Abstract
Structural characterization of powder materials, including those synthesized by mechanochemical methods, remains challenging due to the lack of single crystals suitable for X-ray diffraction. Microcrystal-Electron Diffraction (MicroED) enables structure determination from sub-micrometer crystallites but faces limitations, particularly in locating hydrogen atoms and distinguishing light atoms (C, N, O). We present a general workflow that integrates MicroED with high-resolution mass spectrometry, database mining, solution and solid-state NMR, and DFT-D/GIPAW calculations to resolve atomic structures of complex powders, even with unknown composition. The approach is demonstrated on a pyridoxine-N-acetyl-L-cysteine salt, a mechanochemically synthesized adduct for which large single crystals could not be obtained, and on N-formyl-methionyl-leucyl-phenylalanine (fMLF), a bacterial chemoattractant peptide. This strategy enables comprehensive structure resolution, including identification of molecular components, crystal packing, atom assignments and hydrogen positions. Its modularity and scalability make it suitable for a wide range of powder materials, e.g., pigments, pharmaceutical compounds, etc., especially when conventional crystallography fails.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. The Supplementary Information includes materials and methods details, DART-HRMS spectra, solution and solid-state NMR data, PXRD patterns and Rietveld refinements, database-mining outputs, GIPAW-calculated NMR parameters, and full comparison tables between experimental and computed chemical shifts for both PN–NAC and fMLF. The MicroED crystal structures reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition numbers 2506116 (PN–NAC) and 2506115 (fMLF). These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. The DFT-D-optimized structures are provided as separate supplementary files: Supplementary Data 1 and 2 for PN–NAC (optimized with variable and fixed lattice parameters, respectively); and Supplementary Data 3 for fMLF (optimized with variable lattice parameters).
References
Solares-Briones, M. et al. Mechanochemistry: a green approach in the preparation of pharmaceutical cocrystals. Pharmaceutics 13, 790 (2021).
Braga, D., Maini, L. & Grepioni, F. Mechanochemical preparation of co-crystals. Chem. Soc. Rev. 42, 7638–7648 (2013).
Ying, P., Yu, J. & Su, W. Liquid-assisted grinding mechanochemistry in the synthesis of pharmaceuticals. Adv. Synth. Catal. 363, 1246–1271 (2021).
Hasa, D. & Jones, W. Screening for new pharmaceutical solid forms using mechanochemistry: A practical guide. Adv. Drug Deliv. Rev. 117, 147–161 (2017).
Carneiro, R. L. et al. Mechanochemical synthesis and characterization of a novel AAs–Flucytosine drug–drug cocrystal: a versatile model system for green approaches. J. Mol. Struct. 1251, 132052 (2022).
Kamali, N., Gniado, K., McArdle, P. & Erxleben, A. Application of ball milling for highly selective mechanochemical polymorph transformations. Org. Process Res. Dev. 22, 796–802 (2018).
Do, J.-L. & Friščić, T. Mechanochemistry: a force of synthesis. ACS Cent. Sci. 3, 13–19 (2017).
Caira, M. R., Nassimbeni, L. R. & Wildervanck, A. F. Selective formation of hydrogen bonded cocrystals between a sulfonamide and aromatic carboxylic acids in the solid state. J. Chem. Soc., Perkin Trans. 2, 2213–2216 (1995).
Raheem Thayyil, A., Juturu, T., Nayak, S. & Kamath, S. Pharmaceutical co-crystallization: regulatory aspects, design, characterization, and applications. Adv. Pharm. Bull. 10, 203–212 (2020).
Aitipamula, S. et al. Polymorphs, salts, and cocrystals: what’s in a name? Cryst. Growth Des. 12, 2147–2152 (2012).
Karagianni, A., Malamatari, M. & Kachrimanis, K. Pharmaceutical cocrystals: new solid phase modification approaches for the formulation of APIs. Pharmaceutics 10, 18 (2018).
Yadav, A. V., Shete, A. S., Dabke, A. P., Kulkarni, P. V. & Sakhare, S. S. Co-crystals: a novel approach to modify physicochemical properties of active pharmaceutical ingredients. Indian J. Pharm. Sci. 71, 359–370 (2009).
Vishweshwar, P., McMahon, J. A., Bis, J. A. & Zaworotko, M. J. Pharmaceutical co-crystals. J. Pharm. Sci. 95, 499–516 (2006).
Miroshnyk, I., Mirza, S. & Sandler, N. Pharmaceutical co-crystals-an opportunity for drug product enhancement. Expert Opin. Drug Deliv. 6, 333–341 (2009).
Jones, W., Motherwell, W. D. S. & Trask, A. V. Pharmaceutical cocrystals: an emerging approach to physical property enhancement. MRS Bull. 31, 875–879 (2006).
Schultheiss, N. & Newman, A. Pharmaceutical cocrystals and their physicochemical properties. Cryst. Growth Des. 9, 2950–2967 (2009).
Ross, S. A., Ward, A., Basford, P., McAllister, M. & Douroumis, D. Coprocessing of pharmaceutical cocrystals for high quality and enhanced physicochemical stability. Cryst. Growth Des. 19, 876–888 (2019).
Gokhale, M. Y. & Mantri, R. V. Chapter 4 - API solid-form screening and selection. In Developing Solid Oral Dosage Forms (Second Edition) (eds, Qiu, Y., Chen, Y., Zhang, G. G. Z., Yu, L. & Mantri, R. V.) 85–112 (Academic Press, 2017).
Schultheiss, N. & Henck, J.-O. Role of co-crystals in the Pharmaceutical Development Continuum. In Pharmaceutical Salts and Co-crystals (eds. Wouters, J. & Quéré, L.) 0 (The Royal Society of Chemistry, 2011).
D’Abbrunzo, I. et al. Higher-order multicomponent crystals as a strategy to decrease the IC50 parameter: the case of praziquantel, niclosamide and acetic acid. J. Drug Deliv. Sci. Technol. 109, 106974 (2025).
Pindelska, E., Sokal, A. & Kolodziejski, W. Pharmaceutical cocrystals, salts and polymorphs: advanced characterization techniques. Adv. Drug Deliv. Rev. 117, 111–146 (2017).
Bravetti, F., Hühn, R., Bordignon, S., Reibeling, S. & Schmidt, M. U. Crystal structure and tautomeric state of Pigment Red 48:2 from X-ray powder diffraction and solid-state NMR. Z. f.ür. Kristallographie Crystalline Mater. 239, 283–297 (2024).
Luedeker, D., Gossmann, R., Langer, K. & Brunklaus, G. Crystal engineering of pharmaceutical co-crystals: “NMR crystallography” of niclosamide co-crystals. Cryst. Growth Des. 16, 3087–3100 (2016).
Bravetti, F. et al. Solid-state NMR-driven crystal structure prediction of molecular crystals: the case of mebendazole. Chem. A Eur. J. 28, e202103589 (2022).
Fernandes, J. A., Sardo, M., Mafra, L., Choquesillo-Lazarte, D. & Masciocchi, N. X-ray and NMR crystallography studies of novel theophylline cocrystals prepared by liquid assisted grinding. Cryst. Growth Des. 15, 3674–3683 (2015).
Hodgkinson, P. NMR crystallography of molecular organics. Prog. Nucl. Magn. Reson. Spectrosc. 118–119, 10–53 (2020).
Baias, M. et al. Powder crystallography of pharmaceutical materials by combined crystal structure prediction and solid-state 1H NMR spectroscopy. Phys. Chem. Chem. Phys. 15, 8069–8080 (2013).
Bravetti, F. et al. Zwitterionic or not? Fast and reliable structure determination by combining crystal structure prediction and solid-state NMR. Molecules 28, 1876 (2023).
Khalaji, M., Paluch, P., Potrzebowski, M. J. & Dudek, M. K. Narrowing down the conformational space with solid-state NMR in crystal structure prediction of linezolid cocrystals. Solid State Nucl. Magn. Reson. 121, 101813 (2022).
Dudek, M. K. et al. Crystal structure determination of an elusive methanol solvate – hydrate of catechin using crystal structure prediction and NMR crystallography. CrystEngComm 22, 4969–4981 (2020).
Elena, B., Pintacuda, G., Mifsud, N. & Emsley, L. Molecular structure determination in powders by NMR crystallography from proton spin diffusion. J. Am. Chem. Soc. 128, 9555–9560 (2006).
Harris, R. K. NMR crystallography: the use of chemical shifts. Solid State Sci. 6, 1025–1037 (2004).
Helliwell, J. R. NMR crystallography. In Certifying Central Facility Beamlines for Biological and Chemical Crystallography and Allied Methods (ed. Helliwell, J. R.) 63–64 (Springer Nature, 2025).
Harris, R. K. NMR studies of organic polymorphs & solvates. Analyst 131, 351–373 (2006).
Lahtinen, M., Behera, B., Kolehmainen, E. & Maitra, U. Unraveling the packing pattern leading to gelation using SS NMR and X-ray diffraction: direct observation of the evolution of self-assembled fibers. Soft Matter 6, 1748–1757 (2010).
Kolehmainen, E. et al. Solid state NMR studies of gels derived from low molecular mass gelators. Soft Matter 12, 6015–6026 (2016).
Nonappa, Lahtinen, M., Ikonen, S., Kolehmainen, E. & Kauppinen, R. Solid-state NMR, X-ray diffraction, and thermoanalytical studies towards the identification, isolation, and structural characterization of polymorphs in natural bile acids. Cryst. Growth Des. 9, 4710–4719 (2009).
Guzmán-Afonso, C. et al. Understanding hydrogen-bonding structures of molecular crystals via electron and NMR nanocrystallography. Nat. Commun. 10, 3537 (2019).
Duong, N. T., Aoyama, Y., Kawamoto, K., Yamazaki, T. & Nishiyama, Y. Structure solution of nano-crystalline small molecules using microED and solid-state NMR dipolar-based experiments. Molecules 26, 4652 (2021).
Oikawa, T., Okumura, M., Kimura, T. & Nishiyama, Y. Solid-state NMR meets electron diffraction: Determination of crystalline polymorphs of small organic microcrystalline samples. Acta Crystallogr. Sect. C Struct. Chem. 73, 219–228 (2017).
Nishiyama, Y. Locating hydrogen atoms using fast-MAS solid-state NMR and microED. In NMR Spectroscopy for Probing Functional Dynamics at Biological Interfaces (eds. Bhunia, A., Atreya, H. S. & Sinha, N.), (The Royal Society of Chemistry, 2022).
Rodriguez, J. A. & Gonen, T. Chapter Fourteen - High-resolution macromolecular structure determination by microED, a cryo-EM method. In Methods in Enzymology (ed. Crowther, R. A.) Vol. 579, 369–392 (Academic Press, 2016).
Nannenga, B. L. MicroED methodology and development. Struct. Dyn. 7, 014304 (2020).
Jones, C. G. et al. The CryoEM method MicroED as a powerful tool for small molecule structure determination. ACS Cent. Sci. 4, 1587–1592 (2018).
Gruene, T. et al. Rapid structure determination of microcrystalline molecular compounds using electron diffraction. Angew. Chem. Int Ed. Engl. 57, 16313–16317 (2018).
Kolb, U., Gorelik, T., Kübel, C., Otten, M. T. & Hubert, D. Towards automated diffraction tomography: Part I—Data acquisition. Ultramicroscopy 107, 507–513 (2007).
Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q Rev. Biophys. 28, 171–193 (1995).
Nannenga, B. L. & Gonen, T. MicroED: a versatile cryoEM method for structure determination. Emerg. Top. Life Sci. 2, 1–8 (2018).
Hattne, J. et al. MicroED data collection and processing. Acta Crystallogr. A Found. Adv. 71, 353–360 (2015).
Nannenga, B. L. & Gonen, T. MicroED opens a new era for biological structure determination. Curr. Opin. Struct. Biol. 40, 128–135 (2016).
Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T. High-resolution structure determination by continuous-rotation data collection in MicroED. Nat. Methods 11, 927–930 (2014).
van Genderen, E. et al. Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector. Acta Crystallogr. A Found. Adv. 72, 236–242 (2016).
Shi, D., Nannenga, B. L., Iadanza, M. G. & Gonen, T. Three-dimensional electron crystallography of protein microcrystals. Elife 2, e01345 (2013).
Bernasconi, D. et al. Selective synthesis of a salt and a cocrystal of the ethionamide–salicylic acid system. Cryst. Growth Des. 20, 906–915 (2020).
Chierotti, M. et al. From molecular crystals to salt co-crystals of barbituric acid via the carbonate ion and an improvement of the solid state properties. CrystEngComm 15, 7598–7605 (2013).
Maiorca, B. et al. Investigation of solid-state forms between p-aminosalicylic acid and adenine: Exploring salts, cocrystals and their polymorphism. J. Drug Deliv. Sci. Technol. 115, 107762 (2026).
Gumbert, S. D. et al. Crystal structure and tautomerism of Pigment Yellow 138 determined by X-ray powder diffraction and solid-state NMR. Dyes Pigments 131, 364–372 (2016).
Chierotti, M. R. & Gobetto, R. Solid-state NMR studies of weak interactions in supramolecular systems. Chem. Commun. 1621, 1634 (2008).
Duong, N. T., Gan, Z. & Nishiyama, Y. Selective 1H-14N distance measurements by 14N overtone solid-state NMR spectroscopy at fast MAS. Front. Mol. Biosci. 8, 645347 (2021).
Duong, N. T. et al. Accurate 1H-14N distance measurements by phase-modulated RESPDOR at ultra-fast MAS. J. Magn. Reson 308, 106559 (2019).
Duong, N. T., Raran-Kurussi, S., Nishiyama, Y. & Agarwal, V. Can proton-proton recoupling in fully protonated solids provide quantitative, selective and efficient polarization transfer?. J. Magn. Reson. 317, 106777 (2020).
Geppi, M., Mollica, G., Borsacchi, S. & Veracini, C. A. Solid-state NMR studies of pharmaceutical systems. Appl. Spectrosc. Rev. 43, 202–302 (2008).
Xu, Y., Southern, S. A., Szell, P. M. J. & Bryce, D. L. The role of solid-state nuclear magnetic resonance in crystal engineering. CrystEngComm 18, 5236–5252 (2016).
Berendt, R. T., Sperger, D. M., Munson, E. J. & Isbester, P. K. Solid-state NMR spectroscopy in pharmaceutical research and analysis. TrAC Trends Anal. Chem. 25, 977–984 (2006).
Sardo, M., Rocha, J. & Mafra, L. Solid-state NMR applications in the structural elucidation of small molecules. In Structure Elucidation in Organic Chemistry 173–240 (John Wiley & Sons, Ltd, 2015).
Cossard, A. et al. Advanced feature analysis for enhancing cocrystal prediction. Chemometrics Intell. Lab. Syst. 257, 105318 (2025).
Marasco, W. A. et al. Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. J. Biol. Chem. 259, 5430–5439 (1984).
Salamah, M. F. et al. The formyl peptide fMLF primes platelet activation and augments thrombus formation. J. Thrombosis Haemost. 17, 1120–1133 (2019).
Palatinus, L. et al. Hydrogen positions in single nanocrystals revealed by electron diffraction. Science 355, 166–169 (2017).
Brázda, P., Palatinus, L. & Babor, M. Electron diffraction determines molecular absolute configuration in a pharmaceutical nanocrystal. Science 364, 667–669 (2019).
Stein, M. & Heimsaat, M. Intermolecular interactions in molecular organic crystals upon relaxation of lattice parameters. Crystals 9, 665 (2019).
van de Streek, J. & Neumann, M. A. Validation of experimental molecular crystal structures with dispersion-corrected density functional theory calculations. Acta Cryst. B 66, 544–558 (2010).
Aramini, A. et al. Unexpected salt/cocrystal polymorphism of the ketoprofen–lysine system: discovery of a new ketoprofen–l-lysine salt polymorph with different physicochemical and pharmacokinetic properties. Pharmaceuticals 14, 555 (2021).
Bajaj, V. S., van der Wel, P. C. A. & Griffin, R. G. Observation of a low-temperature, dynamically driven structural transition in a polypeptide by solid-state NMR spectroscopy. J. Am. Chem. Soc. 131, 118–128 (2009).
Nishiyama, Y. et al. Studies of minute quantities of natural abundance molecules using 2D heteronuclear correlation spectroscopy under 100 kHz MAS. Solid State Nucl. Magn. Reson. 66–67, 56–61 (2015).
Giannozzi, P. et al. QUANTUM ESPRESSO: a modular and open-source software project for quantum simulations of materials. J. Phys. Condens Matter 21, 395502 (2009).
Lee, K., Murray, ÉD., Kong, L., Lundqvist, B. I. & Langreth, D. C. A higher-accuracy van der waals density functional. Phys. Rev. B 82, 081101 (2010).
Hamada, I. van der Waals density functional made accurate. Phys. Rev. B 89, 121103 (2014).
Prandini, G., Marrazzo, A., Castelli, I. E., Mounet, N. & Marzari, N. Precision and efficiency in solid-state pseudopotential calculations. npj Comput Mater. 4, 72 (2018).
Charpentier, T. The PAW/GIPAW approach for computing NMR parameters: a new dimension added to NMR study of solids. Solid State Nucl. Magn. Reson. 40, 1–20 (2011).
Dal Corso, A. Pseudopotentials periodic table: from H to Pu. Comput. Mater. Sci. 95, 337–350 (2014).
Franco, F., Baricco, M., Chierotti, M. R., Gobetto, R. & Nervi, C. Coupling solid-state NMR with GIPAW ab initio calculations in metal hydrides and borohydrides. J. Phys. Chem. C. 117, 9991–9998 (2013).
Harris, R. K., Hodgkinson, P., Pickard, C. J., Yates, J. R. & Zorin, V. Chemical shift computations on a crystallographic basis: some reflections and comments. Magn. Reson. Chem. 45, S174–S186 (2007).
Coelho, A. A. TOPAS and TOPAS-Academic: an optimization program integrating computer algebra and crystallographic objects written in C++. J. Appl. Cryst. 51, 210–218 (2018).
Acknowledgements
M.R.C. and C.S. acknowledge support from the project CH4.0 under the MUR program “Dipartimenti di Eccellenza 2023-2027” (CUP: D13C22003520001), the project FLIPPER (PRIN2022 n. 202224KAX8; CUP D53D23010020006) funded by European Union - Next Generation EU, Mission 4 Component 1, the project NICE (PRIN2020 n. 2020Y2CZJ2; CUP D13C22000440001). F.B. acknowledges funding by the Alexander von Humboldt Foundation.
Author information
Authors and Affiliations
Contributions
Y.N. and M.R.C. conceived the study. Y.N., M.R.C. and C.S. designed the work. C.S. performed solution/solid-state NMR. N.M., M.N. and Y.A. collected MicroED data. K.A., K.K. and M.H. collected high-resolution MS data. F.B. and Y.N. performed DFT-D/GIPAW calculations. F.B. performed PXRD analyses. C.S. and Y.N. performed database analysis. All authors contributed to interpretation of data. C.S., Y.N., M.R.C. wrote the first draft of the manuscript. All authors contributed to discussions and to writing and revising the final draft.
Corresponding authors
Ethics declarations
Competing interests
The authors C.S., F.B., and M.R.C. declare no competing interests. Authors N.M., M.N., Y.A., K.A., K.K., M.H. and Y.N. are full-time employees of JEOL Ltd. The company had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Peer review
Peer review information
Communications Chemistry thanks Florian Schulz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Sabena, C., Bravetti, F., Miyauchi, N. et al. An integrated workflow for the structure elucidation of nanocrystalline powders. Commun Chem (2026). https://doi.org/10.1038/s42004-026-01902-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s42004-026-01902-1
