SanDisk Membrain Software Manual do Utilizador

University of Illinois at Urbana-ChampaignBeckman Institute for Advanced Science and TechnologyTheoretical and Computational Biophysics GroupComputati

1 BUILDING A STRUCTURAL MODEL OF KCSA 10cd "< your home directory >/mem-tutorial-files/01-BUILD/"mol new 1K4C.pdbset all [atomselect t

1 BUILDING A STRUCTURAL MODEL OF KCSA 115 Finally, repeat the same procedure to create segment D by typing thefollowing commands:mol delete topmol new

1 BUILDING A STRUCTURAL MODEL OF KCSA 12Figure 3: Top and side views of the KcsA tetramer with an antibo dy complex(1K4C.pdb).In VMD, you should be ab

1 BUILDING A STRUCTURAL MODEL OF KCSA 13step using VMD and psfgen. The first five steps are included in the scriptprepfiles.tcl.1 Be sure that KCSA-ALL.

1 BUILDING A STRUCTURAL MODEL OF KCSA 14set npot [atomselect top "name K and resid 3002 3004 3007"]$npot set name OH2$npot set resname TIP3$

1 BUILDING A STRUCTURAL MODEL OF KCSA 15Selection Drawing Method Coloring Methodprotein and chain C NewCartoon ColorID 6chain C and resname CYS VDW Na

1 BUILDING A STRUCTURAL MODEL OF KCSA 16visualized for residue His25 (Fig. 4 A). Thus we will mo del both as HSE residues.pH- and voltage- dependent g

1 BUILDING A STRUCTURAL MODEL OF KCSA 17crystwatD.pdb. We will use all these files to generate the corresponding PSFand PDB files with VMD, psfgen and t

1 BUILDING A STRUCTURAL MODEL OF KCSA 18filter, and potassium ions.15 Execute the script by typing in a Unix terminal vmd -dispdev text -ebuildpsf.tcl

1 BUILDING A STRUCTURAL MODEL OF KCSA 19Figure 5: Snapshot of KcsA’s selectivity filter obtained using files kcsav.psfand kcsav.pdb. Note the presence o

2A current version of this tutorial is available athttp://www.ks.uiuc.edu/Training/Tutorials/Join the [email protected] mailing list for addition

1 BUILDING A STRUCTURAL MODEL OF KCSA 203 Now we will combine the output PDB file solkcsa.pdb with the rest ofthe system. Do this by entering:vmd -disp

1 BUILDING A STRUCTURAL MODEL OF KCSA 215 Enter the following commands in the Tk Console window:mol new kcsav solv raw.psfmol addfile kcsav solv raw.p

1 BUILDING A STRUCTURAL MODEL OF KCSA 22Figure 6: Solvating exposed areas of a membrane protein. The left panel showsKcsA colored by residue type. An

1 BUILDING A STRUCTURAL MODEL OF KCSA 23mol delete allpackage require psfgenresetpsftopology ../top all27 prot lipid.rtfreadpsf kcsav solv raw.psfcoor

2 PLACING KCSA IN A MEMBRANE 242 Placing KcsA in a MembraneIn this second unit, you will learn how to take your solvated KcsA channel andplace it in a

2 PLACING KCSA IN A MEMBRANE 25Figure 7: Embedding Kcs A in a membrane patch. (A) POPC membrane patch.(B) KcsA protein and location of membrane headgr

2 PLACING KCSA IN A MEMBRANE 26Notice the use of the transaxis function. This is one of a few functionswhich generate transformation matrices of the t

2 PLACING KCSA IN A MEMBRANE 27Grubm¨uller’s Solvate program, we want to find good selection criteria in VMD,and then mark the molecules we want to del

2 PLACING KCSA IN A MEMBRANE 283 Use VMD to look at the selections created and try to find a better selectioncriteria so as to keep as much non-overlap

2 PLACING KCSA IN A MEMBRANE 29Figure 8: Top and side views of KcsA embedded in a membrane patch (ob-tained using files kcsa popc.psf and kcsa popc.pdb

CONTENTS 3Contents1 Building a Structural Model of KcsA 71.1 Downloading and Viewing the Protein . . . . . . . . . . . . . . . 71.2 Examining the PDB

2 PLACING KCSA IN A MEMBRANE 30image and the total number of atoms in your system: having a smallerwater box is a cheap way of making a smaller syste

2 PLACING KCSA IN A MEMBRANE 31Figure 9: Outcome of solvation. Red water molecules are located in the mem-brane re gion and must be deleted.mol delete

2 PLACING KCSA IN A MEMBRANE 32solv remove.tcl.Now we will use the Autoionize Plugin, which creates a specified ionic concen-tration of either KCl or N

2 PLACING KCSA IN A MEMBRANE 33Figure 10: Final simulation system including the KcsA channel, a POPC mem-brane, water, and ions.

3 RUNNING A SIMULATION OF KCSA 343 Running a Simulation of KcsAIn unit 3, you will use the model containing KcsA embedded in a membrane toperform mole

3 RUNNING A SIMULATION OF KCSA 35NBFIX. The standard parameters for ions have been optimized toreproduce bulk properties of ionic solutions, while the

3 RUNNING A SIMULATION OF KCSA 3610 Type in the Tk Console:set wat [atomselect top water]measure minmax $watThe output of measure minmax should now be

3 RUNNING A SIMULATION OF KCSA 37PMEGridSizeX 80PMEGridSizeY 80PMEGridSizeZ 90}Note how we carefully chose the Particle Mesh Ewald grid size so as to

3 RUNNING A SIMULATION OF KCSA 38mol new ../02-MEMBRANE/kcsa popcwi.psfmol addfile ../02-MEMBRANE/kcsa popcwi.pdbset all [atomselect top "all&quo

3 RUNNING A SIMULATION OF KCSA 39Warning. The configuration files provided in this tutorial are de-signed for users with limited computational resources

CONTENTS 4IntroductionThis tutorial is designed to guide users of VMD and NAMD through all thesteps required to set up a membrane protein system for m

3 RUNNING A SIMULATION OF KCSA 405 Check that the line “temperature $temperature” has been commentedout (with a #).Now that velocities (which determin

3 RUNNING A SIMULATION OF KCSA 41Pumping out water. The keep water out.tcl Tcl script auto-matically applies forces to water molecules entering the me

3 RUNNING A SIMULATION OF KCSA 42Figure 13: Achieving a good packing of lipids around the protein. Note thegaps in the membrane–protein interface (lef

3 RUNNING A SIMULATION OF KCSA 433.4 Production RunsNow that the protein has been equilibrated, we are ready to perform productionruns. There will be

CONTENTS 5Required programsThe following programs are required for this tutorial:• VMD: Available at http://www.ks.uiuc.edu/Research/vmd/ (for all pla

1 BUILDING A STRUCTURAL MODEL OF KCSA 61 Building a Structural Model of KcsAIn this unit you will build the KcsA tetramer solvated in water, learning

1 BUILDING A STRUCTURAL MODEL OF KCSA 7Figure 2: Molecules in file 1K4C.pdbNow that the file is loaded, you can explore it by trying out different select

1 BUILDING A STRUCTURAL MODEL OF KCSA 8Coordinates file. The file 1K4C.pdb corresponds to the X-ray struc-ture of KcsA refined at 2.0˚A resolution provid

1 BUILDING A STRUCTURAL MODEL OF KCSA 9REMARK 350 BIOMOLECULE: 1REMARK 350 APPLY THE FOLLOWING TO CHAINS: A, B, CREMARK 350 BIOMT1 1 1.000000 0.000000