Biology subjects

Mullick, D.

Publications and source records attributed to Mullick, D..

3 recordsLinked to original sources

bioRxiv2022

Bridging the light-electron resolution gap with correlative cryo-SRRF and dual-axis cryo-STEM tomography

Cryo-electron tomography (cryo-ET) is the prime method for cell biological studies in three dimensions (3D) at high resolution. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access larger 3D volumes, on the scale of 1 micron, making it ideal to study organelles and their interactions in situ. Here we introduce two relevant advances: a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy (cryo-CLEM). Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to isotropic resolution in the reconstruction without averaging. We show individual protein densities in a mitochondrion-ER contact in a cell region 850 nm thick. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

cell biology↗

bioRxiv2022

3D electron diffraction reveals the chiral nature of the malaria pigment crystal hemozoin

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. X-ray powder diffraction has established that the unit cell contains a cyclic hematin dimer, yet the pro-chiral nature of heme supports formation of four distinct stereoisomers, two centrosymmetric and two chiral enantiomers. Here we apply emerging methods of in situ cryo-electron tomography and diffraction to obtain a definitive structure of biogenic hemozoin. Individual crystals take a striking polar morphology. Diffraction analysis, supported by density functional theory, indicates a compositional mixture of one centrosymmetric and one chiral dimer, whose absolute configuration has been determined on the basis of crystal morphology and interaction with the aqueous medium. Structural modeling of the heme detoxification protein suggests a mechanism for dimer selection. The refined structure of hemozoin should serve as a guide to new drug development.

microbiology↗

bioRxiv2022

Diffraction contrast in cryo-scanning transmission electron tomography reveals the boundary of hemozoin crystals in situ

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasites digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in vivo, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.

biophysics↗