Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors Van Driessche, Alexander Edgard Suzanne Nucleation Proteins Self-assembly Crystallization Precursor phase M.S. acknowledges financial support from the Fonds Wetenschappelijk Onderzoek (FWO) under Project Nos. G0H5316N and 1516215N. This work used the platforms of the Grenoble Instruct-European Research Infrastructure Consortium (ERIC) center (Integrated Structural Biology Grenoble [ISBG]; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INBS-0005-02) and Grenoble Alliance for Integrated Structural and Cell Biology (GRAL), financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) Chemistry, Biology, and Health, European Graduate School (CBHEUR-GS) (ANR-17-EURE-0003). The electron microscope facility is supported by the Auvergne-Rhone-Alpes Region, the Fondation pour la Recherche Medicale (FRM), the Fonds Europeen de Developpement Regional (FEDER), and the Groupement d'Interet Scientifique (GIS)-Infrastructures en Biologie Sante et Agronomie. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG), Le Commissariata l'Energie Atomique et aux Energies Alternatives (CEA). Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into twoand three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed. 2022-04-06T06:32:18Z 2022-04-06T06:32:18Z 2022-01-31 journal article Van Driessche, A. E... [et al.] (2022). Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors. Proceedings of the National Academy of Sciences, 119(7), e2108674119. [https://doi.org/10.1073/pnas.2108674119] http://hdl.handle.net/10481/74151 10.1073/pnas.2108674119 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España National Academy of Sciences