 normal protein and visualize only the AS1 mutant protein in the 3-D
workspace. Color your AS1 mutant protein orange. Assign secondary structure by clicking and holding
the “ribbon representation” button in the display tab and selecting “assign sec. structure”. How many
alpha helices does the mutant AS1 protein have? How many beta-pleated sheets?
Step 5b: Rotate, zoom in and out until the AS1 mutant protein is arranged in the workspace in such a way
that an image representing the structure of the protein can be taken. Take an image and insert the image
here:
Step 6: View the protein as a space filling model, recolor the protein orange and take an image. Insert the
image here:
Step 7: Turn off the space filling model. Select and label the M1, K112 and P223 amino acids. Deselect
the graphical selection; this will remove the green Xs from the display. Take an image and insert it
here:
Step 8: Look at the sequence alignment of the AS1 normal and mutant protein. Do you believe that the 3D
structure of the normal and mutant AS1 proteins will be the same or different in the M1, K112 and P223
regions? Explain.
Step 9: View both the AS1 normal and mutant protein in the 3-D window. Double click on the AS1
normal object title and then Ctrl click the AS1 mutant object title. Both proteins should have all of their
amino acids selected in the 3-D workspace. Now click the superimpose button. Make the AS1 normal the
static object when the box appears asking for this information. Make sure the M1, K112 and P223 are still
labeled. If these labels were turned off, re-label them on both proteins before taking the images below. Of
the amino acids you labeled (M1, K112 and P223), which are located in the same 3-D region when the
mutant and normal proteins are superimposed and which are located in different regions?
Step 10: Take an image of the superimposition and insert it here:
Now, without rotating or zooming the objects in the 3-D window, turn off the AS1 mutant and leave the
AS1 normal on. Take an image and insert it here:
Again, without rotating or zooming the objects in the 3-D window, turn off the AS1 normal and turn on
the AS1 mutant. Take an image and insert it here:
Task 3-3-D modeling assessment
Briefly explain how changing the amino acid sequence of a protein influences the protein’s 3-D structure?
SAMPLE STUDENT HANDOUT: 3-D PROTEIN MODELING (“ADVANCED
INSTRUCTIONS)
Introduction: The goal of today’s laboratory is to compare the three-dimensional (3-D) structures of the
normal and mutant AS1 and AS2 proteins. During these activities, you will use computational molecular
modeling software (ICM-Browser) to view and compare the 3-D models of the AS1 and AS2 proteins.
These activities demonstrate how mutations at the DNA cause changes in the 3-D structure of proteins.
Successful completion of these activities will require careful reading of the instructions. The instructor
has provided you with a document entitled “Top 10 General ICM-Browser 3.8-3 Tips”. It is highly
recommended that you review this document prior to attending the laboratory session because it has
detailed instructions for completing various tasks (selecting/deselecting proteins, changing the color,
taking images, etc.).
Task 1: 3D Analysis of the AS2 normal and mutant proteins
Before you begin this laboratory, download and save this student handout to a computer. Complete the
laboratory by following the instructions provided. When you are finished with the laboratory, upload your
completed handout to the course management system. Also, please have the Top 10 General ICMBrowser 3.8-3 Tips open as you work through the activities. The “Help” feature of the ICM-Browser
program is also a useful resource.
Your instructor will provide you with the .pdb files that were created when the class submitted the
sequences of the normal and mutant AS1 and AS2 proteins to I-TASSER.
1. Upload the .pdb files from I-TASSER that contain the models of the AS2 normal and mutant proteins
into ICM-Browser (Go to File> Open; then select the file to upload). Right click on the title of the AS2
normal protein model in the workspace panel and select “Convert PDB” to convert the model to an ICM
object. When the dialogue box pops up, keep the default boxes checked and in addition check the “replace
the original” box. Repeat for the AS2 mutant protein.
2. To change the background color in the 3-D viewer window, right click in the viewer window and select
“choose background color.”
3. Align the normal and mutant AS2 proteins that you obtained from NCBI in the week 3 activities
entitled “Gel electrophoresis and Bioinformatics”. Enter both the normal and mutant sequences into the
box on this website http://www.genome.jp/tools/clustalw/. Make sure to label the proteins “>wild_type”
and “>mutant” and select the output as FASTA before clicking “Execute multiple alignment.” A different
page will open when the alignment is complete. Click on “clustalw.fasta” and save the file to your
computer. Return to ICM-Browser, go to File>Open, select “alignment in fasta format” from the file type
menu, select the fasta alignment you downloaded to your computer and click “open.” The alignment
should appear at the bottom of the ICM-Browser window. If it does not appear automatically, go to the
top menu in ICM-Browser, select “windows” and make sure “alignments” is checked.
4. Look closely at the alignment of the AS2 normal and mutant proteins. What regions of the AS2 normal
and mutant proteins do you expect will have the same 3-D structure? Explain. Use the amino acid
numbers to refer to the protein regions.
5. Deselect the AS2 mutant protein (if it is selected) and visualize only the AS2 normal protein in the 3-D
workspace. Color your AS2 normal protein green. Assign secondary structure by clicking and holding the
“ribbon representation” button in the display tab and selecting “assign sec. structure.” Rotate, zoom in and
out until the AS2 normal protein is arranged in the workspace in such a way that an image representing
the structure of the protein can be taken. Take an image and insert the image here:
6. Turn on the space filling view and recolor the protein green. Take an image and insert the image here:
7. Turn off the space filling view, to show only the protein backbone of the AS2 normal protein. Turn on
the ball and stick view of the protein. Zoom in and out, while rotating the protein. See if you can identify
chemical interactions between the amino acid side chains in various regions of the protein.
8. Zoom in and out on two amino acids that you think may have some kind of amino acid side chain
interaction. Then, using the pick atom button (Green X) on the menu bar on the right side of the screen
you can click on the atoms of the interacting amino acids. Once you have picked the atoms, click the
residue label button (A5 button) on the top menu bar to label the amino acids. Highlight these two amino
acids in the amino acid sequence located in the workspace panel; this will highlight the entire amino acid
in the 3-D window. Click the invert selection button (green exclamation mark on top menu). Now, all
amino acids, except the two you chose to label, should be selected. Click the ball and stick view button.
Only the two amino acids you wanted to analyze should have the side chains shown. The rest of the
protein should be in protein backbone view. What amino acids did you chose to label and why? Describe
the kind of amino acid side chain interaction that you believe is occurring between these two amino acids.
9. Deselect the normal AS2 protein and view only the mutant protein. Color the mutant protein orange.
Assign secondary structure by clicking and holding the “ribbon representation” button in the display tab
and selecting “assign sec. structure.” Take an image and insert it here:
10. Turn on the space filling view of the AS2 mutant protein and recolor the protein orange. Rotate and
zoom until a good image of the protein is visible. Take an image of the protein and insert it here:
11. Turn off the space filling view of the AS2 mutant and label the same amino acids in the mutant
protein that were labeled in the normal protein in step 8.
12. View both the normal and mutant simultaneously. Make sure the protein backbone view is selected
(space filling and ball and stick view off) and that the amino acids you chose in step 8 are labeled in both
the mutant and the normal protein.
13. Highlight both the normal and mutant AS2 proteins (all the amino acids in both proteins should have
green X’s in the 3-D display). Click the superimpose button.
14. Compare the 3-D structures of the AS2 normal and mutant proteins. How does the overall 3-D
structure of the AS2 normal and mutant compare? Considering the amino acid sequence alignment of the
AS2 normal and mutant proteins did you expect the 3-D structures of these two proteins to be more
similar? Explain.
Task 2: 3-D Analysis of the AS1 normal and mutant proteins
1. Open ICM-Browser and open the AS1 normal and mutant .pdb files. Just as you did for the AS2
protein, convert the AS1 normal and mutant proteins into ICM objects.
2. View only the AS1 normal protein and assign secondary structure to the protein model. Color the
normal protein green and take a good image of the protein. Insert your image here:
3. Turn on the space filling view, recolor the protein green and take an image of the AS1 protein. Insert
your image here:
4. Make a sequence alignment of the AS1 normal and mutant proteins following the same procedure as
you did to make the AS2 normal and mutant sequence alignment. Import this sequence alignment into
ICM-Browser. Analyze the sequence alignment of the AS1 normal and mutant proteins. Highlight an
amino acid at the beginning, middle and end of the sequence that is identical in the AS1 normal and
mutant proteins. Label these amino acids. With these amino acids still selected, click the ball and stick
button. Which amino acids did you select? Do you anticipate that these amino acids will be located in the
same 3-D regions of the mutant and normal proteins once these proteins are superimposed? Explain.
5. Deselect the normal protein and visualize only the AS1 mutant in the 3-D viewer.
6. Color the AS1 mutant protein orange. Arrange the protein in the workspace and take an image. Insert
the image here:
7. Turn on the space filling view of the AS1 mutant protein and recolor the protein orange. Arrange the
protein in the workspace and take an image. Insert the image here:
8. Turn off the space filling view and make sure that only the protein backbone view is on. Highlight and
label the same amino acids that were labeled in the AS1 normal protein in step 4.
9. Visualize both the AS1 normal and mutant proteins. Make sure the protein backbone view is on and
that the amino acids are still labeled. If the amino acid labels were turned off, highlight the amino acids
and re-label them.
10. Highlight both the normal and mutant AS1 proteins (all the amino acids in both proteins should have
green X’s in the 3-D display). Click the superimpose button.
11. Compare the 3-D structures of the AS1 normal and mutant proteins. How does the overall 3-D
structure of the AS1 normal and mutant compare? Considering the amino acid sequence alignment of the
AS1 normal and mutant proteins did you expect the 3-D structures of these two proteins to be more
similar? Of the amino acids you chose to look at, which are located in the same region of the mutant and
normal proteins and which are located in different regions?
Task 3: 3-D modeling assessment
Briefly explain why changing the amino acid sequence of a protein influences the protein’s 3-D structure.