# Estimation of measurement time

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The minimum measurement time of (4,3)D GFT experiments can be reliably predicted from the S/N distribution of a 2D [15N, 1H] HSQC and the rotational correlation time tau_c. First, you need to generate an integrated peaklist.

#### **Peak Integration in 2D [15N, 1H] HSQC**

Having processed 2D (15N, 1H) HSQC as described in data process, one can do the peak picking and integration of 2D (15N, 1H) HSQC manually or semi-automatically as following:

- Peak picking and integration by using program XEASY

- Use command
`ns`to load the spectrum - Use
`ls`to load the corresponding sequence file (optional) - Use
`in`to automatic pick peaks with total peak number slightly more than expected by select adjust contour level; then manually remove side-chain amide peaks - Use
`mn`to measure noise level, and use`tw`to display the noise level value. Normally the value of 2.5 times of standard deviation ( ~250 if the noise level has been normalized ) is taken as noise level. - Use
`ip`to choose peak height (m) as integration mode, and use "ii" to integrate the whole spectrum. An XEASY external program "PeakintI" can be used to obtain more accurate peak height values, which is described below. - Use
`wp`to save the peak list.

- Use command

- More accurate peak height measurement by using program PeakintI.

Type command:

`peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1`

where the`nhsqcsa_b.peaks`is the input peak list and the output file will be`inhsqcsa_b.peaks`. - Combing atom name informaiton in the peak list for S/N distribution (optional).

#### **S/N distribution analysis for 2D (15N, 1H) HSQC**

The SN distribution of resonances in a NMR spectra can be fit to the Gaussian distribution:

`f= a*exp(-0.5*((ln(SN_i)-ln(SN_0))/b)^2) `

where `SN_0` is the most populated S/N observed, `f is the expected population at a certain SN value SN_i`, `a and b` are constants.

The SN of NHSQC `SN_0` can be obtained as following:

- Calculate
`ln(SN)`for each peak from the peak height and noise level, e.g. by using EXCEL - Obtain SN distribution (
`population v.s. ls(SN)`) by using Sigma-Plot - By using Sigma-Plot, fitting SN distribution to Gaussian distribution and obtain
`SN_0`, constant`a and b`.

#### **Calculation of Measurement Time**

The SN distribution of resonances in other NMR spectra can also be fit to the Gaussian distribution as in 2D (15N, 1H) NHSQC:

`f= a*exp(-0.5*((ln(SN_i)-ln(SN_0))/b)^2)`

where `SN_0` is the most populated SN observed, `f` is the expected population at a certain SN value `SN_i`, `a` and `b` are constants. Based on this equation, one can calculate the expected SN_0 for a required peak detection yield.

Assuming that a peak shall have at least SN value of 2 in order to be observed or detected, and the average b for (4,3) GFT experiments is 0.8; if 95% peak detection yield is required, the expected SN_0 is:`SN_0=exp(ln2+1.644*b)=7.4 `

NMR measurement time of (4,3) GFT HNNCABCA, (4,3)D GFT CABCAcoNHN and (4,3)D HABCABCONHN can be calculated from the following equation:

`T_43d= ((SN_43d* Tauc^2)/(SN_2d*A))^2 `

where `T_43d` is the required time for (4,3) GFT experiment, `SN_43d` is the expected SN value of (4,3)D GFT experiments, `SN_2d` is the SN per hour of 2D (15N, 1H) HSQC. =A= is constant, which has value of 0.8639 for (4,3) GFT HNNCABCA, 1.6019 for (4,3)D GFT CABCAcoNHN and 1.0153 for (4,3)D HABCABCONHN.

One can use UBNMR to run the measurement time prediction by the following command:

`predict T2d SN2d Tc`

where

`T2d`is the acquisition time of 2D [15N,1H] HSQC in hours`SN2d`is the SN distribution average of 2D [15N,1H] HSQC`Tc`is the rotational correlation time of protein in nanoseconds.

-- AlexEletski - 03 Mar 2008