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Protein sample production

  1. NESG target selection
  2. DNA cloning protocols
  3. Protein expression and purification protocols 
  4. Sample Optimization
    1. Construct optimization
    2. Buffer optimization
    3. Cofactor optimization
  5. Initial protein analysis
    1. SDS page gel
    2. Protein concentration measurements
    3. Assessment of Oligomerization Status
      1. gel-filtration and light scattering
      2. NMR determined Rotational correlation time
    4. Mass spectrum
    5. NMR screening
      1. 1H 1D screening
      2. Initial [15N,1H] HSQC

Resonance Assignment

  1. Principles and concepts
  2. Practical aspects
    1. Semi-automated protocols
      1. CARA
        1. Spin System Identification in 2D 15N-HSQC and 3D HNNCO
        2. Backbone Resonance Assignment
        3. Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN
        4. Side Chain Assignment
          1. Aliphatic side-chain assignment
          2. Aromatic side-chain assignment
          3. Amide side-chain assignment
      2. Sparky
      3. XEASY
        1. Spin system identification
        2. Backbone resonance assignment
          1. GFT-based spectra
            1. HNCACAB/CABCA(CO)NH
          2. Conventional spectra
            1. HNCACB/CBCA(CO)NH
            2. HNCA/HN(CO)CA
            3. HNCO/HN(CA)CO
            4. NOESY/TOCSY
        3. Side chain resonance assignment
          1. Aliphatic
            1. GFT NMR spectra
              1. (4,3)D GFT HABCAB(CO)NHN
              2. (4,3)D GFT HCCH
            2. Conventional spectra
              1. HAHB(CO)NH
              2. HCCH-COSY
              3. HCCH-TOCSY
              4. Simultaneous NOESY
              5. (H)CC-TOCSY-(CO)NH
              6. H(CC-TOCSY-CO)NH
          2. Aromatic
            1. GFT-based spectra
            2. Conventional spectra
          3. Other
            1. Trp e1 NH and d1 CH
            2. Met e CH3
            3. Asn d2 and Gln e2 NH2
          4. NOESY peak integration
    2. Automated protocols
      1. AutoAssign
      2. AutoAssign server
      3. ABACUS
      4. PINE server
    3. Validation of resonance assignment
      1. Assignment validation suite (AVS)
      2. Linear analysis of chemical shift (LACS)
    4. Depositing chemical shifts

Structure Calculation and Validation

  1. Principles and concepts
  2. Practical aspects
    1. Structure calculation
      1. CYANA
        1. Getting started
        2. FOUND
        3. TALOS
        4. GLOMSA
        5. NOE calibration
        6. Manual structure calculation
        7. Automated NOESY assignment and structure calculation
        8. Structure calculation with residual dipolar couplings (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
        9. Homodimer structure calculations
      2. AutoStructure
        1. Theory
        2. Getting started
        3. CYANA run
        4. XPLOR run
        5. Analyzing the output
        6. RPF/DP scores
        7. Structure calculation using AS-DP
      3. "Consensus" Approaches
        1. Overview of Consensus runs
        2. Finding Consensus NOE assignments
        3. Validation of Consensus runs
      4. CS-ROSETTA
      5. CS-DP ROSETTA
      6. RDC-ROSETTA
      7. RDC-assisted dimer structure calculation
      8. Special topics
        1. Protein-Ligand complexes
        2. Metal ions
        3. Dimers
        4. Residual Dipolar Couplings
        5. REDCAT and REDCRAFT
        6. Paramagnetic constraints
    2. Structure Refinement
      1. CNS refinement
      2. XPLOR-NIH refinement
      3. ROSETTA refinement
    3. Validation and deposition
      1. PdbStat
      2. PSVS
      3. RPF analysis
      4. MolProbity server
      5. PDB and BMRB deposition
      6. ADIT-NMR
      7. HarvestDB
      8. SPINS


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