Preprints
https://doi.org/10.5194/mr-2025-5
https://doi.org/10.5194/mr-2025-5
14 Mar 2025
 | 14 Mar 2025
Status: this preprint is currently under review for the journal MR.

3D-printed microcell for protein NMR at high ionic strengths and small sample volumes

Tayeb Kakeshpour, Martin D. Gelenter, Jinfa Ying, and Ad Bax

Abstract. Standard solution NMR measurements use 5-mm outer diameter (OD) sample tubes that require ca 0.5 mL of solvent to minimize “end effects” on magnetic field homogeneity in the active volume of the sample. Shigemi cells reduce the solvent requirement to ca 0.29 mL. At high ionic strength, or at ultrahigh magnetic fields, smaller OD samples are needed to study samples in conductive, radiofrequency absorbing solvents such as water. We demonstrate an effective and inexpensive alternative for reducing the active sample volume to 0.13 mL by 3D printing of ellipsoidal shaped cells that are inserted into 5-mm OD NMR tubes. Static magnetic susceptibility, χ, of printer resin was measured using a simple slice-selection pulse sequence. We found that the χ of water increases linearly with NaCl concentration, from -9.05 ppm to -8.65 ppm for 0 to 2 M NaCl. The χ of D2O was measured to be -9.01 ppm. The susceptibility difference between the resin (χ= -9.40 ppm) and water can be minimized by paramagnetic doping of the resin. Such doping was found unnecessary for obtaining high quality protein NMR spectra when using ellipsoidal shaped cells that are insensitive to susceptibility mismatching. The microcells offer outstanding RF and good Bo homogeneities. Integrated 600-MHz HSQC signal intensities for the microcell sample in PBS buffer were 6.5±4 % lower than for 0.5 mL of the same protein solution in a regular 5-mm sample tube. The cell is demonstrated for N-acetylated α-synuclein in PBS buffer, and for observing tetramerization of melittin at 2 M NaCl.

Competing interests: AB is a member of the editorial board of Magnetic Resonance.

Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this preprint. The responsibility to include appropriate place names lies with the authors.
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Tayeb Kakeshpour, Martin D. Gelenter, Jinfa Ying, and Ad Bax

Status: final response (author comments only)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • CEC1: 'Editorial Comment on mr-2025-5', Geoffrey Bodenhausen, 14 Mar 2025
    • AC1: 'Reply on CEC1', Ad Bax, 14 Mar 2025
  • CC1: 'Comment on mr-2025-5', Tom Barbara, 19 Mar 2025
  • CC2: 'Comment on mr-2025-5', Tom Barbara, 19 Mar 2025
  • EC1: 'Comment on mr-2025-5', Gottfried Otting, 21 Mar 2025
    • AC2: 'Reply on EC1', Ad Bax, 21 Mar 2025
  • RC1: 'Comment on mr-2025-5', Anonymous Referee #1, 06 Apr 2025
    • CC3: 'Reply on RC1', Tom Barbara, 10 Apr 2025
  • RC2: 'Comment on mr-2025-5', Anonymous Referee #2, 07 Apr 2025
  • EC2: 'Comment on mr-2025-5', Gottfried Otting, 11 Apr 2025
  • RC3: 'Comment on mr-2025-5', Marcel Utz, 12 Apr 2025
  • EC3: 'Comment on mr-2025-5', Gottfried Otting, 13 Apr 2025
Tayeb Kakeshpour, Martin D. Gelenter, Jinfa Ying, and Ad Bax
Tayeb Kakeshpour, Martin D. Gelenter, Jinfa Ying, and Ad Bax

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Short summary
3D printed sample cells, inserted into standard 5-mm NMR tubes, are demonstrated to offer improved sensitivity for biomolecular NMR when the quantity of material is limiting. By using an ellipsoidal shape, magnetic field homogeneity inside the microcell to first order is insensitive to differences in the magnetic susceptibility of printer resin and solvent. The sample cells are particularly useful at high ionic strength and at ultra-high magnetic fields.
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