Peter Engelhardt

Three-Dimensional cryoEM Reconstruction of Native LDL Particles to 16Å Resolution at Physiological Body Temperature

Background Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37°C). Methodology/Principal Findings To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6°C and 37°C resulted in reconstructions at ∼16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6°C than at 37°C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6°C, but not at 37°C. At 37°C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6°C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles. Conclusions/Significance Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.

Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space

ABSTRACT The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.

Reversible Supracolloidal Self-Assembly of Cobalt Nanoparticles to Hollow Capsids and Their Superstructures

The synthesis and spontaneous, reversible supracolloidal hydrogen bond-driven self-assembly of cobalt nanoparticles (CoNPs) into hollow shell-like capsids and their directed assembly to higher order superstructures is presented. CoNPs and capsids form in one step upon mixing dicobalt octacarbonyl (Co2 CO8 ) and p-aminobenzoic acid (pABA) in 1,2-dichlorobenzene using heating-up synthesis without additional catalysts or stabilizers. This leads to pABA capped CoNPs (core ca. 5 nm) with a narrow size distribution. They spontaneously assemble into tunable spherical capsids (d≈50-200 nm) with a few-layered shells, as driven by inter-nanoparticle hydrogen bonds thus warranting supracolloidal self-assembly. The capsids can be reversibly disassembled and reassembled by controlling the hydrogen bonds upon heating or solvent exchanges. The superparamagnetic nature of CoNPs allows magnetic-field-directed self-assembly of capsids to capsid chains due to an interplay of induced dipoles and inter-capsid hydrogen bonds. Finally, self-assembly on air-water interface furnishes lightweight colloidal framework films.

Three-Dimensional Reconstruction of Chromosomes Using Electron Tomography

The evolvement of preparative methods in structural studies has always been as important as the development of sophisticated equipment. Software development is also a significant part for three-dimensional (3D) structural studies using electron tomography methods (ETMs). Advanced computing makes amenable procedures that relatively recently were only visionary, such as the 3D reconstruction of chromosomes with ETM. Morphological guidelines and beauty are occasionally the only standard for a method to be acceptable in the realms of preparative as well as software development. Bulk isolation of metaphase chromosomes using acetic acid is such an apparent accomplishment in preparative methods. Our ETM with maximum entropy and, more so, the ongoing development toward fully automatic alignments, are contributions in the software line. Furthermore, whole mounting of chromosomes on holey-carbon grids makes it possible to use even yesterdays 80-kV transmission electron microscope with a standard goniometer to collect tilt series. These advances in preparing whole-mount metaphase chromosomes enable laboratories that do not have access to a medium- or high-voltage transmission electron microscope to study complex structures like chromosomes in 3D using todays desktop computers.

Whole-mount immunoelectron tomography of chromosomes and cells.

Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.

Small angle X-ray scattering and transmission electron microscopy study of the Lactobacillus brevis S-layer protein

The structure of self-assembly domain containing recombinant truncation mutants of Lactobacillus brevis surface layer protein SlpA in aqueous solution was studied using small-angle X-ray scattering and transmission electron microscopy. The proteins were found out to interact with each other forming stable globular oligomers of about 10 monomers. The maximum diameter of the oligomers varied between 75 A and 435 A.

A three-dimensional Bayesian reconstruction method with the point spread function for micro-rotation sequences in wide-field microscopy

In this paper, we present a three-dimensional (3D) reconstruction algorithm for light microscopy supplied with a microrotation device. In contrast to the traditional optical sectioning techniques where the focal plane is shifted along the optical axis, in micro-rotation imaging, the object rotates where the focal plane is kept in a fixed position. Our reconstruction algorithm is based on Bayesian inversion theory where the imaging model is described by the 3D point spread function (PSF). The results show that the approach is promising for 3D reconstruction of rotating objects in wide-field light microscopy, as an improvement for the effective 3D resolution is provided

On the alignment of transmission electron microscope images without fiducial markers

Accurate image alignment is needed for computing three-dimensional reconstructions from transmission electron microscope tilt series. So far, the best alignment results have been obtained by using colloidal gold beads as fiducial markers. If their use has not been possible for some reason, the only option has been the automatic 2D cross-correlation-based registration methods. However, since the actual motion is three dimensional, this approach is inaccurate and inappropriate for the whole problem. Conversely, we propose a novel method that uses the actual 3D motion model without any fiducial markers in the images. The method is based on matching and tracking corner features by first solving the underlying geometrical constraint of consecutive images in the tilt series. The results show that our method is competitive with the gold marker alignment in the level of accuracy and hence opens the way for new opportunities in the analysis of electron tomography reconstructions.