Powers Wiki - User contributions [en]

SO-FASTprocess

2017-03-20T15:44:55Z

Jcatazaro:

The processing of the So Fast HMQC is similar to processing the standard 1H-15N-HSQC.

However, the HMQC spectra will look squished. Therefore you should double the sweepwidth for the nitrogen dimension in your fid.com to solve this issue.

Running the CLEANEX Experiment

2017-03-20T15:44:12Z

Jcatazaro:

This page will describe the process for running the CLEANEX experiment.

Running CPASS

2017-03-20T15:43:51Z

Jcatazaro:

This page describes the process by which the results of the docking are used as the search query in a CPASS comparison.

Preparing Protein for FAST-NMR

2017-03-20T15:43:28Z

Jcatazaro:

This page will describe the process by which proteins that are to be screened by FAST-NMR are prepared, tested, and added to the database.

Preparing FAST-NMR Mixtures

2017-03-20T15:43:13Z

Jcatazaro:

This is the wiki describing the process by which the mixture plates are created and curated.

FAST-NMR

2017-03-20T15:40:59Z

Jcatazaro:

It's comin, ya'll! This will be the page the describes the overall process of FAST-NMR and links to the protocols of the individual steps involved in FAST-NMR

Docking the Hits in FAST-NMR

2017-03-20T15:40:46Z

Jcatazaro:

This page will describe the process by which compounds that are identified as good binders in the 2D HSQC screen are docked to the binding site identified in the same experiments.

DOCK

2017-03-20T15:40:31Z

Jcatazaro:

This page will describe how to install and use DOCK on the cluster.

Buffer Exchange and Solution Concentration

2017-03-20T15:40:05Z

Jcatazaro:

Preparation of a 10% D2O sample:

1.Take UV absorbance reading of stock sample at 280nm and determine starting concentration.

2.Place 540µL of stock sample in 1.5mL sample tube and add 60µL 100% D2O.

3.Transfer sample to NMR tube and run an HSQC on the 500MHz NMR to check stock sample stability.

4.If stock sample looks good move on to buffer exchange.

5.If sample does not look good, determine/fix problem(s) and repeat steps 1-3 until sample is satisfactory.

Buffer Exchange:

1.Place 500µL of stock sample on the CENTRIPREP column.

2.Add 1000µL of exchange buffer to the column.

3.Attach elute collection tube to bottom of column and place the cap on top.

4.Place in centrifuge and spin down for ~20-30min at 5,000rpm (NOTE: The goal is to decrease the total volume back to original volume (~500µL), depending on your sample you may need to increase/decrease centrifuge time. DO NOT vary centrifuge speed this may cause clogging of column pores or worse!)

5.Take sample out of centrifuge and add another 500µL of exchange buffer.

6.Repeat steps 2-5 about 4 times or until buffer exchange is complete.

7.After exchange is complete remove sample from column and place in 1.5µL sample tube.

8.Take a UV absorbance reading of sample using a 100X dilution to check final concentration (NOTE: 100x dilution is 990µL of exchange buffer and 10µL of sample).

9.Check the % recovery (<math>P</math>) using the following equation:

<math>P=100 \frac{V_f A_f}{V_i A_i}</math>

Where <math>V_i</math>, <math>V_f</math>, <math>A_i</math> and <math>A_f</math> are initial volume, final volume, initial absorbance and final absorbance values.

10.Remove 540µL of sample and place in new 1.5µL sample tube and add 60µL 100% D2O.

11.Transfer sample to NMR tube and run an HSQC on the 500MHz NMR to check stock sample stability.

12.If buffer exchange is used to check long term stability at room temp, store sample(s) at room temperature for the desired length of time while periodically checking stability using HSQC and note any resulting changes.

Solution Concentration:

After optimal buffer conditions are determined the sample is ready to be concentrated. However, by increasing the concentration of protein there is the chance that optimal buffer conditions will change also. Thus further optimization will be needed.

1.Run a number of buffer exchanges (~4) at the experimentally determined optimal conditions. (NOTE: You should collect ~2mL of sample for concentration. Collect and combine each exchange sample in a 2mL sample tube)

2.Check UV absorbance and run an HSQC to prove sample is soluble and stable

3.If sample is soluble and stable than continue with concentration. (NOTE: If sample is not soluble, stable, or both determine/fix problem(s) and start over).

4.Place 1mL of sample on the CENTRIPREP column.

5.Attach elute collection tube to bottom of column and place the cap on top.

6.Place in centrifuge and spin down for ~20-30min at 5,000rpm. (NOTE: The goal is to decrease the total volume back to original volume or about 500µL so depending on sample may need to increase/decrease centrifuge time. DO NOT vary centrifuge speed this may cause clogging of column pores or worse!)

7.Remove 10µL of concentrated sample and place in 990µL of optimal exchange buffer (NOTE: 100X dilution for UV-Vis).

8.Check UV absorbance and run an HSQC to prove sample is soluble and stable.

9.If everything worked well and you have reached the desired sample concentration with relatively few problems such as aggregation or precipitation, consider yourself lucky and prepare for analysis using the 600MHz NMR!

Analysis of 1D Line-Broadening Screen

2017-03-20T15:39:48Z

Jcatazaro:

Using ACD-NMR

==Move Files==

Open a PuTTY session with bionmr-c1 or reboot into Linux on your workstation. Navigate to /home/DATA/FAST_NMR (which is of course /DATA/FAST_NMR from a workstation) and run the ''gambrinus-get'' command.

'''NOTE:''' It is advised that you store all directories created by a 1D screen in a single directory on Gambrinus. For example, proteins already have their own folder on Gambrinus, so place all the ''Protein-YYYYMMDD-mixn'' or ''FREE-YYYYMMDD-mixn'' directories inside ''Protein''/ or ''FREE-YYYYMMDD''/, respectively. (All further examples will use ''Protein'' as the protein name, ''YYYYMMDD'' to represent the date of a new screen, and ''mixn'' to represent the mixture number.)

===Downloading Free Mixtures===

To download free mixtures from Gambrinus:

./gambrinus-get FREE-''YYYYMMDD'' FREE-Mixtures-''YYYYMMDD''

===Downloading Protein Screens===

If previous data exists in the ''Protein''/1D directory, make sure to move it before downloading the new data:

cd ''Protein''/
mv 1D 1D-old-''yyyymmdd''
cd ..

To download 1D screens of a protein from Gambrinus:

./gambrinus-get ''Protein''/1D-''YYYYMMDD'' ''Protein''/1D

==Run Macro==

Open ACD 12 1D NMR Processor

Click the "1D NMR Macro" button

[[Image:ACD-NMR_Menu1.png]]

This will open up the following screen. If there are files listed, clear them.

[[Image:ACD-NMR_1DMacro1.png]]

Then click "Add Group" to choose multiple files for running the macro.

[[Image:ACD-NMR_1DMacro2.png]]

When the window pops up, click on the ellipses on the other side of the "Dir Mask:" category.

[[Image:ACD-NMR_1DMacro3.png]]

'''Be sure the "File Mask:" category had "fid" in the box.'''

Find your protein. As explained in this page: [[1D Line Broadening Screen For FAST-NMR]], your files should be in the correct file type. In order to upload multiple files, you need to select the directory one of your ''fid'' files will be in, and use ''wild cards'' (asterisk: ***) for the correct number of characters representing the various mixture names (i.e. 1*** will show any of the mixtures we currently have).

''Please note if you have different dates, you should use wild cards as needed there.''

[[Image:ACD-NMR_1DMacro4.png]]

[[Image:Dir_Mask.png]]

Now that you have verified your files have all been uploaded, it is time to edit the macro! :) Select macro as such:

[[Image:ACD-NMR_1DMacro5.png]]

''Be sure you have highlighted the "1D Screen" macro.'' Click OK and this next screen should pop up.

[[Image:ACD-NMR_1DMacro6.png]]

This macro (1D Screen) is located in DATA/FAST-NMR/ and is saved again every time someone runs it, so '''be sure to edit the macro first, so you don't write over other files'''.

This is an example of the macro:

[[Image:ACD-NMR_1DMacro_Setup.png]]

File here: [[File:1Dscreen.mcr.txt]]

The area highlighted in the purple color are the areas you need to change. '''If you do not change where these files save, you will overwrite other files.''' In order to change a line, double click on it.

The first line help you reference your TMSP peak. If you wish to run the macro on only one ''fid'', you can look at the processed file to check where the reference is, if you wish.

The second line lets you choose where to put your processed 1D NMR file. You should place them in the PROC folder in the same parent folder the RAW folder you used earlier.

The third line allows you to choose where to place the ASCII file needed for the [http://bionmr-c1.unl.edu/cgi-bin/screens/index Bioscreen] database. Once again, this should be placed in the ASCII directory in the parent folder for the 1D protein screen.

When you have finished editing the macro, go ahead and run it.

When you run it, it will ask you if you wish to run it, say yes. The following figure shows the choices you should make in exporting the ASCII files.

[[Image:ACD-NMR_Export_Step2.png]]

==Check Data==
Files are processed and you have all the right nomenclature and file structures.

Copy the processed files into your PROC folder from the most recent FREE mixture spectra. Be sure to copy and not move.

[[Image:Copyfree.PNG]]

Copy the .com into your PROC folder from the FAST-NMR directory /DATA/FAST_NMR/namechange.csh

Open PuTTY and modify the namechange.csh file
The ONLY thing you should need to change, assuming you have used standard naming protocols while running your NMR experiments, is the protein name. If there are more than 117 mixtures, you will need to change the number of mixtures as well.

[[Image:Puttynamechange.PNG]]

File here: [[File:namechange.csh.txt]]

Print off binding sheet [[File:Binding_Sheets.xls]], and change the protein name to match your protein.

==Compare Data==

Agarose Gel

2017-03-20T15:39:33Z

Jcatazaro:

==Preparing 1% Agarose Gel==

#Measure 0.6 g of agarose
# Pour the agarose powder into microwavable flask with 60 mL of 1XTAE buffer
#Microwave for 1- 3 min (until the agarose is completely dissolved)
#Cool down the agarose and add ethidium bromide to a final concentration of approximately 0.2 to 0.5 ug/mL
# Pour the agarose into a gel try with the well comb in place
#Let sit at room temperature for about half an hour

'''N.B''' Ethidium bromide is a known mutagen.

==Running Agarose Gel==

# add running buffer to the gel apparatus
# carefully load samples in to the well
# load marker
#

Category:Protocols

2017-03-20T15:39:15Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Buffer Exchange and Solution Concentration]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Analysis of 1D Line-Broadening Screen]]
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[Agarose Gel]]
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

FastModelFree

2017-03-20T15:37:35Z

Jcatazaro:

==Optimizing PDB Coordinates before using Fast Model Free==

==='''PDB Inertia'''===
This program calculates the principal moments of inertia for the atoms in a standard pdb file.. By default the program writes the moments of inertia to standard output. Optionally, the program can output a new pdb file in which the molecule is translated so that its center of mass is located at the origin and rotated so that the moments of inertia are aligned with the Cartesian axes. At present, the program only reads lines starting with the 'ATOM' keyword and only recognizes the atoms H, C, N, O, P, S.

* Type the following command in the directory containing your pdb file
** '''pdbinertia -r infile.pdb outfile.pdb'''
** Or '''pdbinertia64 -r infile.pdb outfile.pdb'''

* This will output a translated and rotated pdb file.

==='''R2R1 Diffusion'''===
The program (r2r1_diffusion) uses the apprach of Tjandra, et al. [J. Am. Chem. Soc. 117:12562-12566 (1995)] to determine the diffusion tensors for spherical, and axially-symmetric motional models from experimental nitrogen-15 spin relaxation data.

====''Creating R2R1 file''====

You will need to calculate the <math>\tfrac{R_2}{R_1}</math> ratios ('''R2/R1''') and <math>\tfrac{R_2}{R_1}</math> uncertainities ('''dR2/R1'''). This can be done with any program such as excel, kaleidograph, origin, etc. The required calculations are as follows. To calculate <math>T_1</math> and <math>T_2</math>:

<math>T_1 = \frac{1}{R_1}</math> , and   
<math>T_2 = \frac{1}{R_2}</math>

The variances (squared errors) of these values are found as follows:

<math>\sigma_{T_1}^2 = \frac{\sigma_{R_1}^2}{{R_1}^4}</math> , and   
<math>\sigma_{T_2}^2 = \frac{\sigma_{R_2}^2}{{R_2}^4}</math>

To calculate <math>\tfrac{R_2}{R_1}</math>, just divide <math>R_2</math> by <math>R_1</math>. To calculate the variance of that value, use:

<math>\sigma_{\frac{R_2}{R_1}}^2 = \left (\frac{{R_2}^2}{{R_1}^4}\right ) \sigma_{R_1}^2 + \left (\frac{1}{{R_1}^2}\right ) \sigma_{R_2}^2</math>

If there is an error such as dividing by 0, remove the errors and leave blank. Save the file as a tab deliminated text file.
'''Example File''' 
[[Image:R2-R1example.jpg|500px]] 
 
 
 

====''Setting up Input File''====
*Copy the ubq.in file into the directory containing your translated/rotated pdb file
** The ubq.in file is located in ''${FMF_PATH}''/r2r1_diffusion/linux

'''ubq.in File''' 
[[Image:Ubqinputexample.jpg|500px]] 

*'''Editing the Ubq.in'''
**Second Line
*** The 300 represents the number of spin systems in the R2/R1 data. You will need to change this to the correct number
*** The 500.13 represents the frequency of NMR you used. For example, if you use the 600 Mhz NMR, change to 600.13.
*** The 100 represents the # of simulations. 100 simulations in the minimum needed.
** Third Line
*** The 4.0453e+7 is your initial estimate for D-isotropic tensor. This does not need to be changed unless you have an estimate
*** The 0.800 is the estimated Dpar/Dper tensor. If you do not know an estimate, set this to 1.00
*** The 0.872 and 0.628 are your Theta and Phi values. If you do not know an estimate, set these values to 0.000.
** Fourth Line
*** The 0.8 and 1.2 represents the predicted the High and low ratio set limits on Dpar/Dper. These values do not need to be changed.
*** The 10 represents the number of steps of grid searching to be performed. This does not need to be changed. Increasing the number of steps may help accuracy, however may increase the calculation time
** Fifth Line is your R2R1.txt file
** Sixth Line is your input pdb file generated from pdbinertia
** Seventh Line is your output pdb file

* '''Running the program'''
** type the following command
***'''r2r1_diffusion ubq.in > ubq.out'''
***Or '''r2r1_diffusion64 ubq.in > ubq.out'''
*** When the program is finished, you will need to check the Dpar/Dper value
****Type vi ubq.out
****Type :$
**** This will take you to the bottom of the file. Start scrolling up until you see the final parameters. Here is an example you will be seeing.
[[Image:Ubiqoutput.jpg|500px]]
* If your Dpar/Dper value is the same as your minimum value or maximum value you set on the fourth line in the ubq.in file, you will need to increase the range and rerun the calculation.
* Your predicted Theta and Phi angles will be calcuated. Save those numbers to implement them into FastModelFree.
* The output pdb is what will be used for FastModelFree 
 
 
 

==Fast ModelFree==
===Setting up Files for FastModelFree===
**You will need to copy the FMF.config into your directory containing your R2.txt, R1.txt, NOE.txt, and pdb file
**The most common problem that occurs at this point is the presence of additional white space in the input files. Make sure that additional lines after the data are deleted and additional tabs are also removed.
**FMF.config is located in ${FMF_PATH} 
**You can not have more than 300 spin systems. If there is more than 300 spin systems, the program will just hang and do nothing but act like its calculating.
 
FMF.config
[[Image:FMFconfig.jpg|500px]]
 
*Parameter to modify
** The manual for all the parameters are located in ''${FMF_PATH}''/fmfdox.pdf.
** '''tensor'''- Set as '''Isotropic''' for spherical or '''Axial Symmetric''' for non-spherical
*** Make sure the A and S is capitalized for Axial Symmetric or else it will autorun as Isotropic
*** You do not need a pdb file for Isotropic calculations
**'''cutoff''' set to 0.95
**'''Fcutoff''' set to 0.80
**'''optimize''' set to Yes
**'''maxloop''' set to 10. This means the program will stop after 10 iterations. If your final values have did not converge you may want to set to higher numbers such as 25 or 50 iterations.
**'''almost1''' set to 20. You can set to higher values just like maxloop
**'''S2cutoff''' set to 0.7
**'''seed''' set to any number and change every time.
** '''numsim''' This is the number of simulation. If you want a quick and dirty estimate, you can set it to about 10 simulations. Final calculations should have a minimum of 500 simulations
**'''jobname''' set this as the name of your protein. This is the title for all your output files.
**'''gamma''' set to -2.71
**'''rNH''' set to 1.02
**'''N15CSA''' set to -160
**'''tm''' This is your predicted correlation time. If you are unsure of a correlation time, 10.0 is a good start. The farther away from the actual value the more iterations is needed and longer it takes for calculations
*** You can use HyroNMR to help predict correlation time [[http://leonardo.inf.um.es/macromol/programs/hydronmr/hydronmr.htm]]
**'''tmMin''' Set to 0.0 if first time. You can set this min value near the actual value when you have a general idea what the tm is.
**'''tmMax''' Set to 40.0 if first time. You can set the max value near the actual value when you have a general idea what the tm is.
*** tmMin and tmMax are ranges you set to believe where the actual value is.
**'''tmGrid''' set to 50. Can set to higher value for more thorough calculation
**'''tmConv''' set to 0.001 This is the convergence cutoff. You want this to be small. The accuracy decreases as you increase this value.
**'''Dratio''' This is your Dpar/Dper ratio you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''DratioMin''' Set the value to be smaller than your Dratio
**'''DratioMax''' Set the value to be larger than your Dratio
*** DratioMin and DratioMax are your ranges you set. You want this range to be large when your first start out. After multiple attempts you can decrease the range.
**'''DratioConv''' set to 0.001
**'''Phi''' This is the Phi that you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''PhiMin''' set to 0
**'''PhiMax''' set to 360
**'''PhiGrid''' set to 20. Increase this value increases the thoroughness of the calculation
**'''PhiConv''' set to 0.001
**'''modle1only''' set to No
**'''mdpb''' Name of your pdb file generated from R2R1_diffusion calculations
**'''file{0}{R1}''' Name of your R1 file
**'''file{0}{R2}''' Name of your R2 file
**'''file{0}{NOE}''' Name of your NOE file
***'''file{0}{field}''' Set to 500 if using 500 MHz NMR, 600 if using 600Mhz NMR, etc.
 
 
 

===Running FastModelFree===
*To run the programs type this command
**'''fastMF > mf.log &'''
**Or '''fastMF64 > mf.log &'''
**The & symbol allows fastMF to run in the background. This process may take a minimum of 10 minutes or a few days depending how close or far away your predicted tm, Dratio, and Phi to the converged values
** You will initially see mfmodel, mfinput, mfout files, Jobname.MFDATA, Jobname.MFPAR. These files are output files generated by fastmodelfree, which are parameter sets to be used for ModelFree. The program isn't finished yet.
** You will then see your Jobname.1.pdb, Jobname.1.par. These are iterations being calculated attempting to converge all your values.
** When the calculations are finished check the Jobname.log file to see if your values converged
'''Example Protein.log''' 
[[Image:FMFresultslog.jpg|500px]]
* The tm, Dratio, Theta, and Phi values can be found in this log file
** The Example Protein.log figure show that it took 7 iterations to for the values to converge. Protein is the protein name for the figure The correlation time, Dratio, Theta, and Phi are 23.009, 0.819, -0.006, and -157.900 respectively
*The last pdb generated is the actual pdb generated from the converged values. The name of this file would be call Protein.7.pdb, where the 7 is the 7th iteration.
*Jobname.#.par is the results of your S2, te, Rex values. You will want to use the last .par file created. For example Protein.7.par is the actual results you want to use, in which all the value were converged.
'''Example Protein.7.par''' 

[[Image:FMFparresults.jpg|700px]]
 
 
 
==Generating High Quality Graphs==
===Setting up Gnuplot Scripts===
*Gnuplot is used to generate R1, R2, NOE, S2, Te, Rex graphs
*The files are located in ''${FMF_PATH}''/generate_plot
**There are 6 *.plt scripts to generate each script, and transfer them to your directory consisting of your fastmodelfree results, R1, R2, NOE data.
*The Jobname.#.par file consisting of your final values will need to be edited, because gnuplot does not like empty spaces in the data set. Instead you will have to insert the letter "a" in all the blank spaces. This can be done easily using excel or other programs. 
 
*Alternatively, use the script "addA.py" to quickly insert the letter "a" into all of the blank spaces.
'''Example edited.Protein.7.par''' 
[[Image:editedFMFparresults.jpg|700px]]
 
 
 
*Each .plt script will have to be edited, based on the x,y axis and input files
'''Example what to edit''' 
[[Image:Relaxationgraph.jpg|700px]]
 
 
*Editing R2modelfree.plt, R1modelfree.plt, NOEmodelfree.plt, Temodelfree.plt, Rexmodelfree.plt, s2modelfree.plt
** set your xrange as [0: #ofresidue + 2] Adding +2 to x range will allow the last point to be in the graph instead at the edge
** setting yrange. This is normally commented out (ex.adding # at beginning of line). This allows the y range to be to be set up automatically.
*** If the graph looks bad at y range, Take out the # sign. Change the numbers in the bracket to the range you want. example [0:12]
** setting xtic or ytics. The tics are normally setup automatically.
*** If you do not like the range of tic set up. You can set your own tic range
**** example: '''xtics 3.0 nomirror out font "Times, 16"'''. The 3.0 set the tic range at 3.0 values apart, therefor you will see a tic at 3.0, 6.0, 9.0.
** Setting up horizontal line where y=0. This is for s2modelfree.plt only
*** Where is says set arrow 1 from 190. Change the 190 to the max range you set for your x axis.
** Setting up your input file
*** At the bottom of the script where it says '''plot'''
****The script is designed to compare two different plots, where there is 3 lines involved.
****The first line is the data for your first protein. The second and third line is the data for your second protein. If you only have on protein data set, you can comment out the second and third line with a # sign at the beginning of the line.
*** For R2modelfree.plt use R2.txt data
*** For R1modelfree.plt use R1.txt data
*** For NOEmodelfree.plt use NOE.txt data
*** for Temodelfee.plt, rexmodelfree.plt, and S2modelfree.plt use edited Jobname.par generated by modelfree
 
 

===Running Gnuplot Scripts===
*To run the program, type the name of .plt file to run it.
** example: Type R2modelfree.plt to create R2 graph.
** a .ps file will be generated which contains your graph.
** to generate a pdf. Type '''pstopdf filename of graph'''
 
 
'''Example of a plot'''
[[Image:S2plot.jpg|700px]]

Category:Protocols

2017-03-20T15:36:31Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[FastModelFree]]
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Bioinformatics Tools For Annotating Proteins

2017-03-20T15:35:17Z

Jcatazaro:

This page will describe the various tools and process that can be used to help annotate a protein.

AutoDockFilter

2017-03-20T15:35:01Z

Jcatazaro:

This page will describe the use of AutoDockFilter

AutoDock

2017-03-20T15:34:37Z

Jcatazaro:

This page will describe how to install and use AutoDock on your workstation, the cluster, and the HCC Grid.

Annotating the Protein

2017-03-20T15:34:21Z

Jcatazaro:

This page will describe the final process of using the results from FAST-NMR, CPASS, and other bioinformatics tools to infer the function of a protein.

Adding Compounds To Library

2017-03-20T15:33:15Z

Jcatazaro:

This is a wiki page describing the process by which you add a new compound to the compound library.

== Introduction ==
Before you get started with adding a compound into the physical FAST-NMR library, please [[Adding_Compounds_To_Bioscreen|Add the compound to Bioscreen]] first.

700 MHz NMR checklist

2017-03-20T15:32:05Z

Jcatazaro:

'''Check in for the NMR 700mHz'''

Make sure the system is accessible, e.g. not being used or under maintenance

Check your belongings on your body and remove anything with ferromagnetic properties such as electronic devices and keep them away from the probe

Go to the Sample Jet and change the operating mode to 5mm shuttle, then load the rack of NMR tubes.

Login to the computer

Start topspin software

Load sample by typing in “sx 101” (or if in rack 2: “sx 201”, etc)

Click on the blank document icon

* Name it: initials-title-date

* Experiment: zgesgp (wipes out the water peak)

* Set solvent

* Check the “getprosol” box

Lock the sample by typing “lock d2o”

* The signal should be at around 75% after you lock it. If it is too low, go to BSMS, click “power” and use the scroll on the mouse to get it to around 75%, then click “standby” on BSMS to save the change.

Shim the sample automatically by typing “topshim”

* If the signal is not at 75%, adjust it in the same way as before. Also you can adjust the shimming manually by going to the BSMS and clicking on “z1”, “z2”, “x”, “y”, “xy”, etc. dimensions and using the scroll on the mouse. Don’t forget to click “standby” after any changes.

Autotune the sample by typing “atma”. To manually autotune, type “atmm”.

Next you need to find the 90° pulse.

* Type P1 and enter “8.0” in the pop-up window

* Type “pulprog” and choose “zg”

* Type “ns 1” (number of scans=1), then type “ds 1” (dummy scans), then type “rg 1” (receiver gain).

* Run the sample by typing “zg”

* To transform FID into spectrum, type “efp”

* Type “apk” to autophase

* Not type “p1”, hit enter and try different numbers (usually between 30-44), type “zg” and “efp” in order and see what number (p1) minimizes the peak the most.

* Once you have found the p1 number that minimizes the peak, divide that number by 4. That number is your 90° pulse.

* Type “edprosol” and set the p1 you found into both pulse widths and hit enter in each one

* Click the “copy to solvent” buton and select all relevant

* Click the “copy to probe” and select all relevant again. A popup window will ask you which cryoprobe you want to use. Choose the one that matches the cryoprobe that is in the top left corner.

* Save then “select all relevant”. Click on “yes” and “ok” whenever it asks you and make sure that you calculate all pulses when it asks. Then close out.

To run IconNMR, type “iconnmr” and choose the automation option and enter the password

Go to the holder that your sample is in. For example if your sample is in the 1st rack and is in the first slot in that rack, then go to A1-101.

Type in the name of your experiment, click on the “No.” space to activate that box, choose your solvent, and choose your experiment.

* Experiment for HSQC: C13HSQCS1SP2 (metab)

* Experiment for HMBC: C-13 HMBC

* Experiment for HSQC-TOCSY: SL-hsqcetf3gpml. Metab

Click on the paramters tab and choose “edit all acquisitions parameters”

* In the TD (data points) for the H dimension enter a number that is between 1024 and 2048 although it’s usually 2048. In the C dimension enter a number that is between 64 and 256 although it’s usually 64.

* In the NS (number of scans), type 32 although it can be 64 and even 128 depending on what you want.

* Type d1 and hit enter and input 1.5 for the relaxation time. *If you are doing the HSQC-TOCSY experiment then make sure you also change the d8 (delay time) to whatever number corresponds to the right hand panel

* Type in “rga” to check the automatic receiver gain

* to automation tab and make sure that the AUNM is set to au-zgonly.

* Then click on the button that says “return to iconNMR”

Copy to the samples as needed. (“edit as needed” )

Highlight all of the samples and click submit

Click “Start” and check the “lock/shim has already been completed box” and make sure that you are starting at your sample (for example: 101).

500 MHz NMR checklist

2017-03-20T15:31:46Z

Jcatazaro:

===Check in===
Make sure the system is accessible, e.g. not being used or under maintenance

Check the status of the cryoprobe shown on the laptop screen

Check your belongings on your body and remove anything with ferromagnetic properties such as electronic devices and keep them away from the probe

Login

Load your sample(s)

Start topspin software

Type in “rsp” to save the last dataset

Type in “rsh” (use the newest cryoprobe shim set of your specific solvent)

Type in “lock” and when finished, push the button standby Shim Z1, Z2 manually

Type in “gradshim” (use the proper gradshim settings for a heavily protonated or deuterated solvent)

Wait for it to finish and evaluate the results, if they are not good repeat gradshim

Close gradshim

Make sure the lock light is on Type in “atma” to tune and match the probe Find the proton 90 time (“p1”)

Be sure to use zg and not zg30 Update p1

Note: The command “edprosol” opens the field values 1H pulsewidth then power to change proton 90 degree pulse (for example getprosol 1H 8.3 2 will set the proton 90 time to 8.3 µsec at a power level of 2 dB for your current TOPSPIN session only. Each value is separated by a blank space).

Choose the solvent

Type in “rgacryo” Start running

If you use the BACS automation:

a) Store the necessary gradshim file for automation, understand that ICON willlook for the shim file under a specific name

b) Use administrative password to change default p1

c) Type in “getprosol” to update the parameters

d) Set up the parameters for your first sample in “icon” program

e) Change au_zg to zgonly if you need constant rg value

f) Make sure the sample is not rotating: Go to configuration and click master switches, check the box next to never rotate sample note the selections on the other switches that are important like the lock selections and the standard shim file loading selection. You must be logged in as “nmrsu” (nmr superuser) to change this.

===During the running===

Pay attention to the weather conditions especially any electric outage. Follow the “cryo platform outage procedure” as being described by Joe.

===Check out/end of run===

If any samples are reloaded into the belt, verify that the sample tube is not set to the wrong depth (spinner tightness)

Load the reference CDCl3 sample

Type in “rfshim” and choose the newest CDCl3 cryoprobe shim set

Type in “lock CDCl3” Type in “atma” Change p1 back to the pulse length listed in the back of the log book by the computer

Eject the sample and remove the NMR tube from the spinner Leave the spinner in the black holder at the workstation

Sign up the log book and clear up the area

If you use the BACS automation: First thing is to: Stop the running (when all samples are finished)

Type in “edprosol” and change p1 value back (same procedure above)

===Note===

1. Make sure your tube is clean, the spinner is clean, the sample is 600 µL and the tube is in the right depth of the spinner, holding tightly. If you have multiple samples, they need to have the same volume and fit into the spinners at the same height.

2. Use a container or a rack to carry the NMR tubes from your own lab to the NMR lab. Make sure the sample is not reactive with the solvents and uniformly dissolved in your solvent.

2D NMR Analysis (CCPNMR)

2017-03-20T15:31:16Z

Jcatazaro:

==Opening Spectra==

Go to the “Project” tab and “open spectra”

Change the file format to “Bruker”

Change file type to “All”

Find the spectrum you want to open in your documents folder and choose the “proc” file

Rename the file (ex: HSQC_10)

Click on “Open Spectrum”

Reference the ppm (change the numbers)

Click on “commit” and choose “close-all done”

Repeat these steps for any other spectra that you want opened and you can overlay the spectra

* To toggle between spectra, click on the “spectra” button on the spectra window and then toggle between the spectra that you want shown

* To change the color of the spectra and to toggle the dotted boxed line on and off, go to the “experiment” tab and choose “spectra” and go to the second tab in the pop-up window

==Peak Picking==

Hold control and shift down while clicking and dragging over area that you want peak picked

Then you can delete peaks by clicking on peak (hold shift for multiple) and right-clicking on the peak. Then click on “peak” and “delete selected”.

Click on the “Peak” tab and click “peak lists”. The peak table tab will show you the peaks that you selected on a table

==Assigning Peak Resonance==

Go to peak table and click on Assign F1. Choose “new”, then “select option”. Then “set resonance name” and name it.

Repeat for F2

If they’re the same spin system, then click “set same spin system”

Repeat “assign resonance name” for all other peaks that belong to same spin system but don’t click “set same spin system” again.

Once done, go to “resonance” tab and click on “Resonances”. Highlight all that are in same spin system and click “add to spin system”.

Click the spin system that they belong to and the software will label them as the same spin system.

Repeat for all spin systems

==Exporting Spectra==

Go to the window tab

Click on “print window”

Set your parameters

Click on “save print file”

Now you can drag and drop the file into CorelDraw or other software

==Problems Opening==

If you have problems opening the spectrum and you get an error message that reads: dimtype not....etc., go upstairs and process/phase the data upstairs in topspin instead of using NMRpipe. Then transfer the files using Gambrinus. For some reason sometimes it won't open unless you do it that way.

1H NMR Analysis (SIMCA)

2017-03-20T15:30:24Z

Jcatazaro:

==Excel Processing==
Secondary observation ID labels can be added by inserting a blank second row and filling in the desired labels. This does NOT affect the raw data. It merely makes “viewing” easier in the SIMCA output.

Use the standard method to remove instrument noise. Click here [http://bionmr.unl.edu/wiki/Noise_removal_for_PCA] for details.

For PLS-DA analysis, the y-variable values are placed in a row below the row containing the last NMR integral bucket value. When the spreadsheet is transposed in SIMCA, this last row becomes the last column.

After noise removal, the data should be autoscaled. By default, SIMCA-P will use "UV" autoscale the imported data set.

==PCA Analysis==

1. Start a “new” project by opening the desired integral table spreadsheet

2. Click on new project icon

3. Select the desired Excel file

4. Click “Open”

5. Click on the comma option The data formatting should now look correct.

6. Click “OK”

7. Project type should be SIMCA-P Project

8. Click “next” button

9. Respond “No” to the question regarding the one row that is “empty or contains only text”
It is the row of labels that was inserted using Excel. You can delete the blank row to avoid this.

10. Use the “Commands” button (bottom left) to “transpose” the data set
The rows should now correspond to a given NMR spectrum. Each row is an “observation”. The integral values contained in each row are referred to as “variables”.

11. Click on the button at the top of the column to set the first column as the “primary observation ID’s”
It may already be labeled as primary.

12. Click on the “Observation IDs primary” button
The observation IDs should now be color coded with the observation ID primary color. If secondary observation IDs were inserted using Excel, then click on the second column button and then click on “Observation IDs secondary”. Again, the column should be color coded to the correct color (light yellow).

12. Click on the butoon for the first row
It may already be labeled as primary and then click on the “Variable IDs primary” button to color code the first row (green). Next, repeat for the second row containing the ppm ranges that define each bucket. These will be the “Variable IDs secondary” and are turquoise. Click “Next” button in the lower-right.

13. Click the “Finish” button

14. Exclude the solvent region and “ends” of the spectra

15. Highlight the desired rows by dragging the cursor along the top set of buttons and then click the “exclude” button (along the left edge)

16. Repeat for each desired region

17. Click “Next” then click “Finish”

18. Using the menu bar, click on the “autofit” button.
This should calculate the first and second primary components. Additional components can be calculated using the “Calculate next component” button.

19. View the results, click on the “Create four overview plots” button.
This produces the “Score Scatter” plot in the upper-left hand corner. The lower-left hand corner contains the “Loading Scatter” plot.

20. Expand the score scatter plot for better viewing

21. Click on data point

22. Right-click and choose “Properties”

23. Choose the color tab and choose coloring type by “identifiers”
The default then is to color by secondary observation IDs. This uses the labels inserted using Excel. If desired, change the default colors.

24. Click “Apply”

25. Click “OK”

==PLS Analysis==

1. The data are prepared in an Excel file as described above.

2. The Excel file is opened in SIMCA as described above for PCA analysis.

3. The opened file should then be transposed. Again, the commands button found in the lower left provides access to this command.

4. The label information for observations and variables should be processed as described for PCA analysis above.

5. The difference between the PCA approach and the PLS approach occurs with labeling of the variables (i.e., the bucketed intensities and the discriminator values).

6. The region containing the bucketed intensities is highlighted.
These values are labeled as “x-variables” by clicking on the VARIABLE button in the left hand frame and choosing x-variables.

7. Next, the discriminator values should be set as 1 or 0.

8. Selected data may still be excluded prior to the statistical analysis

9. The dataset is “fit”, additional components can be added/substracted and the results are visualized using the same commands as for PCA analysis described above.

==OPLS-DA Analysis==

1. This approach applies orthogonal signal correction prior to PLS analysis. This manual is for SIMCA-P+ 12.0 or higher version.

2. Start from an Excel file that has NOT been autoscaled and any outliers should be removed prior to OPLS analysis.

3. The data file should be first analyzed using either PCA or PLS as described above to find two unsupervised, separated groups for comparison. Wild-type or control groups should be assigned as "0", mutant or treatment group should be assigned as "1". This column of "0" and "1" should be taken as Y values.

4. Follow the same procedure as PCA to import the data.

5. Click on Y to change the state of these to Y values. Click on the “Next >” button. There may be a message regarding exclusion of variables with no variance. A new Workset window will appear.

6. Click "Yes to All" and then another workset window will pop-up with "model number", "type of model", etc.

7. Double click it and click "Workset" icon on that pop-up window.

8. Select all the variables, and use "Par" for scaling. Model type "OPLS/O2PLS".

9. Click Autofit button on the toolbar

1H NMR Analysis (ACDLab)

2017-03-20T15:30:05Z

Jcatazaro:

=ACDLab/SpecManager=

==Import spectral data==

Use 1D Macro to load the desired series of 1D 1H NMR ".fid" data files. When this is done, by default, the “group treatment” is on and ACD operations will be applied to the entire group.

Steps:

1. Click on the “1D NMR Macro” button.

2. On the “Execute Group Window”, make sure:

a) “Plate” should not be checked.

b) Check “spectra”.

c) Click on “add group”. The wild cards ("*" for numbers, "?" for letters) can be inserted in the directory mask line. The file mask should read “.fid”. Click “OK”.

3. When all of the data files are correctly listed, click “OK” to execute the group macro and load the files into ACDLab/SpecManager. Assuming that the files loaded correctly, click “OK” when the process if finished.

==Pre-process the spectra==

If appropriate, apply a window function. To match what TOPSPIN does automatically when processing data on the 500 MHz console, use a 0.3 Hz exponential window function prior to Fourier transform (FT).

Steps:

1. Click on “WFunctions” in the operations bar.

2. Set LB = 0.3 Hz

3. Choose “Exponential”.

4. Click “Ok”.

5. Fourier transform the FID files

6. Click on “Fourier Tr.” in the operations bar.

7. Phase correct the spectra.

8. Click on the “Phase Corr.” button on the operations bar.

9. Use the “Auto Simple” option.

10. Click on the “checkmark” to accept the result.

11. Set chemical shift reference using the TMSP peak.

12. Click on the “Reference” button on the operations bar.

13. Check the “Options” to verify that TMS is set. Both TMSP and TMS have chemical shift values of 0.000 ppm.

14. Click on “Auto”. Then click “Ok” in response to the “There are no labeled peaks available…” message if it appears.

==Integrate the spectra and export the peak list==

Using the “auto integrate” option:

Steps:

1. Click on the Integration operation.

2. Check the “Options”

a) Set for “Bucket Integration”

b) Set width of buckets to 0.025 ppm

c) Choose intelligent bucketing with width looseness (%) = 50 and method = sum

d) Set reference to “whole spectrum”.

e) Click “Ok”

f) Click on “Auto” to perform the integration

g) Click on the “checkmark” to accept the result.

3. Use the Series pull-down menu to bring up the table of common integrals. A right-click will allow one to save the integral table. The saved table can be imported directly into SIMCA or opened in Excel.

Category:Protocols

2017-03-20T15:29:49Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[2D NMR Analysis (CCPNMR)]]
*[[1H NMR Analysis (SIMCA)]]
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[700 MHz NMR checklist]]
*[[500 MHz NMR checklist]]
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Category:Protocols

2017-03-20T15:27:10Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[1H NMR Analysis (ACDLab)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

Category:Protocols

2017-03-20T15:26:48Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[1H NMR Analysis (ACDLabs)]]
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

2D 15N-HSQC Screens For FAST-NMR

2017-03-20T15:23:19Z

Jcatazaro:

This is the wiki describing the preparation of samples and running the FAST-NMR 2D 15N-HSQC screen.

1D NMR Titrations

2017-03-20T15:19:49Z

Jcatazaro:

== Protein Stock Preparation ==
# Make 2.0 mM (2000 uM) HSA stock solution
## Dissolve 130.8 mg of HSA (MW = 65400 g) in 1.0 mL D2O
# Make a set of serial dilutions starting with the 2.0 mM stock (see table 1) (NOTE: at these stock solutions it only takes 10.0 uL of protein sample to give the desired protein concentration in the NMR tube)
# Spin Samples down in mini centrifuge
# Press short button until max speed then release
# Measure the absorbance and generate a standard curve for the new samples.
# Make 10X dilutions (10.0 uL HSA and 990 uL D2O)

'''Table 1:'''
{| class="wikitable"
|-
! Sample No.
! NMR [HSA] (uM)
! [HSA] (uM)
! Volume (n+1) sample (uL)
! Volume of D2O (uL)
|-
| 1
| 0.000
| 0.00
| 0
| 0
|-
| 2
| 0.100
| 5.00
| 500
| 500
|-
| 3
| 0.200
| 10.00
| 500
| 500
|-
| 4
| 0.400
| 20.00
| 667
| 333
|-
| 5
| 0.600
| 30.00
| 750
| 250
|-
| 6
| 0.800
| 40.00
| 800
| 200
|-
| 7
| 1.000
| 50.00
| 500
| 500
|-
| 8
| 2.000
| 100.00
| 667
| 333
|-
| 9
| 3.000
| 150.00
| 750
| 250
|-
| 10
| 4.000
| 200.00
| 667
| 333
|-
| 11
| 6.000
| 300.00
| 750
| 250
|-
| 12
| 8.000
| 400.00
| 800
| 200
|-
| 13
| 10.00
| 500.00
| 833
| 167
|-
| 14
| 12.00
| 600.00
| 857
| 143
|-
| 15
| 14.00
| 700.00
| 875
| 125
|-
| 16
| 16.00
| 800.00
| 889
| 111
|-
| 17
| 18.00
| 900.00
| 900
| 100
|-
| 18
| 20.00
| 1000.00
| 667
| 333
|-
| 19
| 30.00
| 1500.00
| 750
| 250
|-
| 20
| 40.00
| 2000.00
| 130.8 mg
| 1 mL
|}

== Ligand Stock Preparation ==
# Make 50.0 mM Potassium Phosphate buffer at pH 7.0 in D2O
## For 500.0 mL total solution, measure 2.177 g K2HPO4 (MW = 174.18 g/mol) and 1.70 g KH2PO4 (MW = 136.09g/mol) in separate 250.0 mL volumetric flasks and fill to line with 99.9% D2O.
## Spike with 500 uL of TMSP from 10.0 uM stock solution
# Make 500.0 uL fresh ligand stocks at 20.0 mM concentration
## Calculate the mass of ligand needed and dissolve in 500.0 uL of 100% DMSO
# Make 11.0 mL titration stocks
## Single ligands: In Falcon tube add, in order, 10.5 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55 uL of fresh ligand stock.
## Ligand Mixtures: In a Falcon tube add in order: 10.390 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55μL of each fresh ligand stock solution (The 2 non-binders Choline Bromide and Uridine-5’-monophosphate plus the binding ligand)
# Vortex the titration stock solutions to mix

== NMR Sample Preparation ==
# Label 20 1.5 uL centrifuge tubes with the ligand and protein concentration
# To each tube add 490 uL of the ligand titration stock solution
# To each tube add 10 uL of the specific protein stock to the intended sample (i.e. add 10 uL of 400 uM stock to get protein concentration in NMR tube of 8 uM)
# Spin samples down in mini-centrifuge to make sure all sample is in the bottom of tube
# Pipette sample out of centrifuge tubes and into NMR tubes making sure not to get any bubbles
# Run samples on NMR using the ''zgesgp'' pulse program (''1D-FASTNMR'' parameter set) or other comparable method for water pre-saturation

=== Important Notes ===
* This is a general method for preparing samples for NMR titrations and can be amended as needed.
* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

Category:Protocols

2017-03-20T15:19:22Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[1D NMR Titrations]]

1D Macro

2017-03-20T15:15:16Z

Jcatazaro:

==Macro Script==
[[Image:1D_NMR_Macro.png]]

----

All the lines with a '''>''' in front are the executed lines, all the lines with a '''#''' are commented out.

The first ''three'' lines after the description are used to process the fid data. Line 1 proves to the macro that this is a proton fid.

The second line provides window functions, in the shown macro, it includes an exponential ************

The third **************

----

Next lines are the processing after the fid has been turned into a spectrum, with the first line after ensuring the file is the correct file type.

----

The last three lines are the typically changed lines. '''It is important to change the save location of the processed spectra and ASCII files.''' This

1D Line Broadening Screen For FAST-NMR

2017-03-20T15:14:49Z

Jcatazaro: Blanked the page

Category:Protocols

2017-03-20T15:14:08Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[1D Macro]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]

1D Macro

2017-03-20T15:12:48Z

Jcatazaro:

[[Category:Protocols]]

==Macro Script==
[[Image:1D_NMR_Macro.png]]

----

All the lines with a '''>''' in front are the executed lines, all the lines with a '''#''' are commented out.

The first ''three'' lines after the description are used to process the fid data. Line 1 proves to the macro that this is a proton fid.

The second line provides window functions, in the shown macro, it includes an exponential ************

The third **************

----

Next lines are the processing after the fid has been turned into a spectrum, with the first line after ensuring the file is the correct file type.

----

The last three lines are the typically changed lines. '''It is important to change the save location of the processed spectra and ASCII files.''' This

Category:Protocols

2017-03-20T15:11:13Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]

Category:Protocols

2017-03-20T15:10:03Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[test]]

Category:Protocols

2017-03-20T15:02:40Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]

Category:Protocols

2017-03-20T15:02:14Z

Jcatazaro:

''General Maintenance''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
''Protein Preparation''
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
''Data Collection''
*[[Collecting a 15N Edited HSQC]]
*[[Collecting CEST Data]]
''Data Processing and Analysis''
*[[Processing CEST Data]]
*[[Titration Data Analysis in nmrPipe]]
''Miscellaneous''
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[Creating a New Wiki Page]]

Category:Protocols

2017-03-15T00:06:30Z

Jcatazaro:

*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
*[[Collecting a 15N Edited HSQC]]
*[[Titration Data Analysis in nmrPipe]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[Collecting CEST Data]]
*[[Processing CEST Data]]
*[[Creating a New Wiki Page]]

Category:Protocols

2017-03-15T00:05:48Z

Jcatazaro:

'''Proteins!!!!!'''
*[[Changing the high pressure dewar]]
*[[Filling a Magnet with Nitrogen]]
*[[Autoclaving Laboratory Glassware and Media]]
*[[Chemical Disinfection of Glassware]]
*[[Finding a Protein Target on the NESG website]]
*[[Choosing a Plasmid]]
*[[Plasmid Purification and Transformation Protocol]]
*[[Creating Stock Cultures of Bacteria]]
*[[Luria-Bertani Media]]
*[[M9 Minimal Media]]
*[[Protein Overexpression and Extraction]]
*[[SDS-PAGE Protocol]]
*[[Running a Cobalt Affinity Column]]
*[[Dialysis]]
*[[Centrifugal Protein Concentration and Buffer Exchange]]
*[[Using the Stirred Cell Concentrator]]
*[[Collecting a 15N Edited HSQC]]
*[[Titration Data Analysis in nmrPipe]]
*[[Setting Up a Virtual Screen with AutoDock]]
*[[Simple Protein Crosslinking]]
*[[Collecting CEST Data]]
*[[Processing CEST Data]]
*[[Creating a New Wiki Page]]

Category:Protocols

2017-03-15T00:03:07Z

Jcatazaro:

Changing the high pressure dewar

2017-03-14T22:53:09Z

Jcatazaro:

Changing the High Pressure Dewar (on the 700)

# Make sure that an experiment is not being collected on the 700.
# Close the gas valve on the old/empty dewar.
# Close the two valves on the gas line which goes to into the NMR lab.
# Unscrew the transfer line from the dewar.
# Attach the transfer line to the gas valve of the new dewar.
# Open the two values on the gas line to the NMR lab.
# Open the gas valve on the dewar. It will be noisy at first but will subside quickly.

Changing the high pressure dewar

2017-03-14T22:52:35Z

Jcatazaro:

FastModelFree

2017-03-14T21:50:25Z

Jcatazaro:

[[category:Protocols]]
==Optimizing PDB Coordinates before using Fast Model Free==

==='''PDB Inertia'''===
This program calculates the principal moments of inertia for the atoms in a standard pdb file.. By default the program writes the moments of inertia to standard output. Optionally, the program can output a new pdb file in which the molecule is translated so that its center of mass is located at the origin and rotated so that the moments of inertia are aligned with the Cartesian axes. At present, the program only reads lines starting with the 'ATOM' keyword and only recognizes the atoms H, C, N, O, P, S.

* Type the following command in the directory containing your pdb file
** '''pdbinertia -r infile.pdb outfile.pdb'''
** Or '''pdbinertia64 -r infile.pdb outfile.pdb'''

* This will output a translated and rotated pdb file.

==='''R2R1 Diffusion'''===
The program (r2r1_diffusion) uses the apprach of Tjandra, et al. [J. Am. Chem. Soc. 117:12562-12566 (1995)] to determine the diffusion tensors for spherical, and axially-symmetric motional models from experimental nitrogen-15 spin relaxation data.

====''Creating R2R1 file''====

You will need to calculate the <math>\tfrac{R_2}{R_1}</math> ratios ('''R2/R1''') and <math>\tfrac{R_2}{R_1}</math> uncertainities ('''dR2/R1'''). This can be done with any program such as excel, kaleidograph, origin, etc. The required calculations are as follows. To calculate <math>T_1</math> and <math>T_2</math>:

<math>T_1 = \frac{1}{R_1}</math> , and   
<math>T_2 = \frac{1}{R_2}</math>

The variances (squared errors) of these values are found as follows:

<math>\sigma_{T_1}^2 = \frac{\sigma_{R_1}^2}{{R_1}^4}</math> , and   
<math>\sigma_{T_2}^2 = \frac{\sigma_{R_2}^2}{{R_2}^4}</math>

To calculate <math>\tfrac{R_2}{R_1}</math>, just divide <math>R_2</math> by <math>R_1</math>. To calculate the variance of that value, use:

<math>\sigma_{\frac{R_2}{R_1}}^2 = \left (\frac{{R_2}^2}{{R_1}^4}\right ) \sigma_{R_1}^2 + \left (\frac{1}{{R_1}^2}\right ) \sigma_{R_2}^2</math>

If there is an error such as dividing by 0, remove the errors and leave blank. Save the file as a tab deliminated text file.
'''Example File''' 
[[Image:R2-R1example.jpg|500px]] 
 
 
 

====''Setting up Input File''====
*Copy the ubq.in file into the directory containing your translated/rotated pdb file
** The ubq.in file is located in ''${FMF_PATH}''/r2r1_diffusion/linux

'''ubq.in File''' 
[[Image:Ubqinputexample.jpg|500px]] 

*'''Editing the Ubq.in'''
**Second Line
*** The 300 represents the number of spin systems in the R2/R1 data. You will need to change this to the correct number
*** The 500.13 represents the frequency of NMR you used. For example, if you use the 600 Mhz NMR, change to 600.13.
*** The 100 represents the # of simulations. 100 simulations in the minimum needed.
** Third Line
*** The 4.0453e+7 is your initial estimate for D-isotropic tensor. This does not need to be changed unless you have an estimate
*** The 0.800 is the estimated Dpar/Dper tensor. If you do not know an estimate, set this to 1.00
*** The 0.872 and 0.628 are your Theta and Phi values. If you do not know an estimate, set these values to 0.000.
** Fourth Line
*** The 0.8 and 1.2 represents the predicted the High and low ratio set limits on Dpar/Dper. These values do not need to be changed.
*** The 10 represents the number of steps of grid searching to be performed. This does not need to be changed. Increasing the number of steps may help accuracy, however may increase the calculation time
** Fifth Line is your R2R1.txt file
** Sixth Line is your input pdb file generated from pdbinertia
** Seventh Line is your output pdb file

* '''Running the program'''
** type the following command
***'''r2r1_diffusion ubq.in > ubq.out'''
***Or '''r2r1_diffusion64 ubq.in > ubq.out'''
*** When the program is finished, you will need to check the Dpar/Dper value
****Type vi ubq.out
****Type :$
**** This will take you to the bottom of the file. Start scrolling up until you see the final parameters. Here is an example you will be seeing.
[[Image:Ubiqoutput.jpg|500px]]
* If your Dpar/Dper value is the same as your minimum value or maximum value you set on the fourth line in the ubq.in file, you will need to increase the range and rerun the calculation.
* Your predicted Theta and Phi angles will be calcuated. Save those numbers to implement them into FastModelFree.
* The output pdb is what will be used for FastModelFree 
 
 
 

==Fast ModelFree==
===Setting up Files for FastModelFree===
**You will need to copy the FMF.config into your directory containing your R2.txt, R1.txt, NOE.txt, and pdb file
**The most common problem that occurs at this point is the presence of additional white space in the input files. Make sure that additional lines after the data are deleted and additional tabs are also removed.
**FMF.config is located in ${FMF_PATH} 
**You can not have more than 300 spin systems. If there is more than 300 spin systems, the program will just hang and do nothing but act like its calculating.
 
FMF.config
[[Image:FMFconfig.jpg|500px]]
 
*Parameter to modify
** The manual for all the parameters are located in ''${FMF_PATH}''/fmfdox.pdf.
** '''tensor'''- Set as '''Isotropic''' for spherical or '''Axial Symmetric''' for non-spherical
*** Make sure the A and S is capitalized for Axial Symmetric or else it will autorun as Isotropic
*** You do not need a pdb file for Isotropic calculations
**'''cutoff''' set to 0.95
**'''Fcutoff''' set to 0.80
**'''optimize''' set to Yes
**'''maxloop''' set to 10. This means the program will stop after 10 iterations. If your final values have did not converge you may want to set to higher numbers such as 25 or 50 iterations.
**'''almost1''' set to 20. You can set to higher values just like maxloop
**'''S2cutoff''' set to 0.7
**'''seed''' set to any number and change every time.
** '''numsim''' This is the number of simulation. If you want a quick and dirty estimate, you can set it to about 10 simulations. Final calculations should have a minimum of 500 simulations
**'''jobname''' set this as the name of your protein. This is the title for all your output files.
**'''gamma''' set to -2.71
**'''rNH''' set to 1.02
**'''N15CSA''' set to -160
**'''tm''' This is your predicted correlation time. If you are unsure of a correlation time, 10.0 is a good start. The farther away from the actual value the more iterations is needed and longer it takes for calculations
*** You can use HyroNMR to help predict correlation time [[http://leonardo.inf.um.es/macromol/programs/hydronmr/hydronmr.htm]]
**'''tmMin''' Set to 0.0 if first time. You can set this min value near the actual value when you have a general idea what the tm is.
**'''tmMax''' Set to 40.0 if first time. You can set the max value near the actual value when you have a general idea what the tm is.
*** tmMin and tmMax are ranges you set to believe where the actual value is.
**'''tmGrid''' set to 50. Can set to higher value for more thorough calculation
**'''tmConv''' set to 0.001 This is the convergence cutoff. You want this to be small. The accuracy decreases as you increase this value.
**'''Dratio''' This is your Dpar/Dper ratio you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''DratioMin''' Set the value to be smaller than your Dratio
**'''DratioMax''' Set the value to be larger than your Dratio
*** DratioMin and DratioMax are your ranges you set. You want this range to be large when your first start out. After multiple attempts you can decrease the range.
**'''DratioConv''' set to 0.001
**'''Phi''' This is the Phi that you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''PhiMin''' set to 0
**'''PhiMax''' set to 360
**'''PhiGrid''' set to 20. Increase this value increases the thoroughness of the calculation
**'''PhiConv''' set to 0.001
**'''modle1only''' set to No
**'''mdpb''' Name of your pdb file generated from R2R1_diffusion calculations
**'''file{0}{R1}''' Name of your R1 file
**'''file{0}{R2}''' Name of your R2 file
**'''file{0}{NOE}''' Name of your NOE file
***'''file{0}{field}''' Set to 500 if using 500 MHz NMR, 600 if using 600Mhz NMR, etc.
 
 
 

===Running FastModelFree===
*To run the programs type this command
**'''fastMF > mf.log &'''
**Or '''fastMF64 > mf.log &'''
**The & symbol allows fastMF to run in the background. This process may take a minimum of 10 minutes or a few days depending how close or far away your predicted tm, Dratio, and Phi to the converged values
** You will initially see mfmodel, mfinput, mfout files, Jobname.MFDATA, Jobname.MFPAR. These files are output files generated by fastmodelfree, which are parameter sets to be used for ModelFree. The program isn't finished yet.
** You will then see your Jobname.1.pdb, Jobname.1.par. These are iterations being calculated attempting to converge all your values.
** When the calculations are finished check the Jobname.log file to see if your values converged
'''Example Protein.log''' 
[[Image:FMFresultslog.jpg|500px]]
* The tm, Dratio, Theta, and Phi values can be found in this log file
** The Example Protein.log figure show that it took 7 iterations to for the values to converge. Protein is the protein name for the figure The correlation time, Dratio, Theta, and Phi are 23.009, 0.819, -0.006, and -157.900 respectively
*The last pdb generated is the actual pdb generated from the converged values. The name of this file would be call Protein.7.pdb, where the 7 is the 7th iteration.
*Jobname.#.par is the results of your S2, te, Rex values. You will want to use the last .par file created. For example Protein.7.par is the actual results you want to use, in which all the value were converged.
'''Example Protein.7.par''' 

[[Image:FMFparresults.jpg|700px]]
 
 
 
==Generating High Quality Graphs==
===Setting up Gnuplot Scripts===
*Gnuplot is used to generate R1, R2, NOE, S2, Te, Rex graphs
*The files are located in ''${FMF_PATH}''/generate_plot
**There are 6 *.plt scripts to generate each script, and transfer them to your directory consisting of your fastmodelfree results, R1, R2, NOE data.
*The Jobname.#.par file consisting of your final values will need to be edited, because gnuplot does not like empty spaces in the data set. Instead you will have to insert the letter "a" in all the blank spaces. This can be done easily using excel or other programs. 
 
*Alternatively, use the script "addA.py" to quickly insert the letter "a" into all of the blank spaces.
'''Example edited.Protein.7.par''' 
[[Image:editedFMFparresults.jpg|700px]]
 
 
 
*Each .plt script will have to be edited, based on the x,y axis and input files
'''Example what to edit''' 
[[Image:Relaxationgraph.jpg|700px]]
 
 
*Editing R2modelfree.plt, R1modelfree.plt, NOEmodelfree.plt, Temodelfree.plt, Rexmodelfree.plt, s2modelfree.plt
** set your xrange as [0: #ofresidue + 2] Adding +2 to x range will allow the last point to be in the graph instead at the edge
** setting yrange. This is normally commented out (ex.adding # at beginning of line). This allows the y range to be to be set up automatically.
*** If the graph looks bad at y range, Take out the # sign. Change the numbers in the bracket to the range you want. example [0:12]
** setting xtic or ytics. The tics are normally setup automatically.
*** If you do not like the range of tic set up. You can set your own tic range
**** example: '''xtics 3.0 nomirror out font "Times, 16"'''. The 3.0 set the tic range at 3.0 values apart, therefor you will see a tic at 3.0, 6.0, 9.0.
** Setting up horizontal line where y=0. This is for s2modelfree.plt only
*** Where is says set arrow 1 from 190. Change the 190 to the max range you set for your x axis.
** Setting up your input file
*** At the bottom of the script where it says '''plot'''
****The script is designed to compare two different plots, where there is 3 lines involved.
****The first line is the data for your first protein. The second and third line is the data for your second protein. If you only have on protein data set, you can comment out the second and third line with a # sign at the beginning of the line.
*** For R2modelfree.plt use R2.txt data
*** For R1modelfree.plt use R1.txt data
*** For NOEmodelfree.plt use NOE.txt data
*** for Temodelfee.plt, rexmodelfree.plt, and S2modelfree.plt use edited Jobname.par generated by modelfree
 
 

===Running Gnuplot Scripts===
*To run the program, type the name of .plt file to run it.
** example: Type R2modelfree.plt to create R2 graph.
** a .ps file will be generated which contains your graph.
** to generate a pdf. Type '''pstopdf filename of graph'''
 
 
'''Example of a plot'''
[[Image:S2plot.jpg|700px]]

FastModelFree

2017-03-14T21:44:44Z

Jcatazaro:

[[category:Protocols]]
==Optimizing PDB Coordinates before using Fast Model Free==

==='''PDB Inertia'''===
This program calculates the principal moments of inertia for the atoms in a standard pdb file.. By default the program writes the moments of inertia to standard output. Optionally, the program can output a new pdb file in which the molecule is translated so that its center of mass is located at the origin and rotated so that the moments of inertia are aligned with the Cartesian axes. At present, the program only reads lines starting with the 'ATOM' keyword and only recognizes the atoms H, C, N, O, P, S.

* Type the following command in the directory containing your pdb file
** '''pdbinertia -r infile.pdb outfile.pdb'''
** Or '''pdbinertia64 -r infile.pdb outfile.pdb'''

* This will output a translated and rotated pdb file.

==='''R2R1 Diffusion'''===
The program (r2r1_diffusion) uses the apprach of Tjandra, et al. [J. Am. Chem. Soc. 117:12562-12566 (1995)] to determine the diffusion tensors for spherical, and axially-symmetric motional models from experimental nitrogen-15 spin relaxation data.

====''Creating R2R1 file''====

You will need to calculate the <math>\tfrac{R_2}{R_1}</math> ratios ('''R2/R1''') and <math>\tfrac{R_2}{R_1}</math> uncertainities ('''dR2/R1'''). This can be done with any program such as excel, kaleidograph, origin, etc. The required calculations are as follows. To calculate <math>T_1</math> and <math>T_2</math>:

<math>T_1 = \frac{1}{R_1}</math> , and   
<math>T_2 = \frac{1}{R_2}</math>

The variances (squared errors) of these values are found as follows:

<math>\sigma_{T_1}^2 = \frac{\sigma_{R_1}^2}{{R_1}^4}</math> , and   
<math>\sigma_{T_2}^2 = \frac{\sigma_{R_2}^2}{{R_2}^4}</math>

To calculate <math>\tfrac{R_2}{R_1}</math>, just divide <math>R_2</math> by <math>R_1</math>. To calculate the variance of that value, use:

<math>\sigma_{\frac{R_2}{R_1}}^2 = \left (\frac{{R_2}^2}{{R_1}^4}\right ) \sigma_{R_1}^2 + \left (\frac{1}{{R_1}^2}\right ) \sigma_{R_2}^2</math>

If there is an error such as dividing by 0, remove the errors and leave blank. Save the file as a tab deliminated text file.
'''Example File''' 
[[Image:R2-R1example.jpg|500px]] 
 
 
 

====''Setting up Input File''====
*Copy the ubq.in file into the directory containing your translated/rotated pdb file
** The ubq.in file is located in ''${FMF_PATH}''/r2r1_diffusion/linux

'''ubq.in File''' 
[[Image:Ubqinputexample.jpg|500px]] 

*'''Editing the Ubq.in'''
**Second Line
*** The 300 represents the number of spin systems in the R2/R1 data. You will need to change this to the correct number
*** The 500.13 represents the frequency of NMR you used. For example, if you use the 600 Mhz NMR, change to 600.13.
*** The 100 represents the # of simulations. 100 simulations in the minimum needed.
** Third Line
*** The 4.0453e+7 is your initial estimate for D-isotropic tensor. This does not need to be changed unless you have an estimate
*** The 0.800 is the estimated Dpar/Dper tensor. If you do not know an estimate, set this to 1.00
*** The 0.872 and 0.628 are your Theta and Phi values. If you do not know an estimate, set these values to 0.000.
** Fourth Line
*** The 0.8 and 1.2 represents the predicted the High and low ratio set limits on Dpar/Dper. These values do not need to be changed.
*** The 10 represents the number of steps of grid searching to be performed. This does not need to be changed. Increasing the number of steps may help accuracy, however may increase the calculation time
** Fifth Line is your R2R1.txt file
** Sixth Line is your input pdb file generated from pdbinertia
** Seventh Line is your output pdb file

* '''Running the program'''
** type the following command
***'''r2r1_diffusion ubq.in > ubq.out'''
***Or '''r2r1_diffusion64 ubq.in > ubq.out'''
*** When the program is finished, you will need to check the Dpar/Dper value
****Type vi ubq.out
****Type :$
**** This will take you to the bottom of the file. Start scrolling up until you see the final parameters. Here is an example you will be seeing.
[[Image:Ubiqoutput.jpg|500px]]
* If your Dpar/Dper value is the same as your minimum value or maximum value you set on the fourth line in the ubq.in file, you will need to increase the range and rerun the calculation.
* Your predicted Theta and Phi angles will be calcuated. Save those numbers to implement them into FastModelFree.
* The output pdb is what will be used for FastModelFree 
 
 
 

==Fast ModelFree==
===Setting up Files for FastModelFree===
**You will need to copy the FMF.config into your directory containing your R2.txt, R1.txt, NOE.txt, and pdb file
**FMF.config is located in ${FMF_PATH} 
**You can not have more than 300 spin systems. If there is more than 300 spin systems, the program will just hang and do nothing but act like its calculating.
 
FMF.config
[[Image:FMFconfig.jpg|500px]]
 
*Parameter to modify
** The manual for all the parameters are located in ''${FMF_PATH}''/fmfdox.pdf.
** '''tensor'''- Set as '''Isotropic''' for spherical or '''Axial Symmetric''' for non-spherical
*** Make sure the A and S is capitalized for Axial Symmetric or else it will autorun as Isotropic
*** You do not need a pdb file for Isotropic calculations
**'''cutoff''' set to 0.95
**'''Fcutoff''' set to 0.80
**'''optimize''' set to Yes
**'''maxloop''' set to 10. This means the program will stop after 10 iterations. If your final values have did not converge you may want to set to higher numbers such as 25 or 50 iterations.
**'''almost1''' set to 20. You can set to higher values just like maxloop
**'''S2cutoff''' set to 0.7
**'''seed''' set to 1985
** '''numsim''' This is the number of simulation. If you want a quick and dirty estimate, you can set it to about 10 simulations. Final calculations should have a minimum of 500 simulations
**'''jobname''' set this as the name of your protein. This is the title for all your output files.
**'''gamma''' set to -2.71
**'''rNH''' set to 1.02
**'''N15CSA''' set to -160
**'''tm''' This is your predicted correlation time. If you are unsure of a correlation time, 10.0 is a good start. The farther away from the actual value the more iterations is needed and longer it takes for calculations
*** You can use HyroNMR to help predict correlation time [[http://leonardo.inf.um.es/macromol/programs/hydronmr/hydronmr.htm]]
**'''tmMin''' Set to 0.0 if first time. You can set this min value near the actual value when you have a general idea what the tm is.
**'''tmMax''' Set to 40.0 if first time. You can set the max value near the actual value when you have a general idea what the tm is.
*** tmMin and tmMax are ranges you set to believe where the actual value is.
**'''tmGrid''' set to 50. Can set to higher value for more thorough calculation
**'''tmConv''' set to 0.001 This is the convergence cutoff. You want this to be small. The accuracy decreases as you increase this value.
**'''Dratio''' This is your Dpar/Dper ratio you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''DratioMin''' Set the value to be smaller than your Dratio
**'''DratioMax''' Set the value to be larger than your Dratio
*** DratioMin and DratioMax are your ranges you set. You want this range to be large when your first start out. After multiple attempts you can decrease the range.
**'''DratioConv''' set to 0.001
**'''Phi''' This is the Phi that you calculated using R2R1_diffusion calculations. You will find it in ubq.out file
**'''PhiMin''' set to 0
**'''PhiMax''' set to 360
**'''PhiGrid''' set to 20. Increase this value increases the thoroughness of the calculation
**'''PhiConv''' set to 0.001
**'''modle1only''' set to No
**'''mdpb''' Name of your pdb file generated from R2R1_diffusion calculations
**'''file{0}{R1}''' Name of your R1 file
**'''file{0}{R2}''' Name of your R2 file
**'''file{0}{NOE}''' Name of your NOE file
***'''file{0}{field}''' Set to 500 if using 500 MHz NMR, 600 if using 600Mhz NMR, etc.
 
 
 

===Running FastModelFree===
*To run the programs type this command
**'''fastMF > mf.log &'''
**The & symbol allows fastMF to run in the background. This process may take a minimum of 10 minutes or a few days depending how close or far away your predicted tm, Dratio, and Phi to the converged values
** You will initially see mfmodel, mfinput, mfout files, Jobname.MFDATA, Jobname.MFPAR. These files are output files generated by fastmodelfree, which are parameter sets to be used for ModelFree. The program isn't finished yet.
** You will then see your Jobname.1.pdb, Jobname.1.par. These are iterations being calculated attempting to converge all your values.
** When the calculations are finished check the Jobname.log file to see if your values converged
'''Example Protein.log''' 
[[Image:FMFresultslog.jpg|500px]]
* The tm, Dratio, Theta, and Phi values can be found in this log file
** The Example Protein.log figure show that it took 7 iterations to for the values to converge. Protein is the protein name for the figure The correlation time, Dratio, Theta, and Phi are 23.009, 0.819, -0.006, and -157.900 respectively
*The last pdb generated is the actual pdb generated from the converged values. The name of this file would be call Protein.7.pdb, where the 7 is the 7th iteration.
*Jobname.#.par is the results of your S2, te, Rex values. You will want to use the last .par file created. For example Protein.7.par is the actual results you want to use, in which all the value were converged.
'''Example Protein.7.par''' 

[[Image:FMFparresults.jpg|700px]]
 
 
 
==Generating High Quality Graphs==
===Setting up Gnuplot Scripts===
*Gnuplot is used to generate R1, R2, NOE, S2, Te, Rex graphs
*The files are located in ''${FMF_PATH}''/generate_plot
**There are 6 *.plt scripts to generate each script, and transfer them to your directory consisting of your fastmodelfree results, R1, R2, NOE data.
*The Jobname.#.par file consisting of your final values will need to be edited, because gnuplot does not like empty spaces in the data set. Instead you will have to insert the letter "a" in all the blank spaces. This can be done easily using excel or other programs. 
 
'''Example edited.Protein.7.par''' 
[[Image:editedFMFparresults.jpg|700px]]
 
 
 
*Each .plt script will have to be edited, based on the x,y axis and input files
'''Example what to edit''' 
[[Image:Relaxationgraph.jpg|700px]]
 
 
*Editing R2modelfree.plt, R1modelfree.plt, NOEmodelfree.plt, Temodelfree.plt, Rexmodelfree.plt, s2modelfree.plt
** set your xrange as [0: #ofresidue + 2] Adding +2 to x range will allow the last point to be in the graph instead at the edge
** setting yrange. This is normally commented out (ex.adding # at beginning of line). This allows the y range to be to be set up automatically.
*** If the graph looks bad at y range, Take out the # sign. Change the numbers in the bracket to the range you want. example [0:12]
** setting xtic or ytics. The tics are normally setup automatically.
*** If you do not like the range of tic set up. You can set your own tic range
**** example: '''xtics 3.0 nomirror out font "Times, 16"'''. The 3.0 set the tic range at 3.0 values apart, therefor you will see a tic at 3.0, 6.0, 9.0.
** Setting up horizontal line where y=0. This is for s2modelfree.plt only
*** Where is says set arrow 1 from 190. Change the 190 to the max range you set for your x axis.
** Setting up your input file
*** At the bottom of the script where it says '''plot'''
****The script is designed to compare two different plots, where there is 3 lines involved.
****The first line is the data for your first protein. The second and third line is the data for your second protein. If you only have on protein data set, you can comment out the second and third line with a # sign at the beginning of the line.
*** For R2modelfree.plt use R2.txt data
*** For R1modelfree.plt use R1.txt data
*** For NOEmodelfree.plt use NOE.txt data
*** for Temodelfee.plt, rexmodelfree.plt, and S2modelfree.plt use edited Jobname.par generated by modelfree
 
 

===Running Gnuplot Scripts===
*To run the program, type the name of .plt file to run it.
** example: Type R2modelfree.plt to create R2 graph.
** a .ps file will be generated which contains your graph.
** to generate a pdf. Type '''pstopdf filename of graph'''
 
 
'''Example of a plot'''
[[Image:S2plot.jpg|700px]]

Processing CEST Data

2017-03-14T21:38:28Z

Jcatazaro:

Processing CEST Data

# Install X11, nmrPipe, Python >=2.7, and ChemEx (from the Kay lab)
# Open a c-shell with “csh” and navigate to the directory with your ser file
# Open X11
# In the terminal type “bruker”
# Click “read parameters”
# Switch the F1 and F2 axes
##y should be real with N total and valid points
##z should be the 15N dimension
## zMODE Echo-Antiecho
## yMODE Real
## aq2D States
# Save the script. It should look like the script that follows this protocol.
# Add these lines:
##xyz2pipe –in fid/test%03d.fid –z –ri2c
##| pipe2xyz –out fids/UBQ%03d.fid –y –ov
##/bin/rm –fr data/
# Run the fid.com script you just saved.
# Input file names and phase values into ftcpmg.com (follows this protocol) and run
# Open test spectrum in nmrDraw
# Pick peaks and save the peak list
# Rename the peaks to their corresponding amino acid and residue #. Alternatively, create a peakList.txt with their IDs and run addASS.py.
# Run autoFit.tcl in the c-shell.
##autoFit.tcl –specName fts/UBQ%03d.ft2 –inTab test.tab –series
# Run extract_profiles_bru.py (follows)
##./extract_profiles_bru.py –tbl nlin.tab –par fq3list –out fit/
# Make sure the .out files are named like “A#N-HN.out”
# Then run ChemEx, check their tutorial

Processing CEST Data

2017-03-14T21:37:59Z

Jcatazaro: Created page with "Processing CEST Data # Install X11, nmrPipe, Python >=2.7, and ChemEx (from the Kay lab) # Open a c-shell with “csh” and navigate to the directory with your ser file # O..."

Processing CEST Data

# Install X11, nmrPipe, Python >=2.7, and ChemEx (from the Kay lab)
# Open a c-shell with “csh” and navigate to the directory with your ser file
# Open X11
# In the terminal type “bruker”
# Click “read parameters”
# Switch the F1 and F2 axes
##y should be real with N total and valid points
##z should be the 15N dimension
- zMODE Echo-Antiecho
- yMODE Real
- aq2D States
# Save the script. It should look like the script that follows this protocol.
# Add these lines:
##xyz2pipe –in fid/test%03d.fid –z –ri2c
##| pipe2xyz –out fids/UBQ%03d.fid –y –ov
##/bin/rm –fr data/
# Run the fid.com script you just saved.
# Input file names and phase values into ftcpmg.com (follows this protocol) and run
# Open test spectrum in nmrDraw
# Pick peaks and save the peak list
# Rename the peaks to their corresponding amino acid and residue #. Alternatively, create a peakList.txt with their IDs and run addASS.py.
# Run autoFit.tcl in the c-shell.
##autoFit.tcl –specName fts/UBQ%03d.ft2 –inTab test.tab –series
# Run extract_profiles_bru.py (follows)
##./extract_profiles_bru.py –tbl nlin.tab –par fq3list –out fit/
# Make sure the .out files are named like “A#N-HN.out”
# Then run ChemEx, check their tutorial

Category:Protocols

2017-03-14T21:32:25Z

Jcatazaro:

Collecting CEST Data

2017-03-14T21:29:44Z

Jcatazaro:

Collecting CEST Data

# Open TopSpin >= 3.2.X
# Edit temperature
##“edte”
# Create a dataset and collect a NHSQC to check resolution and NS
# Type “edpy”, select n15cest_gb.py.
# Click “execute” and hit “ok”.
# Carrier Positions
##1H water pos. (O1) = 3290.987 Hz (this can be changed)
##15N carrier pos = 118 ppm
##13C carrier pos = 118ppm (pseudo dimension)
# Click “Ok”
# Calibrated pulse values:
##1H hard pulse = 10.25us @ -7.558748 dB (can be changed)
##15N hard pulse = 35us @ -19.0309 dB
##13C hard pulse = 12us @ -18.60338 dB
# Click “Ok”
# Values related to the weak B1 irradiation:
##15N weak B1 field = 25 Hz (can be changed)
##Upfield irradiation offset limit = 100 ppm (can be changed)
##Downfield irradiation offset limit = 140 ppm (can be changed)
##Offset step size = 25 Hz (can be changed)
# Click “Ok”
# The experiment dataset will now be created. F1 is the pseudo dimension with the TD being the # of offsets that will be collected. The delays for these offsets can be found it FQLIST/FQ3LIST/n15cest_offset_std. This list name is overwritten each time a new CEST experiment is created so it is best to rename.
# Edit F3 and F2 TDs.
# DS must be >= 32.
# NS must be >= 2.
# Edit routing.
# Click “Edit” for CPDPRG, change 90x240y90x to 90x_240y_90x
# Make sure P50, P51, and P52 are all 500us
# Calculate receiver gain
##“rga”
# Edit D2 (t1 time)
# Must also edit D3 so that D3 > D2.
# Check ZGOPTNS
# “zg”
# The planes can be checked with “XFB”
##“xfb s23” plane 1
# Also, collecting the first plane is advisable.

Collecting CEST Data

2017-03-14T21:29:00Z

Jcatazaro:

Collecting CEST Data

# Open TopSpin >= 3.2.X
# Edit temperature
##“edte”
# Create a dataset and collect a NHSQC to check resolution and NS
# Type “edpy”, select n15cest_gb.py.
# Click “execute” and hit “ok”.
# Carrier Positions
**1H water pos. (O1) = 3290.987 Hz (this can be changed)
**15N carrier pos = 118 ppm
**13C carrier pos = 118ppm (pseudo dimension)
# Click “Ok”
# Calibrated pulse values:
**1H hard pulse = 10.25us @ -7.558748 dB (can be changed)
**15N hard pulse = 35us @ -19.0309 dB
**13C hard pulse = 12us @ -18.60338 dB
# Click “Ok”
# Values related to the weak B1 irradiation:
*15N weak B1 field = 25 Hz (can be changed)
*Upfield irradiation offset limit = 100 ppm (can be changed)
*Downfield irradiation offset limit = 140 ppm (can be changed)
*Offset step size = 25 Hz (can be changed)
# Click “Ok”
# The experiment dataset will now be created. F1 is the pseudo dimension with the TD being the # of offsets that will be collected. The delays for these offsets can be found it FQLIST/FQ3LIST/n15cest_offset_std. This list name is overwritten each time a new CEST experiment is created so it is best to rename.
# Edit F3 and F2 TDs.
# DS must be >= 32.
# NS must be >= 2.
# Edit routing.
# Click “Edit” for CPDPRG, change 90x240y90x to 90x_240y_90x
# Make sure P50, P51, and P52 are all 500us
# Calculate receiver gain
##“rga”
# Edit D2 (t1 time)
# Must also edit D3 so that D3 > D2.
# Check ZGOPTNS
# “zg”
# The planes can be checked with “XFB”
##“xfb s23” plane 1
# Also, collecting the first plane is advisable.