Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Step 6: Exploring Protein Sequence and Structure Data

Task(s):  Visually identify sequence variation between IMA1 proteins in our three yeast species, and find the 3D structure of the IMA1 protein for further examination. 

Background on NCBI Resources Used:

NCBI BLAST graphical results options: The web BLAST interface provides many options for visualizing and summarizing the results of a search. Of these, we are going to make the most use of the Alignment and MSA (Multiple Sequence Alignment) viewer options. More about the MSA Viewer, which can be used on its own, can be found starting from the NCBI MSA Viewer landing page. You will use these tools to identify variation between several IMA1 sequences. 

Structure Database: NCBI’s Structure Database stores three dimensional macromolecular structure information. Three dimensional structures provide a wealth of information on the biological function and the evolutionary history of macromolecules. They can be used to examine sequence-structure-function relationships, interactions, active sites, and more. We are going to find and examine a Structure database record for an IMA1 protein.

Macromolecular Modeling Database (MMDB):
Experimentally resolved structures of proteins, RNA, and DNA, derived from the Protein Data Bank (PDB), with value-added features such as explicit chemical graphs, computationally identified 3D domains  that are used to identify similar 3D structures, as well as links to literature, similar sequences, information about chemicals bound to the structures, and more. These connections make it possible, for example, to find 3D structures for homologs of a protein sequence of interest, then interactively view the sequence-structure relationships, active sites, bound chemicals, journal articles, and more. The particular IMA1 record we examine will be a experimentally determined crystal structure record from MMDB.

 

iCn3D: NCBI’s iCn3D, pronounced "I See in 3D", Structure Viewer is a web-based 3D viewer with structure analysis capabilities. The web-based nature of the tool means no downloads are needed to start visualizing and learning! We are going to be using the basics of this tool to start connecting the IMA1 protein structure to its function. 


 

Basic BLAST Alignment Viewing 

interpret information iconBefore we begin, you can make a hypothesis about what variation we might see. Consider what we learned about the biology and genomes of our yeast species - Which protein - the one from S. pastorianus or S. eubayanus - will be more similar to our input S. cerevisiae sequence? 

 

In this step, we are going to take an initial look at differences between the sequences of our query IMA1 protein (S. cerevisiae), and those from S. eubayanus and S. pastorianus

This takes just two steps: 

1. Select several result sequences to compare - you can start with the top three on the table: QID85331.1, QID80070.1, CAI1835472.1
 

Selecting top three blast results to align
2. Click on the Alignment tab! 

 

Sample of default pairwise text alignment between subject and query

By default, the alignment output is a plain Pairwise alignment. Each section of the page represents one of the three proteins we selected aligned to the S. cerevisiae sequence.

 

We can “eyeball” some differences, such as the one I highlighted above, but we can make some adjustments to better spot the differences using the Alignment View Dropdown menu:


Pairwise with dots for identities: Alignments are anchored (shown in relation to) to the query sequence in pairwise fashion with mismatches colored in red. Sbjct will be in red and bold font if a line in the alignment contains mismatches.

Sample of pairwise identities as dots alignment option

 

Query-anchored with dots for identities: Displays all result sequences we selected in relation to the query sequence. Identities are displayed as dots (.), with mismatches displayed as single letter abbreviations. 

Example query-anchored output with dots for identity

 



Exploring an Alignment with NCBI MSA Viewer

 

To enter an interactive MSA viewer with your query and three selected sequences, go back to the "Descriptions" tab, and select the rightmost option: MSA Viewer. You should end up on a screen that looks like this one. A couple of important options are highlighted here: 

multiple sequence alignment viewer default view with labels


One coloring scheme option best suited for looking at variation is "Color by Differences", which highlights the differences between the subjects and the query sequence. 

MSA viewer after coloring by difference


You can also zoom in to sequene level to look at individual residues: 

MSA viewer zoomed into sequence level


Optionally, if you want to download this alignment or a graphic of this alignment - use the Download menu! Or, choose "Share This View" to get a sendable link to this exact view! 

interpret information icon What do you notice about the two S. pastorianus sequences? How might these patterns of variation relate to the hybrid origin of this yeast species? 


 


Finding a 3D Structure for IMA1


First, lets go back to the RefSeq protein page for the original S. cerevisiae IMA1 protein. You may notice a link to a Structure database entry on the righthand side of the page - Click it!



First, save the first result on the list to your collection:
MyNCBI Collection icon
Save the Structure record to the Yeast research folder!

1. Click "Send to" to add to the existing Yeast research folder.
Add to existing collections steps:  Send to, collections, add to existing collection
3. If it worked you'll see this message 
item added to your collection message
or you can click on your name at the top to go to your Dashboard and see your collection there! 

 

Next, click the associated link to go to into the actual Structure record for the Crystal Structure Of Isomaltase In Complex With Maltose

Once again, this a page with a lotttttt of information. The top part of this page provides some background and information to help you determine if you are on the right page, such as an abstract for the citation related to this MMDB entry.

 

interpret information icon
What did the authors learn about IMA1 in this mutant? Can it bind both maltose AND isomaltose?


webpage icon For a hint about what to look for, click here.
abstract of related citation with sentence highlighted

 

 

Download to your computer iconFor future work, you could click on any number of download links
(such as those under Download Structure Data) links
to download the data to a folder on your computer!

 

The molecular components table is also a rich source of information and links out to other databases, and ways to search for similar domains and molecules: 

mmdb molecular components table with labels



For more information about how to make sense of all the data on this page, MMDB has an extremely thorough of these pages in their help documentation.


Advanced: Interactive 3d Structure with iCn3D



Button to go to full icn3d browserTo see a fully interactive, customizeable version of the chemical structure of IMA1, click on this button!

Advanced Exploration: Align an S. pastorianus sequence to this Structure and visualize

  • Click on “full-featured 3D viewer” button.
  • Click on File>Align>Sequence to Structure
  • Provide the Sequence ID:    QID80070.1
  • and click the “Load” button.  (It may take a little time to align & display.)
  • Explore the sequence alignment & the structure!
  • You can get a link to share this interactive view by clicking File>Share Link or you can save a structure or 3D printer file, or an image by clicking File>Save Files> and then select your choice.
If you want to explore, click this link for a pre-modified 3d structure of IMA1 with this protein aligned and the sugar pocket's side chains rendered and visible!
 

This is just the very start of what you can do with iCn3D, and there is a lot of help available: 

Last Reviewed: June 29, 2023