Juergen M. Plitzko

Magnetosome chains are recruited to cellular division sites and split by asymmetric septation

Magnetotactic bacteria navigate along magnetic field lines using well‐ordered chains of membrane‐enclosed magnetic crystals, referred to as magnetosomes, which have emerged as model to investigate organelle biogenesis in prokaryotic systems. To become divided and segregated faithfully during cytokinesis, the magnetosome chain has to be properly positioned, cleaved and separated against intrachain magnetostatic forces. Here we demonstrate that magnetotactic bacteria use dedicated mechanisms to control the position and division of the magnetosome chain, thus maintaining magnetic orientation throughout divisional cycle. Using electron and time‐lapse microscopy of synchronized cells of Magnetospirillum gryphiswaldense, we confirm that magnetosome chains undergo a dynamic pole‐to‐midcell translocation during cytokinesis. Nascent chains were recruited to division sites also in division‐inhibited cells, but not in a mamK mutant, indicating an active mechanism depending upon the actin‐like cytoskeletal magnetosome filament. Cryo‐electron tomography revealed that both the magnetosome chain and the magnetosome filament are spilt into halves by asymmetric septation and unidirectional indentation, which we interpret in terms of a specific adaptation required to overcome the magnetostatic interactions between separating daughter chains. Our study demonstrates that magnetosome division and segregation is co‐ordinated with cytokinesis and resembles partitioning mechanisms of other organelles and macromolecular complexes in bacteria.

Automated screening of 2D crystallization trials using transmission electron microscopy: a high-throughput tool-chain for sample preparation and microscopic analysis.

We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.

3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM

The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein–DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.

Revisiting the Structure of Hemoglobin and Myoglobin with Cryo-Electron Microscopy

Sixty years ago, the first protein structure of myoglobin was determined by John Kendrew and his colleagues; hemoglobin followed shortly thereafter. For quite some time, it seemed that only X-ray crystallography would be capable of determining the structure of proteins to high resolution. In recent years, cryo-electron microscopy has emerged as a viable alternative and indeed in many cases the preferred approach. It is capable of studying proteins that span a size range from several megadaltons to proteins as small as myoglobin and hemoglobin.

In vivo coating of bacterial magnetic nanoparticles by magnetosome expression of spider silk-inspired peptides

Magnetosomes are natural magnetic nanoparticles with exceptional properties that are synthesized in magnetotactic bacteria by a highly regulated biomineralization process. Their usability in many applications could be further improved by encapsulation in biocompatible polymers. In this study, we explored the production of spider silk-inspired peptides on magnetosomes of the alphaproteobacterium Magnetospirillum gryphiswaldense. Genetic fusion of different silk sequence-like variants to abundant magnetosome membrane proteins enhanced magnetite biomineralization and caused the formation of a proteinaceous capsule, which increased the colloidal stability of isolated particles. Furthermore, we show that spider silk peptides fused to a magnetosome membrane protein can be used as seeds for silk fibril growth on the magnetosome surface. In summary, we demonstrate that the combination of two different biogenic materials generates a genetically encoded hybrid composite with engineerable new properties and enhanced potential for various applications.

Chemical Composition and Thickness Retrieval in HRTEM by a Reversed Multislice Process

In general terms, lattice images do not reveal the real atomic structure of the sample directly, can be distorted, and information about the sample thickness and chemical composition is heavily encoded. These drawbacks relate to the presence of lens aberrations and dynamic diffraction. The development of Cs corrected TEM [1, 2] and software that reconstructs the complex electron exit wave function [3-7] aim at removing lens aberrations. Several approaches, such as the 1s state model [8], reversing multislice algorithms [9,10], or simulated annealing and maximum likelihood algorithms [11, 12], have been proposed to remove the dynamical scattering effect and to retrieve the crystal potential from the complex exit wave. In this paper, we present a new method to retrieve the potential map from the exit wave based on reversing multi-slice calculations. This algorithm uses a non-linear optimization scheme to find an optimum phase grating that satisfies two boundary conditions: knowledge of the entrance surface wave and the measured exit surface wave. The exit wave of a wedge shaped Au crystal and an Al (10%Cu) crystal were simulated to test this algorithm. Good agreement between the recovered crystal potentials and input parameters was found up to a thickness where phase reversal occurs because of dynamic scattering. After the phase grating is retrieved, the position of atom columns and their chemical composition can be quantified. Compositional maps Xa(r) and Xb(r) of binary alloys can be deduced from the crystal potential map V(r) using the linear relation V(r) = Xa(r)Va+Xb(r)Vb, where Va and Vb are the mean inner potentials of the element A and B, respectively. Figure 1 (a) and (b) show the phase of an electron exit wave of an Al:Cu bi-crystal and an InGaN/GaN quantum well that were reconstructed from focal series of 20 images each. Figure 2 (a) and (b) shows the retrieved potential maps of the Al and Cu atoms, respectively, and sitespecific Cu segregation to the boundary is revealed. Fig. 3 (a) and (b) depicts Ga and In maps from the quantum well region. It is clear from both cases that chemical differences can be distinguished on a single atom level due to the different atomic number Z of the elements. Limitations of the algorithm due to systematic and statistical errors will be discussed. References: [1] H. Rose, Optik 85 (1) (1990) 19. [2] M. Haider, H. Rose, S. Uhlemann, E. Schwan, B. Kabius, K. Urban, Ultramicroscopy 23 (1998) 768. [3] W.O. Saxton, in: Advances in Electronics and Electron Physics, Computer Techniques for Image Processing in Electron Microcopy, Academic Press, New York, 1978. [4] E.J. Kirkland, B.M. Siegel, N. Uyeda, Y. Fujiyoshi, Ultramicroscopy 17 (1985) 87. [5] M. Op De Beeck, D. Van Dyck, W. Coene, Ultramicroscopy 64 (1996) 167. [6] W.-K. Hsieh, Fu-Rong Chen, Ji-Jung Kai and A. I. Kirkland, Ultramicroscopy 98 (2004) 99 [7] Les Allen et al, Ultramicroscopy 100 (2004) 91-104 [8] M. Op De Beeck and D. Van Dyck, Phys. Stat. Sol., 150 (1995) 587 [9] M. J. Beeching and A. E. C. Spargo, Ultramicroscopy 52 (1993) 243 [10] A. E. C. Spargo, M. J. Beeching and L. J. Allen, Ultramicroscopy 55 (1994) 329 [11] M. Lentzen and K. Urban, Ultramicroscopy, 62 (1996) 89-102 [12] M. Lentzen and K. Urban, Acta Crystallography, A56, (2000), 235-247 Microsc Microanal 11(Suppl 2), 2005 Copyright 2005 Microscopy Society of America DOI: 10.1017/S1431927605509206 2158

Retrieving Potential Maps from Reversed Multislice Calculations

Extended abstract of a paper presented at the Pre-Meeting Congress: Materials Research in an Aberration-Free Environment, at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, July 31 and August 1, 2004.

Reconstitution of a flexible SYCP3-DNA fibre suggests a mechanism for SYCP3 coating of the meiotic chromosome axis

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic crossover. A hallmark of SC formation is the presence of its protein component SYCP3 on the chromosome axis. As SC assembly progresses, SYCP3 is deposited on both axes of the homologue pair, forming the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Taken together, our findings suggest that, upon deposition on the chromosome axis, SYCP3 spreads by polymerising into a fibre that is fastened to the chromosome surface via DNA binding. The resulting layer of SYCP3 coating the chromosome axis might provide a structural basis for LE assembly in meiotic prophase.

Eukaryotically expressed encapsulins as orthogonal compartments for multiscale molecular imaging

We have genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-controlled metal biomineralization. The orthogonal shell protein (EncA) from M. xanthus efficiently auto-assembled inside mammalian cells into nanocompartments to which sets of native (EncB,C,D) and engineered cargo proteins self-targeted. This enabled localized bimolecular fluorescence and enzyme complementation with selective access to substrates via the pores in the nanoshell. Encapsulation of the enzyme tyrosinase lead to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB or EncC) resulted in efficient iron sequestration that produced substantial contrast by magnetic resonance imaging (MRI) and enabled magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells also proved to be excellent genetically encoded markers for cryo-electron tomography (cryo-ET). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Cryo-FIB Lift-out Sample Preparation Using a Novel Cryo-gripper Tool

In recent years, biological specimen preparation using a cryo-focused ion beam (cryo-FIB) has become a key technique for investigating complex cellular structures in situ [1-3]. Cryo-FIB has enabled cryoelectron tomography (cryo-ET) of plunge-frozen hydrated cells, revealing the cellular interior with sufficient resolution and contrast to study membrane-bound macromolecules in their native state [4-7].