Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Low-energy Electron Holography Microscope for Imaging of Single Molecules

Sven Alexander Szilagyi

Structure determination techniques are an essential tool to investigate and understand molecules, especially in biology, where functionality crucially depends on structure. The dominant high-resolution methods, such as transmission electron microscopy and X-ray diffraction, rely on ensemble averaging, making them unsuitable for evidencing structural variations of flexible molecules. Common single-molecule imaging techniques like scanning probe microscopy achieve high resolution but lack access to 3D objects. A possible way to overcome these limitations is offered by low-energy electron holography (LEEH). So far, LEEH achieved molecular imaging at resolutions in the range of 7-8 Å on the single-molecule level. A resolution in the range of a few Å, which would give access to atomic details of molecular structures, is in theory attainable. Obtaining a resolution near the theoretical limit requires a fully optimized LEEH setup and workflow, for which in particular sample and emitter preparation are crucial. Here, I present a novel LEEH microscope with a unique experimental workflow, which is capable of resolving molecular substructures with an unprecedented resolution for LEEH experiments. The newly constructed LEEH microscope takes advantage of an efficient vibration isolation system and is remote-controlled to minimize external perturbations. The setup is capable of low-temperature (50 K) measurements, which enhances the stability of the system. A reliable way for preparing highly coherent electron emitters constituted by sharp tungsten tips is presented along with different methods for improving their performance via functionalization and UHV treatments. I show an optimized and reliable substrate preparation protocol resulting in ultra-clean free-standing single-layer graphene (SLG) samples. Native electrospray ion beam deposition (ES-IBD) allows for the transfer of non-volatile molecules from solution into gas phase and onto the substrate. By employing a quadrupole mass selection, highly pure molecular ion beams are obtained, which are soft-landed onto the SLG substrate in a controlled and reproducible manner while retaining an intact structure. This versatile preparation method allows us to study a great variety of molecules by LEEH. Data on proteins with masses ranging from 12-820 kDa reveal molecular shapes consistent with model structures. Locally, structural details as small as 5 Å were identified, thus we are approaching our goal of a resolution in the range of a few Å. The application of our methodology to flexible proteins, exemplified by antibodies, allowed for the imaging of their conformational variability and for determining the influence of the sample preparation on their structure. This gave us the possibility to directly image gas-phase related conformations. Data of compact molecular systems with masses below 3 kDa reveal that our LEEH microscope is able to investigate small molecules, such as porphyrines, at high imaging contrast. The presented results indicate that an optimized LEEH setup and workflow in combination with ES-IBD is a versatile microscopy method, which is filling the gap between high-resolution and single-molecule techniques by elucidating structural details of various molecular species.

EPFL2022

KSm(MoO4)2, an incommensurately modulated and partially disordered scheelite-like structure

Alla Arakcheeva, Gervais Chapuis, Philip Pattison

The incommensurately modulated scheelite-like KSm(MoO4)2 structure has been refined in the monoclinic superspace group I2/b(ab0)00 by the Rietveld method on the basis of synchrotron radiation powder diffraction data. The systematic broadening of satellite reflections has been accounted for by applying anisotropic microstrain line-broadening. The microstructure has been studied by transmission electron microscopy (TEM). The partial disorder of the K and Sm cations in the A position is best approximated by a combination of harmonic and complex crenel functions with (0.952Sm + 0.048K) and (0.952K + 0.048Sm) atomic domains. This combination yields a compositional wave distribution from {KMoO4} to {SmMoO4} observed in the ab structure projection along q. The specific features of KSm(MoO4)2 and degree of the A-cation ordering are discussed in comparison with the previously reported structure of KNd(MoO4)2.

2008

Electron probing of oxygen-evolving oxide catalysts

Tzu-Hsien Shen

Water splitting is one of the cleanest ways to store energy. The production of hydrogen and oxygen gases can be utilized in fuel cells to generate electricity, power, and heat. In the water splitting process, the oxygen evolution reaction (OER), taking place at the anode, is the sluggish reaction limiting the efficiency. Electrocatalysts are used to reduce the kinetic barrier of OER. Development of high-performance and stable OER catalysts requires fundamental understanding of their electrocatalytic properties and, hence, their holistic characterization is indispensable. The objective of this thesis is to probe Co-based oxide catalysts for OER in an alkaline medium using advanced (scanning) transmission electron microscopy ((S)TEM) techniques. Starting with the surface characterization of a highly active OER oxide catalyst, Ba0.5Sr0.5Co0.8Fe0.2O3-d (BSCF), it is shown that the surfaces of the as-synthesized BSCF particles are Co/Fe rich and adopt a spinel-like structure with a reduced valence of Co ions. The Co/Fe spinel structure is further linked to the formation of the highly active Co(Fe)OOH at the BSCF surface. Further real-time probing of BSCF oxide catalysts using electrochemical liquid-cell TEM shows a switchable wetting behavior of Co-based oxide catalysts by analyzing the liquid movement around oxide particles. The interfacial interactions at the surface-electrolyte under potential cycling are correlated to electrowetting and hydrophilic surface transformation. Molecular oxygen evolution is detected qualitatively under OER conditions by identifying the O2 feature in electron energy-loss spectra. Quantification of the O2 using electron energy-loss spectroscopy is also demonstrated, pending further improvements.The work herein offers new insights into the characterization framework of oxide catalysts for OER using (S)TEM. The bulk to surface structure of the catalysts can be analyzed, and the interfacial interactions of single-particle catalysts can be probed in real-time. The findings aid in understanding the performance and stability of oxygen-evolving oxide catalysts with further prospects envisioned towards a complete quantitative probing of electrocatalytic reactions in real-time. Ultimately, quantitative characterization in electrochemical liquid-phase (S)TEM can provide understanding of surface-active sites and reaction kinetics of oxygen evolution catalysts.

EPFL2022

Low-energy Electron Holography Microscope for Imaging of Single Molecules

Sven Alexander Szilagyi

EPFL2022

Electron probing of oxygen-evolving oxide catalysts

Tzu-Hsien Shen

EPFL2022