Find protein sequences associated with a given gene
Find the most closely related structure to these proteins
Use the structure and CD to explore a SNP known to cause a disease phenotype
Import and align a transcript variant to the template alignment.
Steps
Retrieve a gene record using Entrez
From the NCBI home page, search for "LHX3[sym] AND human[orgn]".
Click on the Gene hits to view them.
Review the gene record
Scroll down to graphic under Genomic regions, transcripts, and products. How many proteins is this gene expected to produce?
Make a note of the NP accession numbers of these proteins.
Load a related OMIM record describing a disease phenotype
Follow the link to OMIM in the Links menu.
Click on the accession of the record labeled "LIM Homeobox Gene 3"
Find a SNP in LHX3 resulting in a disease phenotype
Click on "Allelic Variants" in the left bar of the page.
Concentrate on variant .0001, Y116C.
What are the clinical consequences of this SNP?
Find the SNP site in an annotated protein sequence
Scroll up to the top of the OMIM page, and follow the link to Gene in the Links menu.
Click on the accession of NP_055379 (isoform b) to the right of the graphic under Genomic regions, transcripts, and proteins.
Open the Genpept format and confirm that the sequence has a tyrosine at
position 116 (Y116).
Find a structural template for the region of the protein including
the SNP
Click 'Related Structure' in the Links menu in the upper right of the protein record.
Use the ruler above the graphic to find the best-matching structures that contain a sequence
match at the position of the SNP (116).
Locate the SNP position in the sequence-similar structure
Click on the red bar representing a best-matching structure containing position 116.
Find the SNP position in the text alignment. The green numbers indicate residue
positions in each sequence. What position is the SNP in the structure record?
Build a VAST alignment
Click on the MMDB link (to the right of Reference) in the top section of the page.
On the structure summary page, click anywhere in the blue bar (domain 2) that contains the SNP position.
In the row to the right of the List button, change Graphics to Table, and click List.
View the VAST alignment
Check the boxes to the left of the top three structure neighbors.
Click View 3D Alignment to launch Cn3D.
Import NP_055379
In the alignment window, choose Edit / Enable Editor.
Choose Imports / Show Imports.
In the Imports window, choose Edit / Import Sequences.
Choose Network via accession and click OK.
Type NP_055379 in the box and click OK.
Align NP_055379
Choose Algorithms / BLAST/PSSM and click anywhere on the pair of sequences.
If there are red-shaded regions, try the Block Aligner. Otherwise, skip the next two steps.
Choose Algorithms / Block Align Single and click on the sequences.
In the dialog, uncheck Global Alignment, then click OK.
Choose Alignments / Merge All to merge your new alignment.
Close the Imports window.
Find the SNP position in the alignment
Find the SNP position in NP_055379 (the bottom row) in the alignment window by moving your
mouse across the sequence and monitoring the location in the lower left corner of the
alignment window.
In the alignment window, choose Mouse Mode / Select Columns.
Click on the alignment column at the SNP position.
Locate the SNP position in the structure
Locate the wild type residue in the structure. What metal binding site is it near?
Clear your highlighting by clicking in whitespace in the alignment window.
Discover what amino acids contact the metal ion by double-clicking the metal ion from any of the
structures and
searching by distance with a radius of 4 Angstroms. What residues are they?
Investigate the possible changes caused by the mutation
Using the Compare menus in the left bar of the page, compare the wild type (Tyr) and
mutant (Cys) residues using Text.
What are the significant differences between these residues?
Compare them using Graphics to assess the relative sizes and shapes of the side chains.
Find residues in contact with the SNP position
Return to Cn3D.
Again select the column at the SNP position as you did before.
In the structure window, choose Show/Hide / Select by Distance / Residues Only.
Set the radius to 3.0 Angstroms.
Predict the consequences of the SNP
Are any of the residues that contact the ligand within 3.0 Angstroms of the SNP position?
Knowing what the mutant residue is, what additional interactions might occur in the mutant
protein? What might be the biochemical and biological consequences of these interactions?
