Mapping HSQC Chemical Shift Changes To Protein Structures

2012-02-15T22:09:25Z

Shulei:

==Mapping HSQC Chemical Shift Changes To Protein Structures==

'''NOTE:''' You must first have your chemical shift assignments completed and a full structure or homology model before running this.

'''NOTE:''' For some homology models it will take more massaging of the “mapping_chem_shifts.awk” file for this to work; if there are any questions ask for help.

1.Open Linux or IRIX running environment

2.Move structure or homology model file ('''model.pdb''') and “mapping_chem_shifts.awk” file into the same directory

3.Identify the chemical shift changes in your HSQC ('''NOTE:''' it helps to sort by residue number)

4.Open with vi editor the “mapping_chem_shifts.awk” file

5.In the “mapping_chem_shifts.awk” file you will see a list of if statements (see example). These are saying if the residue number in the pdb file matches the residue changing in the HSQC; insert a 10 to the 11<sup> th </sup> column. For most .pdb files the residue number is in the 6<sup> th </sup> column of the file. Append “mapping_chem_shifts.awk” so that all residues which correspond to a change in chemical shift ($6==37) add “10” to this 11th column or in other words residue 37 changed in the HSQC so it get a 10.

6.The VMD Explorer reads the numbering scheme as gradient of changes (10=change, 0=no change, or –10=opposite change). Therefore, there must be a -10 somewhere in the .pdb file. Find a residue distant from the bulk of changes and add the -10 to this residue ($6==120).

Example: Modifying mapping_chem_shifts.awk

if ($6==37) $11 =10.0

if ($6==45) $11 =10.0

if ($6==46) $11 =10.0

if ($6==66) $11 =10.0

if ($6==120)$11 =-10.00

7.After making the necessary modifications save and exit the awk script

8.Run “mapping_chem_shifts.awk” (awk –f mapping_chem_shifts.awk model.pdb >> '''out.pdb''') ('''NOTE:''' this will run the awk script on your structure file and output a new pdb file.)

9.Open the newly created pdb file ('''out.pdb''') with VMD Explore ('''NOTE:''' ask how to run VMD if you need help)

10.Generate a surface model or MS/MS model for the file and color by occupancy. For all residues labeled with a “10” in the new .pdb file (out.pdb) the color will change to blue. The blue regions of the protein indicate where the changes in the HSQC have taken place. ('''A'''=root protein structure '''B'''=mapped changes in HSQC mapped to protein surface blue=10 red=-10)

[[Image:protocol mapping chemical shift changes to protein structures.png]]

[[Category:Protocols]]

UV-Vis tutorial

2012-02-15T21:20:01Z

Shulei:

==UV-Vis tutorial==
#Turn on all hardware, Computer, UV-Vis and Printer. The switch for UV is on the back left side, feel around for it you’ll find it. The printer is on the front right side, again feel around you’ll find it.
#After the CPU is done warming up there will be a DOS prompt “C:/” and it might be flashing. TYPE UV and PRESS ENTER.
#After you press enter, a blue screen will pop up and have a list of things you can do. I have been using GENERAL SCAN, the first thing on the list. PRESS ENTER to start
#Once you are working in the GENERAL SCAN software, you want to use the F_ keys, the program calls them softkeys but I call them F_ keys.
#Press F4 to set up your acquisition parameters and use the U/D arrows keys to highlight the parameter you want to change and hit ENTER. I have only changed the scanning range (274-284nm for HSA) and have left everything else alone. When finished use ESC key to exit
#To start a run place blank in cuvette holder, shut lid and hit the F2 key (measure blank). This will measure your blank sample and bring you to a new “window” with your spectrum displayed and new F_key possibilities. It is VERY IMPORTANT to note that this instrument does not save your blank. Make sure after each blank follow #7&8
#To rescale your spectrum hit F3. This will walk you through the X and Y ranges you want to look at.
#After you have rescaled mark interesting peaks by hitting F2 (cursor). Use L/R arrow keys to place the little arrow on the peak you want then hit F1 (mark).
#To exit the cursor mode hit F10. This will only bring you back to the measure blank “window” from step 6.
#Place sample in instrument and hit F1 to measure sample. You can follow steps 8-9 again to find interesting peaks
#To print, hit F9 (hardcopy) while you are still in the measure window.
#Hit F10 to exit back to main window and then exit program when finished
[[Category:Protocols]]

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Mapping HSQC Chemical Shift Changes To Protein Structures

UV-Vis tutorial