3. Open the electron density map using “File> Auto Open
MTZ. . . .” from the COOT menu bar, and select the appropriate MTZ file. Use “HID> ScrollWheel> Attach scroll wheel to
which map? > 1. . . .FWT PHWT . . .” to change the direct
electron density map root-mean-square deviation (rmsd) to
1.0 using the mouse scroll wheel (the rmsd contour level is
displayed in the top of the graphics window). Use “HID>
ScrollWheel> Attach scroll wheel to which map? > 2. . . .
DELFWT PHDELWT” to change the difference electron density map rmsd to 3.0 using the mouse scroll wheel.
4. Click on the “Calculate” tab in COOT and select “RCrane
launch.” An “RCrane” menu appears next to the “Extensions”
menu in COOT.
5. Press [CNTRL] + left mouse button to manually move the
pointer in COOT to the position of a phosphate.
6. Select “New Trace 5
0
!3
0
. . .” from the “RCrane” menu to start
an RNA trace.
7. Click “Accept” to accept the phosphate position or select an
alternative predicted starting phosphate position using the
“Next,” “Previous,” or “Manually Adjust” buttons. The starting phosphate position is depicted by an orange cube and
represents the closest predicted phosphate to the pointer position in COOT. An RCrane window will appear for tracing the
nucleotide chain (see Fig. 6a).
8. Select the appropriate base type—adenine (A), guanine (G),
cytosine (C), or uracil (U)—from the drop-down list in the
RCrane window. Click “Accept Nt” to accept the nucleotide
position or use the “Next Phos,” “Previous Phos,” or “Manually Adjust” buttons to cycle through alternative positionings
for the nucleotide. Click “Switch” to reverse the tracing, i.e.,
3
0
!5
0 , if the chain direction is wrong (see Fig. 6b).
9. Repeat step 8 to trace additional nucleotides in the chain (see
Fig. 6c).
10. Click “Build Backbone” to calculate the backbone atomic
coordinates for the traced nucleotides. During minimization,
the bases and phosphates are allowed to move slightly while
imposing torsion and sugar pucker restraints. An “Overview”
popup window will appear with a ranked list of the most likely
conformers (see Fig. 6d). Typically, the highest ranking
conformer is correct. Conformer suites highlighted in orange
or red indicate that there is a problem with the nucleotide
conformation.
11. Use “Previous Conformer” or “Next Conformer” to calculate
backbone coordinates for alternate conformers, which can be
440
Andrew P. Turnbull and Xiaoqiu Wu
MTZ. . . .” from the COOT menu bar, and select the appropriate MTZ file. Use “HID> ScrollWheel> Attach scroll wheel to
which map? > 1. . . .FWT PHWT . . .” to change the direct
electron density map root-mean-square deviation (rmsd) to
1.0 using the mouse scroll wheel (the rmsd contour level is
displayed in the top of the graphics window). Use “HID>
ScrollWheel> Attach scroll wheel to which map? > 2. . . .
DELFWT PHDELWT” to change the difference electron density map rmsd to 3.0 using the mouse scroll wheel.
4. Click on the “Calculate” tab in COOT and select “RCrane
launch.” An “RCrane” menu appears next to the “Extensions”
menu in COOT.
5. Press [CNTRL] + left mouse button to manually move the
pointer in COOT to the position of a phosphate.
6. Select “New Trace 5
0
!3
0
. . .” from the “RCrane” menu to start
an RNA trace.
7. Click “Accept” to accept the phosphate position or select an
alternative predicted starting phosphate position using the
“Next,” “Previous,” or “Manually Adjust” buttons. The starting phosphate position is depicted by an orange cube and
represents the closest predicted phosphate to the pointer position in COOT. An RCrane window will appear for tracing the
nucleotide chain (see Fig. 6a).
8. Select the appropriate base type—adenine (A), guanine (G),
cytosine (C), or uracil (U)—from the drop-down list in the
RCrane window. Click “Accept Nt” to accept the nucleotide
position or use the “Next Phos,” “Previous Phos,” or “Manually Adjust” buttons to cycle through alternative positionings
for the nucleotide. Click “Switch” to reverse the tracing, i.e.,
3
0
!5
0 , if the chain direction is wrong (see Fig. 6b).
9. Repeat step 8 to trace additional nucleotides in the chain (see
Fig. 6c).
10. Click “Build Backbone” to calculate the backbone atomic
coordinates for the traced nucleotides. During minimization,
the bases and phosphates are allowed to move slightly while
imposing torsion and sugar pucker restraints. An “Overview”
popup window will appear with a ranked list of the most likely
conformers (see Fig. 6d). Typically, the highest ranking
conformer is correct. Conformer suites highlighted in orange
or red indicate that there is a problem with the nucleotide
conformation.
11. Use “Previous Conformer” or “Next Conformer” to calculate
backbone coordinates for alternate conformers, which can be
440
Andrew P. Turnbull and Xiaoqiu Wu