Mike's NMR Page Group Wisdom
NMR assignment sequences/processing Relaxation Stuff Varian vnmr faq's Example NMRPipe scripts for Varian NMRview stuff non-linear sampling		Info on linux clusters Mike's PubMed Publications Mike's NMRPipe tutorial Scheduling jobs over muliple cpu's NMR flash animations  Some useful scripts
Protein Expression protocols: Minimal Media Notes on expression in E.coli (powerpoint presentation) NMR Manager: Mike Osborne

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Most common commands

Mikey's NMR Page        

Basic NMR experiments for assignment of proteins

Tried and tested for the 600 Inova spectrometer (as of May 21^st 2005)

2D Heteronuclear Experiments


3D Triple Resonance Experiments
(for relative sensitivity - click here)

3D Experiments Giving Dipole-Dipole/Chemical exchange information
including:
3D Time-Shared NOESY for optimal resolution


4D experiments

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Relaxation Experiments

Varian Setup

Heteronuclear NOE

For setup and analysis of T1 and T2 data from Varian

Click Here for 2D 1H-15N relaxation Experiments (bruker)

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NMRPipe Stuff

NMRPipe processing of Varian experiments

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Other Stuff


Mike's Translational diffusion analysis programme


Mike's scripts for converting NMRView formats to aria formats



Problems phasing in the indirect dimension?



Click for converting NMRPipe format to NMRView, SPARKY and XEASY


Click here for NMRVIEW stuff


Click here for pulse programmes

Other useful tips

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

For smaller proteins use coherence selection: for larger proteins you may lose sensitivity, in which case do not use coherence selection.

• HNCACB

• CBCA(CO)NH
• with example for dealing with bad points in a plane
  
• HNCO
• HN(CO)CA
• HBHA(CBCACO)NH
  
• HCCO(TOCSY)NH
  
• CC(CO)TOCSYNH
  
• 
  

• HNCACB

• Bruker

• Correlates Ca(i-1), Cb(i-1), with Ca(i), Cb(i) for NH resonance (i)

• i-1 are negative, i are positive (by convention)

• click here for a ¹H-¹³C projection
• click here for NMRPipe processing script
  

• 
  
• Varian

  • click here for  ¹H-¹³C projection

  • click here for ¹H-¹⁵N projection

  • nmrPipe script

• CBCA(CO)NH

• Bruker
  

  • Correlates Ca(i-1) and Cb(i-1) for NH resonance (i)

  • Click here for 1H-13C projection
  • Click here for NMRPipe processing

  varian
  

  • click here for ¹H-¹³C projection

  • click here for ¹H-¹⁵N projection

  • nmrPipe script (note - the processing shows how to LP a corrupted point)

  
  

• HNCO
• Bruker
• correlates CO(i-1) to NH(i)
  
• click here for 1H-13C projection
• click here for processing script
  

• HN(CA)CO
  

• Bruker   
  

• correlates CO(i, i-1) to NH(i)
• click here for projection
  
• click here for processing script
  

• HBHA(CBCACO)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

• Bruker   
  

• click here for 1H-1H plane
• click here for processing script
• For set up make sw to ~ 6PPM
  
• 
  
• Varian
  

• (note uses o1 jumping during t2)
  

• • 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 cause sibstantial heating

• The Bruker coldprobes are pretty tolrent but do not set up unless you are certain you know what you are doing.

• C(CCO)NH-TOCSY - gc_co_nh

• Bruker

• 1H-13C plane
• processing script
  

• • Shows 13C side chains past alpha and beta of residue NH(i-1)

• H(CCO)NH-TOCSY

• Bruker

• Use SW(1H) of ~ 8ppm
  
• 1H-1H plane
• procsessing script
  
• • 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)

A number of other experiments need to be optimised. Ask Mike if you have a particular experiment you wish to run

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NMR experiments for dipolar information

• 3D Simultaneous (13C/15N) NOESY for ILV proteins (see paper here)  
  

• 
  
• You get get 2 very high resolution 3D spectra (noesy peaks are in direct dimension)
  
• 1. 15N separated NOESY, which will give HN - HN and HN - Methyl cross peaks
• 2. 13C separated NOESY, which will give Methyl - Methyl and Methyl- HN cross peak

•  Ideally for ILV and 12C backbone labelling.
  

•  You can use 13C uniformly labelled ILV BUT need to check resolution in 13C/15N less the the C-N coupling constant
  

• As the NOE is in the direct dimension you get separation of peaks normally overlapping. You can also implement NUS schemes to improve resolution in the other dimsnsion
  

• You get get 2 very high resolution 3D spectra (noesy peaks are in direct dimension)
  
• 1. 15N separated NOESY, which will give HN - HN and HN - Methyl cross peaks
• 2. 13C separated NOESY, which will give Methyl - Methyl and Methyl- HN cross peaks
• 
  
• SETUP (not straightforward)
  
• Use Poisongap  scheduler to generate a sampling schedule (can use the web page)
  
• Paper used 10% of XX by XX
• Here is an example of how to setup  20% of a 50 (13C/15N) x 120 (1H) complex point spread (1200 out of a total of 6000) using the web page.
• Save and copy the file (e.g. sched) to /opt/topspin3.5.plXX/exp/stan/nmr/lists/vc
• In eda make sure vclist is set to this. Also set nuslist to the same.
• 
  

• 
  
• 
  
• 
  
• Process with any NUS software: probably istHMS best espscially if use lower sampling schedules
  

• • Click here for first plane (13C/15N)

  • Click here for nmrpipe processing script

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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

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¹H-¹⁵N Relaxation Experiments

Varian

15N Heteronuclear NOE on Varian inova

Setup

It is best to acquire these in an interleaved manner by doing the following steps (anything in red must be typed into vnmr)

Bruker

•     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