Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.

CCP4 Research Complex at Harwell Rutherford Appleton Laboratory Didcot OX11 0FA U K

Department of Biomolecular Sciences Weizmann Institute of Science 76100 Rehovot Israel

Department of Chemical and Biological Physics Weizmann Institute of Science 76100 Rehovot Israel

Department of Chemical Research Support Weizmann Institute of Science 76100 Rehovot Israel

Department of Chemistry Carnegie Mellon University Pittsburgh Pennsylvania 15213 United States

Department of Inorganic Chemistry Faculty of Chemistry University of Vienna Vienna 1090 Austria

Department of Materials Science and Engineering Carnegie Mellon University Pittsburgh Pennsylvania 15213 United States

Department of Microbiology and Molecular Genetics Institute for Medical Research Israel Canada and The Kuvin Center for the Study of Infectious and Tropical Diseases The Hebrew University Hadassah Medical School Jerusalem 9112010 Israel

Department of Molecular Chemistry and Materials Science Weizmann Institute of Science 76100 Rehovot Israel

Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0DE U K

Division of Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Oxford OX3 7BN U K

Faculty of Geosciences and MAPEX Center for Materials and Processes University of Bremen Klagenfurter Str 2 28359 Bremen Germany

Institute of Physics of the Czech Academy of Sciences Na Slovance 2 182 21 Prague 8 Czechia

STFC Rutherford Appleton Laboratory Didcot OX11 0FA U K

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