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  11 <td ALIGN=LEFT VALIGN=TOP WIDTH=280><br><h2>Getting started - Protein unfolding</h2>
  12 <font size=-1><A HREF="../online.html">Main Table of Contents</A></font><br><br></td>
  13 </TABLE></TD><TD WIDTH="*" ALIGN=RIGHT VALIGN=BOTTOM><p><B>VERSION 4.0<br>
  14 Sun 18 Jan 2009</B></td></tr></TABLE>
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  16
  17 <h3>Protein unfolding</h3>
  18 <p>
  19 In this exercise we will study a protein unfolding simulation that was
  20 done before. The protein is the C-terminal fragment of the L7/L12
  21 ribosomal protein (see below). It consists of 68 residues, and is
  22 known to be quite stable (in simulations). It is dissolved in a box
  23 filled with 3777 water molecules a structural Sulfate ion and four
  24 Sodium ions. A simulation was performed for 10 ns at 400 K.  The
  25 trajectory and other relevant files can be found in
  26 <TT>~david/ctf</tt>.
  27 </p>
  28 <table>
  29   <tr>
  30     <td><img src="../images/1ctf-0.jpg" border=0></td>
  31     <td><img src="../images/1ctf-0.2.jpg" border=0></td>
  32   </tr>
  33   <tr>
  34      <td align="center">Native structure</td>
  35      <td align="center">200 ps</td>
  36   </tr>
  37   <tr>
  38     <td><img src="../images/1ctf-0.5.jpg" border=0></td>
  39     <td><img src="../images/1ctf-1.jpg" border=0></td>
  40   </tr>
  41   <tr>
  42      <td align="center">500 ps</td>
  43      <td align="center">1 ns</td>
  44   </tr>
  45   <tr>
  46     <td><img src="../images/1ctf-4.jpg" border=0></td>
  47     <td><img src="../images/1ctf-10.jpg" border=0></td>
  48   </tr>
  49   <tr>
  50      <td align="center">4 ns</td>
  51      <td align="center">10 ns</td>
  52   </tr>
  53 </table>
  54
  55 <br><hr><br>
  56 <P><H3><A NAME="analysis">Analysis</A></H3>
  57 <oL>
  58 <li><p>
  59 Start by making a new working directory, and then move there.
  60 <br><br>
  61 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
  62 <tr NOSAVE>
  63 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
  64 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
  65
  66   <tt> cd ~/tutor</tt>
  67 </td>
  68 </tr>
  69 <tr NOSAVE>
  70 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
  71 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
  72   <tt> mkdir unfold</tt>
  73 </td>
  74 </tr>
  75 <tr NOSAVE>
  76 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
  77 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
  78
  79   <tt> cd unfold </tt>
  80 </td>
  81 </tr>
  82 </table>
  83 <br>
  84
  85 </p>
  86 </li>
  87
  88 <LI><p> View the trajectory on your own X-screen (program
  89 <a href="ngmx.html">ngmx</a>).
  90 <br><br>
  91 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
  92 <tr NOSAVE>
  93 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
  94 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
  95
  96 <tt> ngmx -s ~david/ctf/unfold.tpr -f ~david/ctf/unfold.xtc
  97 </tt>
  98 <td></td>
  99 </tr>
 100 </table>
 101 <br>
 102 <i>Hint 1: In the filter it may be advantageous to select Mainchain
 103 rather than Protein.<br>
 104 Hint 2: Go to the display menu and select options. Then set skip frames
 105 to 9 before you start the animation.</i>
 106
 107 <font color="red">What happens to the protein?</font>
 108 </p>
 109 </li>
 110
 111 <li><p>The Root Mean Square Deviation (RMSD) with respect to the crystal
 112 structure (program
 113 <a href="g_rms.html">g_rms</a>) is a measure of how well the
 114 crystal (starting) structure is maintained in the simulation.
 115 <br><br>
 116 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 117 <tr NOSAVE>
 118 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 119 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 120
 121 <tt> g_rms -s ~david/ctf/unfold -f ~david/ctf/unfold -o rmsd
 122 </tt>
 123 <td></td>
 124 </tr>
 125 </table>
 126 <br>
 127 Select the 1 for the number of groups, and select C-alpha (group 3) for fitting
 128 and for computing the RMSD. View the output graph with xmgrace.
 129 <br><br>
 130 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 131 <tr NOSAVE>
 132 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 133 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 134
 135 <tt> xmgrace rmsd.xvg
 136 </tt>
 137 <td></td>
 138 </tr>
 139 </table>
 140 <br>
 141 <font color="red">Does the RMSD
 142 converge within the simulation? If not, what does this indicate?</font>
 143 </p>
 144
 145 <LI><p>The Radius of Gyration (Rg, program
 146 <a href="g_gyrate.html">g_gyrate</a>)) is a measure of the size of the
 147 protein.
 148 <br><br>
 149 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 150 <tr NOSAVE>
 151 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 152 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 153
 154 <tt> g_gyrate -p -s ~david/ctf/unfold -f ~david/ctf/unfold -o gyrate
 155 </tt>
 156 <td></td>
 157 </tr>
 158 </table>
 159 <br>
 160 Select protein when asked. View the graph with xmgrace:
 161 <br><br>
 162 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 163 <tr NOSAVE>
 164 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 165 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 166
 167 <tt> xmgrace -nxy gyrate.xvg
 168 </tt>
 169 <td></td>
 170 </tr>
 171 </table>
 172 <br>
 173 <font color="red">Does the radius of gyration change during the
 174 simulation?</font> The x, y, and z components indicate the
 175 overall shape of the molecule (like the axes of an ellipsoid).
 176 i.e. if they are all equal,
 177 the molecule has spherical shape, if one is much long than
 178 the other two, the molecule is elongated.
 179 <font color="red">Based on this graph and the animation
 180 does the protein change shape?</font>
 181 </P>
 182
 183 <LI><p>The Ramachandran Plot shows whether the backbone torsion angles
 184 (&phi;/&psi;) of your
 185 peptide are within the allowed region.
 186 (program <a href="g_rama.html">g_rama</a>).
 187 We will compare the start structure and the final structure by running
 188 the program twice.
 189 <br><br>
 190 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 191 <tr NOSAVE>
 192 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 193 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 194
 195 <tt> g_rama -s ~david/ctf/unfold -f ~david/ctf/unfold -o rama-start -e 1
 196 </tt>
 197 <td></td>
 198 </tr>
 199 <tr NOSAVE>
 200 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 201 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 202
 203 <tt> g_rama -s ~david/ctf/unfold -f ~david/ctf/unfold -o rama-end -b 9999
 204 </tt>
 205 <td></td>
 206 </tr>
 207 </table>
 208 <br>
 209 View the graphs with xmgrace:
 210 <br><br>
 211 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 212 <tr NOSAVE>
 213 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 214 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 215
 216 <tt> xmgrace rama-start.xvg rama-end.xvg -legend load
 217 </tt>
 218 <td></td>
 219 </tr>
 220 </table>
 221 <br>
 222 In black we have the backbone angles from
 223 the starting structure, in red those from the final structure.
 224 <i>Hint 3: click on the red graph, and a dialog box will plop up.
 225 Select linetype none for the second graph, and select a circle as a symbol.</i>
 226 <font color="red">Are all the angles in the allowed region?
 227 What kind of structures do the angles indicate in the folded respectively unfolded conformation?</font>
 228 </P>
 229
 230 <li><p>
 231 Now we will analyse the number of hydrogen bonds the protein makes.
 232 First with itself, then with the solvent.
 233 <br><br>
 234 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 235 <tr NOSAVE>
 236 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 237 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 238
 239 <tt> g_hbond -s ~david/ctf/unfold -f ~david/ctf/unfold -num hbnum-pp
 240 </tt>
 241 <td></td>
 242 </tr>
 243 </table>
 244 <br>
 245 Select protein as the first group and second group. Then redo the
 246 analysis for protein with solvent (change the output file name to
 247 hbnum-ps, and select first the protein, and then solvent). <br>
 248 View the output file:
 249 <br><br>
 250 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 251 <tr NOSAVE>
 252 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 253 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 254
 255 <tt> xmgrace hbnum-pp.xvg hbnum-ps.xvg
 256 </tt>
 257 <td></td>
 258 </tr>
 259 </table>
 260 <br>
 261 <font color="red">Does the number of hydrogen bonds change for either of these?
 262 </font>
 263
 264 </p>
 265 </li>
 266
 267 <li><p>
 268 Here we will analyse the solvent accessible surface area of the protein.
 269 We will be looking at both hydrophobic surface area and hydrophilic surface
 270 area.
 271 <br><br>
 272 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 273 <tr NOSAVE>
 274 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 275 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 276
 277 <tt> g_sas -s ~david/ctf/unfold -f ~david/ctf/unfold -n ~david/ctf/index -skip 25
 278 </tt>
 279 <td></td>
 280 </tr>
 281 </table>
 282 <br>
 283 (Select protein again). View the output file:
 284 <br><br>
 285 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 286 <tr NOSAVE>
 287 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 288 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 289
 290 <tt> xmgrace -nxy area.xvg
 291 </tt>
 292 <td></td>
 293 </tr>
 294 </table>
 295 <br>
 296 <font color="red">How do the two components of the solvent accessible surface
 297 area change? How does the total change?</font>
 298
 299 </p>
 300 </li>
 301
 302 <LI> Secondary Structure analysis (program
 303 <a href="my_dssp.html">my_dssp</a>).
 304 This analysis uses the dssp (dictionary of secondary structure in proteins,
 305 <A HREF=#kabsch83>Kabsch & Sander, 1983</A>)  software.
 306 <br><br>
 307 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 308 <tr NOSAVE>
 309 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 310 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 311
 312 <tt> my_dssp -s ~david/ctf/unfold -f ~david/ctf/unfold -dt 50
 313 </tt>
 314 <td></td>
 315 </tr>
 316 </table>
 317 <br>
 318 Select protein when asked to select a group.
 319 You can postprecess the output file with:
 320 <br><br>
 321 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 322 <tr NOSAVE>
 323 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 324 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 325
 326 <tt> xpm2ps -f ss.xpm -o ss.eps
 327 </tt>
 328 <td></td>
 329 </tr>
 330 </table>
 331 <br>
 332 This will give you a postscript file which you can either print or
 333 view with xpsview.
 334 <br><br>
 335 <table BORDER=0 CELLSPACING=0 CELLPADDING=8 COLS=3 WIDTH="100%" NOSAVE >
 336 <tr NOSAVE>
 337 <td WIDTH="2%" NOSAVE><font color="#000000"></font></td>
 338 <td WIDTH="80%" BGCOLOR="#000066" NOSAVE><font color="#FFFFFF">
 339
 340 <tt> xpsview ss.eps
 341 </tt>
 342 <td></td>
 343 </tr>
 344 </table>
 345 <br>
 346 <font color="red">What happens to the Alpha helix (in blue)? What happens to the Beta sheets? Which secondary structure element is more stable?</font>
 347
 348 </p>
 349 </li>
 350 <li><p> <font color="red">Give a summary of what happens during the
 351 unfolding process.  What happens first to the structure? How do the
 352 structure and shape of the protein develop? Try to formulate relevant
 353 conclusions for the protein folding problem based on this simulation.
 354 </font>
 355 </p></li>
 356
 357 </ol>
 358
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