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Editor: damberger
Time: 2014/11/07 21:48:06 GMT+1 |
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Note: |
changed: - 3. Project !ProteinA: Determine the maximum ChainId number in the Sequence and the maximum SpinId in the SpinList. - 3. Project !ProteinA: Use the SequenceExplorer to determine the "maximum residue number":MaximumResidueNumber in the Sequence and the maximum SpinId in the SpinList.
Determining the structures of complexes or oligomers requires some additional procedures. Typically isotope-labeling of individual components helps to simplify the problem. The project is therefore divided into subprojects where only the resonance assignments of the isotope-labeled component are determined. Later the assignments of the individual components must be merged so that the NOEs between components can be analysed using experiments which use isotope-filtering and -editing to obtain only intermolecular NOEs.
CARA provides support for this analysis by allowing the definition of samples with different isotope-labeling patterns Moreover, "SpinLinks:SpinLink which represent NOEs? between spins can be defined to only appear in a subset of NOESY spectra. Via the SpinLink? visibility the subset of NOEs? visible in isotope-filtered/edited experiments can be represented. (See release notes for how to do this, further information is forthcoming on the wiki).
Here is a step-by-step procedure for determining the structure of a complex between two molecules A and B (or a heterodimer, or homodimer).
- Project ProteinA: assign protein A in a project using samples with labeled protein A and unlabeled protein B. In this project you will only have the sequence of protein A. You assign in the usual way for a single protein project.
- Project ProteinB: assign protein B in a different project using samples with unlabeled protein A and labeled protein B. In this project you will only have the sequence of protein B. You assign in the usual way for a single protein project.
- Project ProteinA: Use the SequenceExplorer? to determine the "maximum residue number":MaximumResidueNumber? in the Sequence and the maximum SpinId? in the SpinList?.
- Project ProteinB (note for a homodimer, create a copy of ProjectA? naming it ProjectB?. This can be used for chain B of the homodimer. Otherwise follow the steps below)
- SequenceTable?: renumber Chain of protein B starting with a number that is higher than the highest Chain number of protein A in project A. Right-click on the first residue of protein B and select "renumber from here" entering the starting ChainId? number. (e.g. 200).
- Load the version 11 or later of WriteAssignments?.lua into the repository containing Project B.
- Run version 11 of WriteAssignments?.lua
- Select Project ProteinB
- checkbox 'number by ChainId?'
- Enter a number in the "Add offset to SpinId?" field that is larger than the maximum SpinId? determined in step 4 (e.g. 5000)
- Select format CYANA.
- Click OK (this writes out a sequence file starting at the ChainNr? of the first residue of protein B, and a chemical shift list .prot where each spin has an Id in the output file which is +5000 compared to the Id it has in Project B. We do this so that the sequence and chemical shift list can be merged with that of project A.
- Duplicate project ProteinA (renaming it something like ProteinAandB?).
- Project ProteinAandB: Merge the two projects:
- In the projects SequenceExplorer? assign the sequence of protein A the chain identifier A (right-click "set chain" and enter "A".
- Append the Sequence of protein B: "right-click Append Chain, select the sequence file of protein B generated in step 5, and assign the ChainId? "B".
- read in the chemical shift list of protein B: right-click on the project Inter_A_lab_B_unlab and "Import-Atom List" selecting the option AtomList? references "Residues"
- Step 7 appends the sequence of protein B with its assigned chemical shifts to protein A. Next you can define two samples with either residues of protein A or residues of protein B 12C,14N-labeled.
- Now load the filtered-edited NOESY spectra with either protein A or protein B 13C,15N-labeled and assign the corresponding samples to these spectra.