Goals of tutorial. Introduce NMRbox platform
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1 Introduce NMRbox platform Goals of tutorial Showcase NMRbox with NUS tools A dozen different NUS processing tools installed and configured more coming. Demonstrate potential of NMRbox Now that the platform is maturing we can focus attention to enhancing tools, providing training materials, etc. Wrapping NUS processing tools into nmrdraw processing mode, NMRFx- Processor, Connjur Workflow Builder Learn some information about NUS NUS can improve: Time if there is sensitivity to spare Resolution Collect to longer time increments without increased experiment time Sensitivity per unit time Focus more sampling on early points with smaller time increments without sacrificing resolution
2 NUS Processing Tools in NMRbox Tool MaxEnt (RNMRTK) LONER (RNMRTK) hmsist NMRPipe IST NESTA-NMR NMRPipe NESTA SMILE MDD Tool Cambridge CS SEER nmr_wash (SCRUB) nmr_wash (CLEAN) CAMERA (MaxEnt) NMRFx-Processor IST NMRFx-Processor NESTA MDD compressed sensing Items in green are now implemented in the processing mode of NMRDraw
3 Differences in NUS techniques maxent & loner: Output frequency no DFT of indirect dimensions scrub & clean: Start with nudft spectrum and scrub sampling artifacts hmsist, NMRPipe-IST, CAMERA, NESTA-NMR, SMILE, NMRFX-Processor IST & NESTA, MDD & MDD-CS,: fill-in missing time domain data data processed with DFT afterwards Keep or replace experimental data? Phases: Some tools are independent of phase, some need to know the phase during reconstruction, and some will not work properly with a first order phase correction. Extending data beyond last collected point Deconvolution: MaxEnt can deconvolve linewidth and J-couplings Non-linearity
4 HNCACB Sample Schedule Point Spread Function
5 Processing HNCACB-nus Before starting Copy the NUS-processing directory to /scratch/username cp -r NUS-processing /scratch/username Example: cp -r NUS-processing /scratch/mmaciejewski All processing should be done in /scratch for better performance. cd NUS-processing/HNCACB-nus bruker Check Run NMRDraw in Process Mode After Conversion Select Read Parameters button Select Save Script button and press Continue Press Quit more fid.com Script uses nusexpand to expand the NUS data to a full uniform data set with zeros filling in for the missing data./fix-fid.com Script adds -extranus to the end of the nmrdraw line and uncomments the nmrdraw command more fid.com./fid.com Data is converted and opened in NMRDraw window in process mode
6 Processing HNCACB-nus In the NMRDraw processing window Press Update and Execute FT buttons Press h to get horizontal slice and move to intense peak Adjust P0 to phase peak (should be close to 90.0) Press Update and Execute FT buttons again Press Show XY, Show XZ buttons. In 15N and 13C dimension signals are pushed to the outside of the spectrum Check Rotate Halves (-alt) in both 15N and 13C dimension to fix Check Exchange Y and Z Axis checkbox to put 13C along y axis Press Update and Execute FT buttons Press Show XY, Show XZ buttons. Peaks are now centered, but 15N dimension is reversed Check Reverse (-neg) in 15N Add 9.5ppm for Region Start and 6.5ppm for Region End Press Update and Execute FT buttons Spectrum should now be processed with correct FT parameters and phases. Of course, window functions, solvent suppression, ZF size, and baseline correction can also be tweaked, but for demo we will keep things simple.
7 Processing HNCACB-nus Now let s process with NUS Reconstruction techniques Select SMILE form NUS Reconstruction pull-down menu (use right mouse button) Deselect Extrapolate Press Update and Execute NUS Press the Show NUS buttons to view 3D and 2D projections Repeat for NIH NESTA and RNMRTK MEM NOTE: Be sure to deselect Extrapolate for RNMRTK MEM due to bug that will be fixed in next release. We can compare all three NUS reconstructions as a strip plot by pressing the Strips button. You can adjust the contour level by pressing the HN/15N labels at the bottom of any given strip and scrolling with the scroll button of a mouse.
8 Auto MaxEnt Reconstruction Workflow The maximum entropy algorithm in RNMRTK has three adjustable parameters; def, aim, and lambda. Data can be processed with constant aim mode or constant lambda mode. Workflow for 3D processing with msa2d Choose Def and Aim (Based on empirical noise) Process spectrum in Constant Aim mode Look up the converged Lambda value Process the whole dataset in Constant Lambda mode Choosing def and aim aim should be a small integer multiple of the rms noise in the data def should be a value smaller than that of the smallest expected peak too small and the baseline noise distribution becomes spikey too large and the noise level will be too high lambda is determined by running a preliminary calculation in constant aim mode to determine the converged lambda value automated Determing def, aim, and lambda now automated
9 Exercises Generate quality sample schedules and process a HNCACB, 15N-NOESYHSQC, and HNCO each collected with uniform sampling, but which can be down-sampled to test different sample schedules. What are the qualities that will make a good sample schedule? Any bias, number of elements in the sample schedule, gap sizes? Do the different data sets require different sample schedule sizes? Who can do the best with the least? Feel free to use SMILE, NESTA, or RNMRTK MEM (MaxEnt). I would avoid IST as it is slow in the current incarnation of NMRPipe.
10 Workflow for HNCACB-try-nus (Bruker) Exercises cp -r HNCACB-try-nus HNCACB-unique-name Ex: cp -r HNCACB-try-nus HNCACB-test1 cd HNCACB-unique-name Ex: cd HNCACB-test-1 Create a zero-indexed sample schedule named nuslist being careful to not exceed the maximum collected increments which are 32 in t1 and 64 in t2. Note: Thus the sample schedule, being zero indexed, cannot exceed 31 or 63 in t1 and t2 respectively Uniform DFT is already present and named uniform-* for comparison../make-nus.com Use the bruker command and follow the general procedure for the HNCACB-nus experiment to process the spectra. For speed follow these guidelines Stick with single zero fill Turn off extrapolation Extract 10.6ppm to 6.6ppm Use -neg, -alt in both dimensions p0 along t3 = All others phases are 0.0 Exchange y and z axis
11 Exercises Workflow for 15N-NOESYHSQC-try-nus (Varian) For each sample schedule: cp -r 15N-NOESYHSQC-try-nus 15N-NOESYHSQC-unique-name cd HNCACB-unique-name Create a zero-indexed sample schedule named sampling.sch being careful to not exceed the maximum collected increments which are 180 in t1 and 70 in t2. Note: Thus the sample schedule, being zero indexed, cannot exceed 179 or 69 in t1 and t2 respectively Uniform DFT is already present and named uniform-* for comparison../make-nus.com Use the varian command and follow the general procedure for the HNCACB-nus experiment to process the spectra. For speed follow these guidelines Stick with single zero fill Turn off extrapolation Extract 9.6ppm to 7.0ppm p0 along t3 = All others phases are 0.0 p0 along t1 = 44.8 except RNMRTK MEM where is should be -45.2
12 Exercises The HNCO is designed for rapid testing of sample schedules. Rather than creating a sample schedule, down-sampling, converting to NMRPipe, and then manual processing with nmrdraw processing window, the HNCO can be simply processed following the directions below cd HNCO-try-nus The HNCO was run with t1 and t2 collected out to 128 points../rnmrtk.com will process the uniform data set with DFT for comparison. Files are saved with ft-. Create a sample schedule that is one indexed and does not exceed 128 in t1 or t2./msa2d.com SampleSchedule.scd BaseOutputName SampleSchedule.scd and BaseOutputName names are your choice. After running the data will be saved with the filename Base_output_name.ft3 Data is also processed with a nudft for comparison
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