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Editor: damberger
Time: 2016/09/05 16:49:30 GMT+2 |
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Note: updated instructions |
changed: - 1. Complete the sequence-specific resonance assignments. Check your assignments using the LUA script "AssignmentReport.lua":CALUA. This will help you identify the following types of inconsistencies before starting structure calculation: 1. Complete the sequence-specific resonance assignments. Check your assignments using the LUA script "!AssignmentReport.lua":AllCaluaScripts. This will help you identify the following types of inconsistencies before starting structure calculation: changed: - 3. Generate .seq file and .prot files using the script "WriteAssignments.lua":CALUA. - if you have systematic differences in the chemical shifts of some classes of spins relative to others you can use "!ShiftSpinsInCatagory.lua":AllCaluaScripts to adjust them. - if you must calibrate (or recalibrate) spectra after the assignment process is completed use the script "!ShiftSpinsInCatagory.lua":AllCaluaScripts to shift spins in a given class as a group. a. The NOESY spectra used for structure calculation should be calibrated to DSS externally. I.e. if you use Topspin to process the NOESYs these spectra should have chemical shift scales which are properly referenced to DSS. When you load them into the CARA project with the assignments, the NOESY tower positions should agree with the positions of crosses CARA shows for these towers. If this is not the case, they should not be recalibrated inside CARA. Instead, the chemical shifts of the project should be adjusted so the crosses generated by CARA agree with the position of NOESY signals. Do this as follows: b. Determine the offset in position between actual signal position and cross in each dimension of the NOESY. (e.g. in the direct acquisition dimension of a 3D 15N NOESY the cross positions are systematically 0.1 ppm higher than the real signal position). c. Apply this correction to all 1H atoms using the script "!ShiftSpinsInCatagory.lua":AllCaluaScripts d. Afterwards run the script "!RecalibrateSpectra":AllCaluaScripts.lua to adjust the calibration values of all spectra in the project. e. Finally reset the cal values of the NOESY spectra used for the structure calculation to zero using the calibrate spectrum context menu in CARA's spectra-explorer. f. You will need to do this for each type of shift appearing in the NOESY spectra. (1H, 13C, 15N). 3. Generate .seq file and .prot files using the script "!WriteAssignments.lua":AllCaluaScripts. changed: - - Run the script: "WriteAssignments.lua":CALUA (Why use this script and not the menu item ""project-export atomlist"? See "FAQ":FAQ) - Run the script: "!WriteAssignments.lua":AllCaluaScripts (Why use this script and not the menu item ""project-export atomlist"? See "FAQ":FAQ) changed: - - *select oxidized or reduced for the CYS residues*: This can be determined from the CB shift. >35ppm is oxidized. !WriteAssignments.lua only allows you to select all reduced or all oxidized. If you have a mixture, you will need to edit the .seq file to reflect the correct redox states for each CYS. - *select oxidized or reduced for the CYS residues*: This can be determined from the CB shift. >35ppm is oxidized. "!WriteAssignments.lua":AllCaluaScripts only allows you to select all reduced or all oxidized. If you have a mixture, you will need to edit the .seq file to reflect the correct redox states for each CYS. changed: - 4. Check to be sure the covalent check gives a sufficiently good chemical shift agreement. Typical values are 80-90%. If your values are lower, try to improve the position of your NOE towers. Your chemical shift assignments should agree well with the NOESY spectra used in the structure calculation. 4. Check to be sure the covalent check of AtnosCandid gives a sufficiently good chemical shift agreement. Typical values are 80-90%. If your values are lower, it may mean there is disagreement between the chemical shifts in your project and the position of the corresponding resonances in the NOESY spectra. - Be sure that your calibrations were written to the spectrum file which you supplied to AtnosCandid. I.e. the when the NOESY spectra you supplied to AtnosCandid are loaded into the project without any calibration being applied inside CARA (cal values in Spectrum-Viewer are all zero) the position of the NOESY towers in the CARA project should agree with the crosses CARA displays. - If this is the case, try to improve the position of your NOE towers. Your chemical shift assignments should agree well with the NOESY spectra used in the structure calculation.
HB2
and HB
assigned in an AMX system like SER. Either delete the HB2
& HB3
leaving only HB
or delete HB
keeping only HB2
and HB3
. Note that a stereospecific assignment is indicated by an exclamation point in front of the label: !HB2
.a. select the corresponding 3D NOESY spectrum in the Strips-Select Spectrum" menu
b. select a system either by clicking on it in the projection (2D plane) or by typing the goto residue command "gr" and typing the residue number.
c. the noesy tower will appear in the strip. use the arrow keys to move the system position in the 2D projection so that the NOESY tower is centered well on the grey line in both strip views XZ and YZ.
d. when the position matches, type the "move spins" command "ms".
Note that if you have many large systematic shift differences between NOESY spectra, this indicates that the solution conditions or proteins may differ. It is probably better to make a fresh sample or use the same sample for all NOESY measurements to ensure that all are taken under identical conditions than to try to analyse inconsistent data.
a. The NOESY spectra used for structure calculation should be calibrated to DSS externally. I.e. if you use Topspin to process the NOESYs? these spectra should have chemical shift scales which are properly referenced to DSS. When you load them into the CARA project with the assignments, the NOESY tower positions should agree with the positions of crosses CARA shows for these towers. If this is not the case, they should not be recalibrated inside CARA. Instead, the chemical shifts of the project should be adjusted so the crosses generated by CARA agree with the position of NOESY signals. Do this as follows:
b. Determine the offset in position between actual signal position and cross in each dimension of the NOESY. (e.g. in the direct acquisition dimension of a 3D 15N NOESY the cross positions are systematically 0.1 ppm higher than the real signal position).
c. Apply this correction to all 1H atoms using the script ShiftSpinsInCatagory.lua
d. Afterwards run the script RecalibrateSpectra to adjust the calibration values of all spectra in the project.
e. Finally reset the cal values of the NOESY spectra used for the structure calculation to zero using the calibrate spectrum context menu in CARA's spectra-explorer.
f. You will need to do this for each type of shift appearing in the NOESY spectra. (1H, 13C, 15N).