Edit detail for StructureCalculationOfComplexes revision 32 of 40
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Editor: damberger
Time: 2014/11/08 00:49:28 GMT+1
Note: 
changed:
-  a. SequenceTable: renumber Chain of protein B starting with a number that is +1 relative to the maximum residue number of protein A in project A. Right-click on the first residue of protein B and select "renumber from here" entering the starting ChainNr. (158 in the example shown here).
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-  b. Load the version 11 or later of "WriteAssignments.lua":CALUA into the repository containing Project B.
  - SequenceTable: renumber Chain of protein B starting with a number that is +1 relative to the maximum residue number of protein A in project A. Right-click on the first residue of protein B and select "renumber from here" entering the starting ChainNr. (158 in the example shown here).

  - Load the version 11 or later of "WriteAssignments.lua":CALUA into the repository containing Project B.

changed:
-  a. In the projects SequenceExplorer assign the sequence of protein A the chain identifier A (right-click "set chain" and enter "A").
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-  b. 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".
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-  c. read in the chemical shift list of protein B: right-click on the project !ProteinAandB and "Import-Atom List" selecting the option AtomList references "Residues"
  - 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 !ProteinAandB and "Import-Atom List" selecting the option AtomList references "Residues"

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? 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) Screenshots of procedure

  1. 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.
  2. 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.
  3. Project ProteinA: Use the SequenceExplorer? to determine the maximum residue number in the Sequence and the maximum SpinId? in the SpinList?.
  4. 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 +1 relative to the maximum residue number of protein A in project A. Right-click on the first residue of protein B and select "renumber from here" entering the starting ChainNr?. (158 in the example shown here).
    • Load the version 11 or later of WriteAssignments.lua into the repository containing Project B.
  5. Run version 11 or later 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.
    • Your WriteAssignment.lua dialog should look something like this
  6. Duplicate project ProteinA (renaming it something like ProteinAandB).
  7. 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 ProteinAandB and "Import-Atom List" selecting the option AtomList? references "Residues"
  8. 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.
  9. 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.