title: Two types of amorphous protein particles facilitate crystal nucleation.
abstract: Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.
authors.affiliation: Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan.|Department of Earth Science, Graduate School of Science, Tohoku University, Sendai, Miyagi 980-8578, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan; ykimura@lowtem.hokudai.ac.jp.
authors.forename: Yuki
authors.identifier: 0000-0002-9218-7663
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204-4004.|Department of Chemistry, University of Houston, Houston, TX 77204-4004.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Vekilov
authors.affiliation: Department of Earth Science, Graduate School of Science, Tohoku University, Sendai, Miyagi 980-8578, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Furukawa
authors.affiliation: Hitachi High-Technologies Corporation, Ibaraki 312-0057, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Shirai
authors.affiliation: Hitachi High-Technologies Corporation, Ibaraki 312-0057, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Matsumoto
authors.affiliation: Institut des Sciences de la Terre (ISTerre), CNRS, Université Grenoble Alpes, F-38000 Grenoble, France.
authors.forename: Alexander E S
authors.initials: AE
authors.lastname: Van Driessche
authors.affiliation: Department of Earth Science, Graduate School of Science, Tohoku University, Sendai, Miyagi 980-8578, Japan.
authors.forename: Katsuo
authors.initials: K
authors.lastname: Tsukamoto
keywords: in situ observation; lysozyme; nucleation; protein; transmission electron microscopy