Basic NMR experiments for assignment of proteins

2D Heteronuclear Experiments

• 2D ¹⁵N-¹H HSQC with gradient selection and sensitivity enhancement
  




• Pulse programme: gNhsqc/gNhsqcA
  
• click here for image 
• click here for nmrpipe script
  

• 2D ¹⁵N-¹H HSQC "fast" hsqc with gradient selection
  




• Pulse programme: gNfhsqc/gNfhsqcA
  
• click here for image 
• click here for nmrpipe script

• 2D 13C-1H HSQC 

• Pulse programme gChsqcA
  
• click here for image 
• click here for nmrpipe script
  

• 2D 13C-1H HSQC Constant time

• click here for image 

3D Triple Resonance Experiments

• HNCACB
  


• Set up parameters using ghn_cacb/ghn_cacbA
  
• Correlates Ca(i-1), Cb(i-1), with Ca(i), Cb(i) for NH resonance (i)
• i-1 are negative, i are positive.
  
• 4 scans on 1 mM sample (78 residues)
  
• click here for  ¹H-¹³C projection
• click here for ¹H-¹⁵N projection
• nmrPipe script
  

• CBCA(CO)NH
• Set up parameters using gcbca_co_nh/gcbca_co_nhA
  
• Correlates Ca(i-1) and Cb(i-1) for NH resonance (i)
• click here for ¹H-¹³C projection
• click here for ¹H-¹⁵N projection
• nmrPipe script (note - the processing shows how to LP a corrupted point)
  

• HBHA(CBCACO)NH - ghbha_co_nh
  
• Set up parameters using ghbha_co_nh
  
• Similar to CBCA(CO)NH. Shows Ha(i-1) and Hb(i-1) of NH (i)
• Very useful for connecting backbone and side chain information
• note: Halpha and Hbeta have different sign
• 2 scans on 1 mM sample (78 residues)
• click here for first ¹HN-HAHB projection
• click here for ¹HN-¹⁵N projection
• click here for nmrPipe script
  

• The next two experiments are invaluable for assigning side chains, and so easy to analyse.
• However, substantial heating is associated with the Spin-lock sequences and can fry the cryoprobe!
• Do not set up unless you are certain you know what you are doing.
  

• C(CCO)NH-TOCSY
• Set up parameters found at:
• ozzy chab 7 1
  
• Shows 13C side chains past alpha and beta of residue NH(i-1)
• click here for first 1H-13C projection. NOTE the many side chain resonances

• H(CCO)NH-TOCSY
• Set up parameters found at:
• ozzy chab 6 1
  
• Shows 1H side chains of residue NH(i-1). Preferable to  3D 15N edited TOCSY for larger protein

Note: for a protein of < 15kDa all these experiments should give reasonable spectra. These 5 spectra represent the minimum number of experiments (~ 2-3 days) required to yield complete assignments (except aromatic ring atoms)

NMR experiments for dipolar information

• 3D Simultaneous (13C/15N) NOESY
• Click here for first plane (13C/15N)
• Click here for nmrpipe processing script
  

4D experiments

•   4D  13C-15N-HMQC-NOESY-HSQC
• Set up parameters found at 
• ozzy 4dyggx 10 1  
• nmrPipe conversion script
• nmrPipe processing script (using more than one CPU - in this case 4) 
• How to analyse using NMRView scripts
  

1H-15N Relaxation Experiments

•     Heteronuclear NOE
• notes for setup and processing
• click here for nmrPipe processing script
  

NMRView info

• A powerpoint presentation for beginners
• The official website
  
• Script for updating a xpk file based on chemical shifts
• Scripts for analysing dipolar couplings
• Scripts for analysis of HNHA experiments
  
• Awk script for converting NMRView shifts to BMRB. Also allows you to add/subtract constant values for incorrect referencing!
• 4D analysis scripts
  

Other tips

       Temp calibration

        DSS standard