David Joseph (Max Planck Institute for Multidisciplinary Sciences, Germany)
X: @DaJo_1729
Abstract: Improving the sensitivity of nuclear magnetic resonance (NMR) spectroscopy requires advancements in both instrument technology and experimental methodology. In this study, we introduce the first proton-detected large volume cryoprobe designed for 1.2 GHz instruments, leveraging optimal control pulse sequences to enhance performance (Sci. Adv. 9,eadj1133, 2023). Our results demonstrate up to a 56% increase in sensitivity and more than a twofold reduction in experimental time compared to the small volume cryoprobes in use at the moment. Additionally, we systematically optimized the experimental conditions to fully exploit the capabilities of GHz-class magnets. To further extend the benefits of our approach, we developed a library of optimal control triple resonance experiments, enabling boosted sensitivity for advanced NMR applications.
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When comparing the results from the 5 mm TCI probe at 1.2 GHz with the 5 mm TCI probe at 950 MHz, what is the most surprising/interesting/useful insight that you have personally encountered? In the future, what do you think might be the most useful/interesting insights enabled by performing experiments at 1.2 GHz?
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The most useful insight is that bio-NMR experiments perform much better using optimal control pulses. A 5 mm TCI at 950 MHz approaches the power availability limit for broadband pulses, particularly for the 13C and 15N channels. At 1.2 GHz, a 5 mm TCI can only be used with optimal control pulses. However, using optimal control pulses with fields starting from 800 MHz would provide free signal enhancement and save valuable experimental time.
The most interesting insights would come from performing experiments at 1.2 GHz to study biomolecular dynamics. All B₀-dependent parameters, such as CSA and alignment, reach their maximum values at this frequency, enabling access to data on motions that would otherwise be impossible to observe with lower field magnets. Increased resolution at 1.2 GHz would also be useful for studying larger proteins and intrinsically disordered proteins.
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Thank you for your response, David.
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These are important reference data.
1) Wouldn’t one expect that the sensitivity obtained with a Shigemi tube is either the same or less than that obtained with a conventional 5 mm tube?
2) Which compound and signal did you use to measure the sensitivities in the presence of different salt concentrations – ubiquitin or sucrose?
3) Does CSA relaxation of ubiquitin amide protons broaden their 1H NMR signals noticeably more than at, say, 950 MHz?-
1) The sensitivity of a Shigemi depends on the amount of sample available. It is especially sensitive when a lower volume of sample is available. There is also an optimal height that provides the best signal-to-noise ratio when using a Shigemi tube. Our concern here was B_1 inhomogeneity, which is lower with a Shigemi tube. However, since the pulses also compensate for ±20% inhomogeneity, we only see only a slight improvement in sensitivity when using a Shigemi tube.
2) It was p53 1-73, a disordered protein, in a Tris-Bis buffer, using optimal control HNCA sequence.
3) Thanks for the question! I just looked it up, and for an HNCO experiment, the difference is around 3 Hz, while for an HSQC, it’s around 1 Hz (along the proton dimension). It is broader at 1.2 GHz.
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1) The sensitivity of a Shigemi depends on the amount of sample available. It is especially sensitive when a lower volume of sample is available. There is also an optimal height that provides the best signal-to-noise ratio when using a Shigemi tube. Our concern here was B_1 inhomogeneity, which is lower with a Shigemi tube. However, since the pulses also compensate for ±20% inhomogeneity, we only see only a slight improvement in sensitivity when using a Shigemi tube.
2) It was p53 1-73, a disordered protein, in a Tris-Bis buffer, using optimal control HNCA sequence.
3) Thanks for the question! I just looked it up, and for an HNCO experiment, the difference is around 3 Hz, while for an HSQC, it’s around 1 Hz (along the proton dimension). It is broader at 1.2 GHz.
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Hi David, brilliant presentation. Clear, concise, and insightful.
You mentioned a useful tip about using buffers with lower conductivity and larger ions. Could you please elaborate on why this is beneficial and how exactly it helps in practice?-
Hi, thank you! This has to do with noise contribution from the sample, which is especially problematic for the cryoprobe. The noise from the sample is proportional to its conductivity and dielectric properties. Using a buffer with larger ions will lower the mobility, thus lowering the conductivity of the buffer and reducing the noise from the sample. This increases the signal-to-noise ratio of the spectrum.
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Hi, thank you! This has to do with noise contribution from the sample, which is especially problematic for the cryoprobe. The noise from the sample is proportional to its conductivity and dielectric properties. Using a buffer with larger ions will lower the mobility, thus lowering the conductivity of the buffer and reducing the noise from the sample. This increases the signal-to-noise ratio of the spectrum.
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