Cryo-tomography and 3D Electron Diffraction Reveal the Polar Habit and Chiral Structure of the Malaria Pigment Crystal Hemozoin

The authors used advanced cryo-electron tomography and three-dimensional electron diffraction to determine the structure of hemozoin crystals, which are produced by malaria parasites to detoxify heme released from digested hemoglobin. Understanding hemozoin is important because its formation is a key target for antimalarial drugs. Previous studies relied on synthetic analogs and powder diffraction, but this work analyzed native hemozoin directly from ruptured parasite cells, providing higher resolution and more accurate results.
The study revealed that biogenic hemozoin crystals have a distinct polar shape: one end of the crystal is smooth and chisel-like, while the other end is irregular and variable. This polar morphology is not compatible with a perfectly symmetrical (centrosymmetric) crystal structure and suggests the presence of chirality in the unit cell. The authors found that the unit cell contains heme dimers, which can exist as four different stereoisomers—two centrosymmetric and two chiral enantiomers. Through electron diffraction and theoretical calculations, they determined that native hemozoin is made from a specific mixture of one centrosymmetric and one chiral dimer. This selective combination explains both the polar shape and the efficient, rapid crystal growth necessary for the parasite’s survival.
The absolute configuration of the crystals was determined using both morphological analysis and a novel method called exit-wave reconstruction. The study also found that atomic disorder appears asymmetrically on certain crystal facets, and this can be explained by how water molecules bind to the crystal surface. Furthermore, structural modeling of the heme detoxification protein suggests that it acts as a chiral agent, favoring the formation of a single crystalline phase and supporting rapid detoxification.
These findings not only clarify the detailed structure and growth of hemozoin in malaria parasites but also provide a structural blueprint that could help in the design of new antimalarial drugs that specifically target this detoxification pathway.
The members of the FZU team performed the analysis of the electron diffraction data using the dynamical refinement, utilizing unique methods and software developed by the team. The results contributed significantly to the understanding of the intriguing crystal structure.

Description

Groups exposed at the surfaces of hemozoin crystals composed of a mixture of R/S′ + R/R′ dimers. (A). Array of unit cells as a visual guide. The mixture appears as disorder on the (001̅) and (1̅01̅) surfaces, as represented in Figure 2C, but the (001) and (101) surfaces are completely ordered. (B) A detailed view of the methyl-vinyl pairs on the various exposed surfaces. Additionally, the carboxyl and carboxylate groups (dotted red circles) are exposed to the aqueous medium at the (001̅) surface, rendering it more hydrophilic than the (101) and (1̅01̅) surfaces where the available H-bonds are saturated and buried (red circles, fading represents depth). Crystal growth of the relatively hydrophilic (001̅) face will be inhibited by competitive binding of water. (C) Phase reconstruction from a through focus series (gray) of a crystal oriented precisely along the –a direction. Alternating columns of channels between the heme dimers appear either “filled” (red arrow, carboxyl links) or “empty” (green arrow, methyl-vinyl). The red overlay is a computed phase image based on the diffraction result for the model combining R/S′ + R/R′ dimers, shown overlaid in ball and stick form. The faint purple circle shows one methyl-vinyl channel; note the asymmetric density around the dashed line, whose origin lies in the chiral contribution. After indexing as indicated, the chiral dimer is identified as R/R′.