Browsing the Protein Data Bank and Viewing 3-D structures

The goal of this tutorial is to learn how to download PDB files and manipulate them with the graphics program PYMOL.

PYMOL= Graphics Program to display and to show atomic molecular structures in three dimensions.

PDB = Protein Data Bank = Web Based Depository of 3Dimensional atomic data solved by CryoEM, X-ray crystallographyi and NMR.

We will use the GAL4 transcription activator structure for this demo.

Yeast protein GAL4 is a transcription activator for genes that encode galactose-metabolizing proteins. GAL4 binds to a 17bp DNA palindrome sequence as a homodimer. The 881-amino acid GAL4 protein contains various function for its different segments, including DNA binding residues (1-65), dimerization region (65-94 and weak dimerization 50-64) and three acidic activating regions (94-106, 148-196, 768-881) Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that binds DNA as a homodimer and can activate transcription by interacting with the mutant Gal11P protein. Although structures have been reported of the Gal4 dimerization domain and the binuclear cluster domain bound to DNA as a dimer, the structure of the "complete" Gal4 dimer bound to DNA has not previously been described. Here we report the structure of a complete Gal4 dimer bound to DNA and additional biochemical studies to address the molecular basis for Gal4 dimerization in DNA binding. We find that Gal4 dimerization on DNA is mediated by an intertwined helical bundle that deviates significantly from the solution NMR structure of the free dimerization domain. Associated biochemical studies show that the dimerization domain of Gal4 is important for DNA binding and protein thermostability. We also map the interaction surface of the Gal4 dimerization domain with Gal11P.

See attached PDF file DNA recognition by GAL4: structure of a protein-DNA complex Structural basis for dimerization in DNA recognition by Gal4. Hong M, Fitzgerald MX, Harper S, Luo C, Speicher DW, Marmorstein R. Structure. 2008 Jul;16(7):1019-26.

A) DOWNLOADING PDB FILES FROM THE NET.

Download the PDB file for GAL4 here GAL4 PDB FILE ,

The code 3COQ is also called the PDB accession number.

Using the mouse right hand button click "SAVE LINK AS" button, this will save the pdb file in your computer with the name 3coq.pdb. (NOTE: make a note of the directory in the local disk where the pdb file is saved)

B) BASIC PYMOL COMMANDS

Mouse actions:

Right Mouse Click = ZOOM (Move mouse up or down)

Left Mouse Click = ROTATION

Middle Mouse Click = Recenter on Atom

Start the PYMOL program and read the file you just downloaded from the PDB server. Go to the FILE menu and click on OPEN and find the file 3coq.pdb .

Open the PYMOL COMMAND LINE at the bottom of the screen.

Click on the atoms and watch the PYMOL COMMAND LINE window, and identify atoms at the begining of each protein and DNA chain.

1) Highlight the Protein in blue :

select PROTEIN, chain A+B

color blue , PROTEIN

show cartoon, PROTEIN

2) Highlight the DNA in red

select DNA, chain D+E

color red, DNA

show ribbon, DNA

3)Highlight the ZINC Metal Binding Site

select ZINC, resname ZN

show spheres, ZINC

color yellow, ZINC

set sphere_scale = 1.0

4) Highlight Metal Binding domain in green

select METALBINDING, resid 1:40 and chain A+B

color green , METALBINDING

show cartoon , METALBINDING

5) Highlight extender linker in cyan

select LINKER, resid 41:49 and chain A+B

color cyan, LINKER

show cartoon, LINKER

6) Highlight helical dimerization domain in red

select DIMERIZATION, resid 50:65 and chain A+B

show cartoon, DIMERIZATION

color red, DIMERIZATION

7) The Metal Binding Domain in detail

The metal binding domain is held together by two zinc ions , tetrahedrally coordinated by six cystines. Two of the cystines ligate both metals creating a binuclear cluster.

select CYSTEINES, resname CYS

show sticks, CYSTEINES

color yellow, CYSTEINES

8) The Dimerization Element in detail

Residues 50-65 form an ampipathic a-helix. The dimer interface is formed by the packing of the two parallel a-helices in a coiled-coil formation, similar to those found in the 'luecine zipper' group of transcription factors

select ZIPPER, residue 50:65 and resname VAL+LEU

color red, ZIPPER

9)To make a short movie to import in Powerpoint:

mset 1 x360

util.mroll(1,360,1)

10) Also you can make more complicated movements with the mdo command

mset 1

mdo 1: turn x,1; turn y,1;

mplay

Click on "Play" to play the movie and "Stop" to stop it. Click on the ">" and "<" buttons to move forward or backward a single frame.

Download the PYMOL script for GAL4 here GAL4 PYMOL SCRIPT ,

Download the PYMOL HOMEWORK here HOMEWORK ,