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== Protein Target Selection, Sample Preparation, and Initial Screening  ==
== Protein Target Selection, Sample Preparation, and Initial Screening  ==


#[[Target selection|NESG target selection]] - overview of target selection in PSI III
#[[Target selection|NESG target selection]] <br>
#[[Bioinformatics with protein sequence]]
#[[DNA cloning protocols|DNA cloning protocols]]<br>  
#[[DNA cloning protocols|DNA cloning protocols]]<br>  
#[[Protein purification|Protein expression and purification protocols]]&nbsp;<br>  
#[[Protein purification|Protein expression and purification protocols]]&nbsp;<br>  
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##[[Oligomerization Status|Assessment of Oligomerization Status]]  
##[[Oligomerization Status|Assessment of Oligomerization Status]]  
###[[Gel filtration and light scattering|gel-filtration and light scattering]]  
###[[Gel filtration and light scattering|gel-filtration and light scattering]]  
###[[Sedimentation equilibrium|Sedimentation equilibrium]]
###[[NMR determined Rotational correlation time]]  
###[[NMR determined Rotational correlation time]]  
##[[MassSpectrometry|Mass spectrum]]  
##[[MassSpectrometry|Mass spectrum]]  
##NMR screening
##[[NMR screening]]  
###[[1D screening|<sup>1</sup>H 1D screening]]  
<br>
###[[Nhsqc screen|Initial [<sup>15</sup>N,<sup>1</sup>H] HSQC]]<br>


== NMR Data Collection  ==
== NMR Data Collection  ==
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#NMR data acquisition for protein structure determination  
#NMR data acquisition for protein structure determination  
##[[Common NMR experiment sets]]  
##[[Common NMR experiment sets]]  
##Custom NMR experiment setup scripts for VNMRJ  
##[[NMR experiment setup scripts for VNMRJ|Custom NMR experiment setup scripts for VNMRJ]]
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC  
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC  
##[[Estimation of rotational correlation time]]  
##[[Estimation of rotational correlation time]]  
##Estimation of measurement time  
##[[Estimation of measurement time]]
##NMR experiments for spin system identification  
##NMR experiments for spin system identification  
##2D and 3D NOESY  
##2D and 3D NOESY experiments
###[[Simultaneous 13C,15N-resolved NOESY]]
##Double and triple NMR experiments  
##Double and triple NMR experiments  
###3D CBCA(CO)NH and HNCACB  
###3D CBCA(CO)NH and HNCACB  
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###(4,3)D HCCH  
###(4,3)D HCCH  
##Other NMR experiments  
##Other NMR experiments  
###2D [13C, 1H]-HSQC for 5% 13C-labeled samples  
###[[2D (13C, 1H) HSQC for fractionally 13C-labeled samples|2D [13C, 1H]-HSQC for fractionally 13C-labeled samples]]
###2D [15N, 1H]-long-range-HSQC for determination of histidine protomer state  
###[[Long-range 15N-1H correlation experiments for histidine rings]] - determination of histidine isoprotomer state  
###MEXICO  
###MEXICO  
###CLEANEX  
###CLEANEX  
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#Advanced problems for data collection  
#Advanced problems for data collection  
##[[Setting up non-uniformly sampled spectra]]  
##[[Setting up non-uniformly sampled spectra]]  
###[[NUS guide for Varian|Guide for Varian/BioPack]]  
###[[Setting up non-uniformly sampled spectra/NUS guide for Varian|Guide for Varian/BioPack]]  
###[[NUS guide for Bruker according to Arrowsmith group in Toronto|Guide for Bruker/Topspin according to Arrowsmith group]]  
###[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|Guide for Bruker/Topspin according to Arrowsmith group]]  
#Maintenance  
#Maintenance  
##VARIAN  
##VARIAN  
###Installing and updating <span class="twikiNewLink">BioPack</span>
###[[Installing and updating BioPack]]
###Full Probefile calibration  
###[[Full probefile calibration]]
###Rebooting the console  
###[[Rebooting spectrometer console]]
###Cryoprobe conditioning
###[[Conditioning procedure for cryogenic probes]]
##BRUKER
##BRUKER


== NMR Data Processing  ==
== NMR Data Processing  ==


#General Priciples and Concepts
#Processing Procedures for Routine Experiments<br>
##Fourier transformation
##NMRPipe
###Zero-filling
###[[Brief description of philosophy, commands, and functions of NMRPipe|Brief description of philosophy, commands, and functions of NMRPipe]]
###Apodization
###[[Routine 2D Experiment|Routine Processing Procedure for 2D Experiment]]<sup></sup>
###Phasing
###[[Routine Processing Procedure for 3D 15N and 13C-edited Experiments|Routine Processing Procedure for 3D <sup>15</sup>N and <sup>13</sup>C-edited Experiments]]
###Linear prediction
###[[HSQCTROSY RDC Measurement|2D ]]<sup>[[HSQCTROSY RDC Measurement|15]]</sup>[[HSQCTROSY RDC Measurement|N HSQC-TROSY experiment for RDC measurement]]
###G-matrix Fourier transformation (GFT)
###[[Jmodulation Experiment RDC|2D J-modulation experiment for RDC measurement]]
##Alternatives to Fourier transformation
###[[Processing NMR spectra with UB PERL scripts|Routine processing of spectra using Buffalo-developed PERL&nbsp;scripts]]
###Maximum entropy reconstruction
##[[Processing NMR spectra with PROSA|PROSA]]  
###[[Processing non-uniformly sampled spectra with Multidimensional Decomposition]]  
##[[Bruker Data Processing|TOPSPIN]]
###...
##[[AGNuS/AutoProc|AUTOPROC]]
#Practical Aspects
##[[UBNMR|UBNMR]]
##NMRPIPE
#Processing Procedures for Alternative Data Collection Methods
###<font color="#000000">General information</font>
##G-Matrix Fourier transformation (GFT)
###<font color="#0000ff">Buffalo's Processing Protocol using NMRpipe</font>
##[[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Processing non-uniformly sampled spectra with Multidimensional Decomposition]]
##PROSA
#Spectra Format Conversion
##TOPSPIN
##[[Spectra Format Conversion from NMRPipe Data|NMRPipe processed data conversion to Sparky, CARA, XEASY, and NMRViewJ]]
##[[AGNuS/AutoProc|AGNuS/AutoProc]]  
##TOPSPIN processed data conversion to Sparky, CARA, XEASY, and NMRViewJ<br>
##UBNMR
##Spectral format conversion
 
<br>  


== Resonance Assignment  ==
== Resonance Assignment  ==


This chapter would focus on individual data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.  
This chapter focuses on data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.  


<br>  
<br>  


#[[Principles and concepts]]
#[[Resonance Assignment/Principles and concepts|Principles and concepts]]  
##[[Stable isotope labeling schemes]]
#[[Resonance Assignment/Practical aspects|Practical aspects]]  
##[[NMR experiments]]
##Semi-automated protocols  
###[[Through-bond]]
###[[Resonance Assignment/CARA|CARA]]  
###[[Through space]]
##[[Spin systems]]
###[[Definitions]]
###[[Identification]]
###[[Linking spin systems]]
###[[Matching onto covalent structure]]  
#[[Practical aspects]]  
##[[Semi-automated protocols]]
###[[CARA]]  
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]  
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]  
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]  
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]  
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#####[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]  
#####[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]  
###[[Sparky]]  
###[[Sparky]]  
###[[XEASY]]  
###[[Resonance Assignment/XEASY|XEASY]]
####[[XEASY Introduction|Introduction]]
####[[XEASY Spin system identification|Spin system identification]]  
####[[XEASY Spin system identification|Spin system identification]]  
####Backbone resonance assignment'''<br>'''  
####Backbone resonance assignment'''<br>'''  
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######[[HNCACAB/CABCA(CO)NH]]  
######[[HNCACAB/CABCA(CO)NH]]  
#####Conventional spectra  
#####Conventional spectra  
######[[HNCACB/CBCA(CO)NH]]  
######[[HNCACB/CBCA(CO)NH]]<br>
######HNCA/HN(CO)CA
####Side chain resonance assignment  
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic  
#####Aliphatic  
######GFT NMR spectra  
######GFT-based spectra  
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]  
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]  
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]  
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]  
######Conventional spectra  
######Conventional spectra  
#######HAHB(CO)NH  
#######HAHB(CO)NH  
#######HCCH-COSY  
#######HCCH-COSY<br>
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]  
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]  
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]  
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]  
######GFT-based spectra
######Conventional spectra
#####Other  
#####Other  
######Trp e1 NH and d1 CH  
######Trp e1 NH and d1 CH  
######[[Met methyl assignment with NOESY|Met e CH3 ]]  
######[[Met methyl assignment with NOESY|Met e CH3 ]]  
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]  
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]  
#####NOESY peak integration  
####NOESY peak integration  
##Automated protocols  
##Automated protocols  
###[[AutoAssign|AutoAssign]]  
###[[AutoAssign|AutoAssign]]  
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###[[AVS|Assignment validation suite (AVS)]]  
###[[AVS|Assignment validation suite (AVS)]]  
###[[LACS|Linear analysis of chemical shift (LACS)]]  
###[[LACS|Linear analysis of chemical shift (LACS)]]  
##Depositing chemical shifts
##[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]


<br>  
<br>


== Structure Calculation and Validation  ==
== Structure Calculation and Validation  ==
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####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA|<br>]]  
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA|<br>]]  
###AutoStructure  
###AutoStructure  
####[[AutoStructure/RPF Theory|Theory]]
####[[AutoStructure|Getting started]]  
####[[AutoStructure|Getting started]]  
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]  
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]  
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####[[Validation of Consensus Run|Validation of Consensus runs]]  
####[[Validation of Consensus Run|Validation of Consensus runs]]  
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]  
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]  
###[[Structure Calculation Using CS-CP ROSETTA|CS-DP ROSETTA]]  
###[[Structure Calculation Using CS-DP ROSETTA|CS-DP ROSETTA]]  
###[[Structure Calculation Using RDC-ROSETTA|RDC-ROSETTA]]  
###[[Structure Calculation Using CS-RDC-ROSETTA|CS-RDC-ROSETTA]]  
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]]<br>  
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]]<br>  
###Special topics  
###Special topics  

Latest revision as of 20:51, 14 December 2009

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

  • It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
  • There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
  • We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
  • "Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
  • Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page


HTP NMR structure determination

Protein Target Selection, Sample Preparation, and Initial Screening

  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


NMR Data Collection

  1. Routine operation
    1. NMR sample tubes
    2. NMR Sample Preparation
    3. Inserting NMR Sample
    4. Tuning and matching
    5. Deuterium Lock
    6. Shimming
    7. Pulse width calibration
    8. Temperature calibration
    9. Chemical shift referencing
  2. Advanced operation
    1. Deuterium pulse width calibration and decoupling
  3. NMR data acquisition for protein structure determination
    1. Common NMR experiment sets
    2. Custom NMR experiment setup scripts for VNMRJ
    3. 1D 1H NMR spectra and 2D [15N, 1H]-HSQC
    4. Estimation of rotational correlation time
    5. Estimation of measurement time
    6. NMR experiments for spin system identification
    7. 2D and 3D NOESY experiments
      1. Simultaneous 13C,15N-resolved NOESY
    8. Double and triple NMR experiments
      1. 3D CBCA(CO)NH and HNCACB
      2. 3D HNCA and HN(CO)CA
      3. 3D HAHB(CO)NH
      4. (4,3)D CABCA(CO)NH and HNCACB
      5. (4,3)D HABCAB(CO)NH
      6. (H)CCH
      7. (H)CCH-TOCSY
      8. H(C)CH
      9. H(C)CH-TOCSY
      10. (4,3)D HCCH
    9. Other NMR experiments
      1. 2D [13C, 1H]-HSQC for fractionally 13C-labeled samples
      2. Long-range 15N-1H correlation experiments for histidine rings - determination of histidine isoprotomer state
      3. MEXICO
      4. CLEANEX
      5. H-D exchange experiment
      6. 15N spin relaxation parameters
  4. Advanced problems for data collection
    1. Setting up non-uniformly sampled spectra
      1. Guide for Varian/BioPack
      2. Guide for Bruker/Topspin according to Arrowsmith group
  5. Maintenance
    1. VARIAN
      1. Installing and updating BioPack
      2. Full probefile calibration
      3. Rebooting spectrometer console
      4. Conditioning procedure for cryogenic probes
    2. BRUKER

NMR Data Processing

  1. Processing Procedures for Routine Experiments
    1. NMRPipe
      1. Brief description of philosophy, commands, and functions of NMRPipe
      2. Routine Processing Procedure for 2D Experiment
      3. Routine Processing Procedure for 3D 15N and 13C-edited Experiments
      4. 2D 15N HSQC-TROSY experiment for RDC measurement
      5. 2D J-modulation experiment for RDC measurement
      6. Routine processing of spectra using Buffalo-developed PERL scripts
    2. PROSA
    3. TOPSPIN
    4. AUTOPROC
    5. UBNMR
  2. Processing Procedures for Alternative Data Collection Methods
    1. G-Matrix Fourier transformation (GFT)
    2. Processing non-uniformly sampled spectra with Multidimensional Decomposition
  3. Spectra Format Conversion
    1. NMRPipe processed data conversion to Sparky, CARA, XEASY, and NMRViewJ
    2. TOPSPIN processed data conversion to Sparky, CARA, XEASY, and NMRViewJ

Resonance Assignment

This chapter focuses on data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.


  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. Introduction
        2. Spin system identification
        3. Backbone resonance assignment
          1. GFT-based spectra
            1. HNCACAB/CABCA(CO)NH
          2. Conventional spectra
            1. HNCACB/CBCA(CO)NH
        4. Side chain resonance assignment
          1. Aliphatic
            1. GFT-based 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. Simultaneous NOESY
          2. Aromatic
          3. Other
            1. Trp e1 NH and d1 CH
            2. Met e CH3
            3. Asn d2 and Gln e2 NH2
        5. 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. Getting started
        2. CYANA run
        3. XPLOR run
        4. Analyzing the output
        5. RPF/DP scores
        6. 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. CS-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

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009