TY - JOUR
T1 - A mechanism of ferritin crystallization revealed by cryo-STEM tomography
AU - Houben, Lothar
AU - Weissman, Haim
AU - Wolf, Sharon G.
AU - Rybtchinski, Boris
N1 - This work was supported by the Helen and Martin Kimmel Center for Molecular Design. We thank S. Albeck, M. Peretz and J. Jacobovitch for assistance with GPC experiments, I. Biran for help acquiring cryo-STEM tomograms, and B. Palmer and R. Diskin for discussions. The electron microscopy studies were partially supported by the Irving and Cherna Moskowitz Center for Nano and BioNano Imaging (Weizmann Institute of Science). S.G.W. acknowledges support for cryo-STEM tomography studies from the Israel Science Foundation (grant number 1285/14). Author contributions B.R., L.H. and H.W. conceived the project. H.W. performed the crystallization experiments and the 2D cryo-TEM imaging; S.G.W., H.W. and L.H. performed the cryo-STEM tomography imaging; and L.H performed the cryo-STEM tomography data analysis. B.R. and L.H. wrote the manuscript. All authors contributed to the discussion and commented on the manuscript.
PY - 2020/3/25
Y1 - 2020/3/25
N2 - Protein crystallization is important in structural biology, disease research and pharmaceuticals. It has recently been recognized that nonclassical crystallization—involving initial formation of an amorphous precursor phase—occurs often in protein, organic and inorganic crystallization processes1–5. A two-step nucleation theory has thus been proposed, in which initial low-density, solvated amorphous aggregates subsequently densify, leading to nucleation4,6,7. This view differs from classical nucleation theory, which implies that crystalline nuclei forming in solution have the same density and structure as does the final crystalline state1. A protein crystallization mechanism involving this classical pathway has recently been observed directly8. However, a molecular mechanism of nonclassical protein crystallization9–15 has not been established9,11,14. To determine the nature of the amorphous precursors and whether crystallization takes place within them (and if so, how order develops at the molecular level), three-dimensional (3D) molecular-level imaging of a crystallization process is required. Here we report cryogenic scanning transmission microscopy tomography of ferritin aggregates at various stages of crystallization, followed by 3D reconstruction using simultaneous iterative reconstruction techniques to provide a 3D picture of crystallization with molecular resolution. As crystalline order gradually increased in the studied aggregates, they exhibited an increase in both order and density from their surface towards their interior. We observed no highly ordered small structures typical of a classical nucleation process, and occasionally we observed several ordered domains emerging within one amorphous aggregate, a phenomenon not predicted by either classical or two-step nucleation theories. Our molecular-level analysis hints at desolvation as the driver of the continuous order-evolution mechanism, a view that goes beyond current nucleation models, yet is consistent with a broad spectrum of protein crystallization mechanisms.
AB - Protein crystallization is important in structural biology, disease research and pharmaceuticals. It has recently been recognized that nonclassical crystallization—involving initial formation of an amorphous precursor phase—occurs often in protein, organic and inorganic crystallization processes1–5. A two-step nucleation theory has thus been proposed, in which initial low-density, solvated amorphous aggregates subsequently densify, leading to nucleation4,6,7. This view differs from classical nucleation theory, which implies that crystalline nuclei forming in solution have the same density and structure as does the final crystalline state1. A protein crystallization mechanism involving this classical pathway has recently been observed directly8. However, a molecular mechanism of nonclassical protein crystallization9–15 has not been established9,11,14. To determine the nature of the amorphous precursors and whether crystallization takes place within them (and if so, how order develops at the molecular level), three-dimensional (3D) molecular-level imaging of a crystallization process is required. Here we report cryogenic scanning transmission microscopy tomography of ferritin aggregates at various stages of crystallization, followed by 3D reconstruction using simultaneous iterative reconstruction techniques to provide a 3D picture of crystallization with molecular resolution. As crystalline order gradually increased in the studied aggregates, they exhibited an increase in both order and density from their surface towards their interior. We observed no highly ordered small structures typical of a classical nucleation process, and occasionally we observed several ordered domains emerging within one amorphous aggregate, a phenomenon not predicted by either classical or two-step nucleation theories. Our molecular-level analysis hints at desolvation as the driver of the continuous order-evolution mechanism, a view that goes beyond current nucleation models, yet is consistent with a broad spectrum of protein crystallization mechanisms.
U2 - https://doi.org/10.1038/s41586-020-2104-4
DO - https://doi.org/10.1038/s41586-020-2104-4
M3 - مقالة
C2 - 32214264
SN - 0028-0836
VL - 579
SP - 540
EP - 543
JO - Nature
JF - Nature
IS - 7800
ER -