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Phasing 1H-15N HSQC Kinetics data from Bruker Instrument

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  • nicole_milkovic
    Hello group! I should mention that my lab specializes in X-Ray Crystallography and my thesis project has me learning NMR. So, I apologize for the perhaps
    Message 1 of 3 , Aug 4, 2011
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      Hello group!

      I should mention that my lab specializes in X-Ray Crystallography and my thesis project has me learning NMR. So, I apologize for the perhaps vague, often descriptive, and possible incorrect terminology of my question. That aside, I could really use some help!

      I have been using NMR to collect HSQC's for my protein for awhile now, with no problems. It wasn't until recently when I used NMR as a way to collect kinetics data on my protein that I began to experience phasing issues. I basically collected 22 1H-15N HSQC's over ~60hrs at 310K on a Bruker instrument (with cryoprobe). I am attempting to phase my first spectrum using nmrDraw, and this is where I am having issues. The top half of my spectrum contains positive peaks and the bottom half contains negative peaks (please note I am NOT referring to a single peak contain both positive and negative areas, but literally the top half and bottom half of the spectrum). As I begin to phase, it seems nearly impossible to get all of my peaks to be positive.

      Is there something wrong with the processing file I am using? Is there another way to phase this data? I will attempt to post an image of my nmrDraw screen for the initial un-phased spectrum.

      Thank you for your help!
      Nicole Milkovic

      For your convenience, these are my (generic) processing files (with phases set to 0):

      #!/bin/csh
      bruk2pipe -in /Users/name/file/2/ser \
      -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12 -grpdly -1 \
      -xN 2048 -yN 256 \
      -xT 1024 -yT 128 \
      -xMODE DQD -yMODE Echo-AntiEcho \
      -xSW 7309.942 -ySW 2027.164 \
      -xOBS 500.132 -yOBS 50.684 \
      -xCAR 4.821 -yCAR 120.136 \
      -xLAB 1H -yLAB 15N \
      -ndim 2 -aq2D States \
      -out /Users/name/file/HSQC_2.fid -verb -ov

      #!/bin/csh
      nmrPipe -in HSQC_2.fid \
      | nmrPipe -fn SOL \
      | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
      | nmrPipe -fn ZF -size 2048 \
      | nmrPipe -fn FT -auto \
      | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
      | nmrPipe -fn EXT -left -sw -verb \
      | nmrPipe -fn POLY -auto \
      | nmrPipe -fn TP \
      | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
      | nmrPipe -fn ZF -size 512 \
      | nmrPipe -fn FT -auto \
      | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
      | nmrPipe -fn POLY -auto \
      | nmrPipe -fn TP \
      -verb -ov -out HSQC_2.ft2 -inPlace
    • DeRose, Eugene (NIH/NIEHS) [C]
      Hi Nicole, If the first data point in the indirect dimension started at half the dwell time, the phase correction in the indirect dimension should be -p0 -90
      Message 2 of 3 , Aug 5, 2011
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        Hi Nicole,

        If the first data point in the indirect dimension started at half the dwell time, the phase correction in the indirect dimension should be -p0 -90 -p1 180. Depending on the pulse sequence, sometimes an additional 45 degrees must be added or subtracted to the p0 correction, see the sample 3D HN processing scripts in the "macro edit" pull down in NMRDraw. In addition, when using half dwell acquisition, any aliased peaks will have opposite sign. Note also, the first point multiplier in the SP statement should be -c 1.0 in this case, again, see the sample 3D HN scripts in NMRDraw for examples.

        Best Regards,
        Eugene

        Eugene DeRose, Ph.D.
        NIEHS NMR Laboratory Manager
        Contractor to NIEHS/DIR
        National Institute of Environmental Health Sciences
        Research Triangle Park, NC 27709
        919.541.1981 voice
        derose@...


        On 8/4/11 1:23 PM, "nicole_milkovic" <nmmilkov@...> wrote:






        Hello group!

        I should mention that my lab specializes in X-Ray Crystallography and my thesis project has me learning NMR. So, I apologize for the perhaps vague, often descriptive, and possible incorrect terminology of my question. That aside, I could really use some help!

        I have been using NMR to collect HSQC's for my protein for awhile now, with no problems. It wasn't until recently when I used NMR as a way to collect kinetics data on my protein that I began to experience phasing issues. I basically collected 22 1H-15N HSQC's over ~60hrs at 310K on a Bruker instrument (with cryoprobe). I am attempting to phase my first spectrum using nmrDraw, and this is where I am having issues. The top half of my spectrum contains positive peaks and the bottom half contains negative peaks (please note I am NOT referring to a single peak contain both positive and negative areas, but literally the top half and bottom half of the spectrum). As I begin to phase, it seems nearly impossible to get all of my peaks to be positive.

        Is there something wrong with the processing file I am using? Is there another way to phase this data? I will attempt to post an image of my nmrDraw screen for the initial un-phased spectrum.

        Thank you for your help!
        Nicole Milkovic

        For your convenience, these are my (generic) processing files (with phases set to 0):

        #!/bin/csh
        bruk2pipe -in /Users/name/file/2/ser \
        -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12 -grpdly -1 \
        -xN 2048 -yN 256 \
        -xT 1024 -yT 128 \
        -xMODE DQD -yMODE Echo-AntiEcho \
        -xSW 7309.942 -ySW 2027.164 \
        -xOBS 500.132 -yOBS 50.684 \
        -xCAR 4.821 -yCAR 120.136 \
        -xLAB 1H -yLAB 15N \
        -ndim 2 -aq2D States \
        -out /Users/name/file/HSQC_2.fid -verb -ov

        #!/bin/csh
        nmrPipe -in HSQC_2.fid \
        | nmrPipe -fn SOL \
        | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
        | nmrPipe -fn ZF -size 2048 \
        | nmrPipe -fn FT -auto \
        | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
        | nmrPipe -fn EXT -left -sw -verb \
        | nmrPipe -fn POLY -auto \
        | nmrPipe -fn TP \
        | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
        | nmrPipe -fn ZF -size 512 \
        | nmrPipe -fn FT -auto \
        | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
        | nmrPipe -fn POLY -auto \
        | nmrPipe -fn TP \
        -verb -ov -out HSQC_2.ft2 -inPlace







        Eugene DeRose, Ph.D.
        NIEHS NMR Laboratory Manager
        Contractor to NIEHS/DIR
        National Institute of Environmental Health Sciences
        Research Triangle Park, NC 27709
        919.541.1981 voice
        derose@...
      • nicole_milkovic
        De. DeRose, Thank you for directing me to the macro edit pull down in NMRDraw. This, in addition to your comment regarding dwell times, has seemed to solve
        Message 3 of 3 , Aug 9, 2011
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          De. DeRose,

          Thank you for directing me to the "macro edit" pull down in NMRDraw. This, in addition to your comment regarding dwell times, has seemed to solve my problem.

          In appreciation,
          Nicole

          --- In nmrpipe@yahoogroups.com, "DeRose, Eugene (NIH/NIEHS) [C]" <derose@...> wrote:
          >
          > Hi Nicole,
          >
          > If the first data point in the indirect dimension started at half the dwell time, the phase correction in the indirect dimension should be -p0 -90 -p1 180. Depending on the pulse sequence, sometimes an additional 45 degrees must be added or subtracted to the p0 correction, see the sample 3D HN processing scripts in the "macro edit" pull down in NMRDraw. In addition, when using half dwell acquisition, any aliased peaks will have opposite sign. Note also, the first point multiplier in the SP statement should be -c 1.0 in this case, again, see the sample 3D HN scripts in NMRDraw for examples.
          >
          > Best Regards,
          > Eugene
          >
          > Eugene DeRose, Ph.D.
          > NIEHS NMR Laboratory Manager
          > Contractor to NIEHS/DIR
          > National Institute of Environmental Health Sciences
          > Research Triangle Park, NC 27709
          > 919.541.1981 voice
          > derose@...
          >
          >
          > On 8/4/11 1:23 PM, "nicole_milkovic" <nmmilkov@...> wrote:
          >
          >
          >
          >
          >
          >
          > Hello group!
          >
          > I should mention that my lab specializes in X-Ray Crystallography and my thesis project has me learning NMR. So, I apologize for the perhaps vague, often descriptive, and possible incorrect terminology of my question. That aside, I could really use some help!
          >
          > I have been using NMR to collect HSQC's for my protein for awhile now, with no problems. It wasn't until recently when I used NMR as a way to collect kinetics data on my protein that I began to experience phasing issues. I basically collected 22 1H-15N HSQC's over ~60hrs at 310K on a Bruker instrument (with cryoprobe). I am attempting to phase my first spectrum using nmrDraw, and this is where I am having issues. The top half of my spectrum contains positive peaks and the bottom half contains negative peaks (please note I am NOT referring to a single peak contain both positive and negative areas, but literally the top half and bottom half of the spectrum). As I begin to phase, it seems nearly impossible to get all of my peaks to be positive.
          >
          > Is there something wrong with the processing file I am using? Is there another way to phase this data? I will attempt to post an image of my nmrDraw screen for the initial un-phased spectrum.
          >
          > Thank you for your help!
          > Nicole Milkovic
          >
          > For your convenience, these are my (generic) processing files (with phases set to 0):
          >
          > #!/bin/csh
          > bruk2pipe -in /Users/name/file/2/ser \
          > -bad 0.0 -noaswap -DMX -decim 24 -dspfvs 12 -grpdly -1 \
          > -xN 2048 -yN 256 \
          > -xT 1024 -yT 128 \
          > -xMODE DQD -yMODE Echo-AntiEcho \
          > -xSW 7309.942 -ySW 2027.164 \
          > -xOBS 500.132 -yOBS 50.684 \
          > -xCAR 4.821 -yCAR 120.136 \
          > -xLAB 1H -yLAB 15N \
          > -ndim 2 -aq2D States \
          > -out /Users/name/file/HSQC_2.fid -verb -ov
          >
          > #!/bin/csh
          > nmrPipe -in HSQC_2.fid \
          > | nmrPipe -fn SOL \
          > | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
          > | nmrPipe -fn ZF -size 2048 \
          > | nmrPipe -fn FT -auto \
          > | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
          > | nmrPipe -fn EXT -left -sw -verb \
          > | nmrPipe -fn POLY -auto \
          > | nmrPipe -fn TP \
          > | nmrPipe -fn SP -off 0.5 -end 0.98 -pow 2 -c 0.5 \
          > | nmrPipe -fn ZF -size 512 \
          > | nmrPipe -fn FT -auto \
          > | nmrPipe -fn PS -p0 0.0 -p1 0.0 -di \
          > | nmrPipe -fn POLY -auto \
          > | nmrPipe -fn TP \
          > -verb -ov -out HSQC_2.ft2 -inPlace
          >
          >
          >
          >
          >
          >
          >
          > Eugene DeRose, Ph.D.
          > NIEHS NMR Laboratory Manager
          > Contractor to NIEHS/DIR
          > National Institute of Environmental Health Sciences
          > Research Triangle Park, NC 27709
          > 919.541.1981 voice
          > derose@...
          >
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