Visualization of three-dimensional protein structure

 

Three-dimensional structure:         

          Visualization of single sequence

          Identification of conserved regions (multiple sequences)

 

Tools:

          *SIB (Student Interface to Biology workbench)

           educational-friendly interface to the Biology Workbench

 (http://deltas.animal.uiuc.edu/sib/servlet/SIB)

*Chime

 web browser plugin that depicts the image of molecules

 (http://www.umass.edu/microbio/chime/)

 must install Chime in computer prior to using Protein Explorer

*Protein Explorer

           user interface to visualization plugin software (Chime)

           recommended: 1 hour Quick tour and tutorial

 (www.proteinexplorer.org)

*ConSurf

           visualization of conserved regions

 uses:

single sequence 3D structure and

multiple sequence alignment (SWISSPROT or user entered)

protein explorer

(http://bioinfo.tau.ac.il/ConSurf)

 


Example

 

Insulin-like growth factor II (IGF2)

also known as somatomedin A

single-chain polypeptide

shares amino acid sequence homology with insulin and relaxin

maps to human chromosome 11p15 next to insulin locus

functions include:

stimulation of cell growth,

modulator of muscle growth and differentiation

gene is widely expressed during prenatal development

overexpressed in Wilms tumors

activity is regulated by genomic imprinting

(gene being inactive on the maternal chromosome)

 

Activities

1) In SIB, enter your Username and Password and create a NEW session named “IGF2” and go to Protein Tools




 


2) Use Ndjinn in SIB to search for IGF2 sequences (keyword igf2) in the SWISSPROT database

 

 


Select and import the following sequences:

SWISSPROT IGF2: Human, Sheep, Mouse, Pig and Rat

 


3a) Repeat the Ndjinn search using a different database

  Search for an IGF sequence (keyword igf) with PDBFinder (PDB database)

 

3b) Select and import the following sequences:

PDBFINDER: 1GF2

 

Check that your IGF2 session now has the following sequences

 

 

 


4) Select and align all sequences using CLUSTALW


 

 



IGF2 related sequences share the following conserved pattern:

C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-C

 

Results from CLUSTALW show the conserved region CC…………C

 


5) Import alignment

    In Alignment Tools, select and view the alignment (click on View)

 

6) Check that your alignment is in Fasta format. Save alignment to your computer using the first “document” icon in the top right corner. You may be prompted to enter your login and password. Name the file alignm.txt (locate the file in an easy to access folder)

 


7) Go to Protein Tools and visualize the IGF2 3D structure

 

Select the PDBFINDER_1GF2 sequence and click on View Records

 

 

 


The record information is:

 

 

8) Use the Protein Explorer link (middle upper right) to visualize the 3D structure

 

9) Click on Start Explorer Session

Wait until the Busy red box (lower right corner) changes to Ready green box

Click the OK button (upper left corner)

 

Click on the Toggle Spinning button (FirstView left frame) to freeze image


10) Scroll down the FirstView frame

      Click Explore More link for further manipulation of sequence

 

 


The Select, Display and Color options allow to identify particular segments

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11) For example, identify the alpha-helices with a cartoon display in blue:

 

The Advanced Explorer allows to further explore the sequence


IGF2 related sequences share the following conserved pattern:

C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-C

 

12) Select the Advanced Explorer link (QuickViews frame) and then Seq3D

Select Spacefill and Green in the white boxes (upper frame)

Find in the lower frame sequence the first pair of cysteines (CC) and the final cysteine (C) the mark the start and end of the conserved IGF2 region.

Click on the first CC (cysteines) and the last C (thirteen positions below)

 

 

 

 

 

 

 

 

 

13) Click on the Advanced Explorer frame link, MSA3D: Multiple Sequence

Click on the link: Visualizing Regions of Conservation in 3D Protein Structures and then click on ConSurf

 

 

 


14) Type as PBD ID: 1gf2

Type as chain identifier: none

Click on the Submit button

 

The alignment may take a few minutes (click on Click Here link for updates)

 

 

 

 

 

15) Click on the View ConSurf Results with Protein Explorer link

      Click on the Start Protein Explorer link

      Wait until Protein Explorer is “ready

16) Click on Spin and Spacefill None to change rotation and display

To highlight more or less conserved regions of the sequence, click on the 9 to 1 colored boxes


17) Click on ConSurf Seq3D to identify positions of interest

Select first and last amino acid characteristic of IGF (use spacefill and Sec’y structure colors)

 

 

 

 

Conserved extremes are inside the molecule

 

For further display changes, use the Explore link as in Protein Explorer

18) Note, if ConSurf cannot find matches, use your own CLUSTALW alignment file (alignm.txt) saved in your computer

Type the PDB ID: 1gf2; chain Identifier: none

Type your own MSA file: alignm.txt

Type the target sequence name in the MSA file: PDBFINDER_1GF2__

Click on Submit