Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions

The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a “block-based” reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes

Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

Geometric analysis of macromolecule organization within cryo-electron tomograms

Cryo-electron tomography (CET) provides unprecedented views into the native cellular environment at molecular resolution. While subtomogram analysis yields high-resolution native structures of molecular complexes, it also determines the precise positions and orientations of these macromolecules within the cell. Analyzing the geometric relationships between adjacent macromolecules can offer structural insights into molecular interactions and identify supramolecular ensembles. However, computation..

Application of new reconstruction strategies to enhance the interpretable resolution in 3D Electron Microscopy - Studies of Rigor and ADP binding complexes of Actin and Myosin V

Subject of these studies was the strong binding complex of actin and myosin V. How the molecular motor protein myosin powers muscles by generating mechanical force out of chemical energy at high efficiency fascinates researchers already for a long time. Myosin V offers the unique possibility to study not only the rigor state but also its preceding strong binding ADP state, which is only transiently present in other myosins. Since the actomyosin complex could not be studied by protein crystallography so far, transmission electron microscopy (TEM) of vitrified protein complexes is the method of choice. New reconstruction strategies were applied, to enhance the interpretable resolution in 3D electron microscopy, together with subsequent molecular dynamics (MD) simulations of protein crystal structures. Such a combined approach allows for almost the same resolution as X-ray diffraction facilitates. As a new – and in principle superior – reconstruction scheme a filtered least squares re- construction algorithm was applied in order to obtain a better defined density localization. Thorough analysis combined with electron tomography studies revealed flexible specimen properties that limit the applicability of the current implementation of the least squares method due to necessary ad hoc assumptions. As a first step to reduce these assumptions iterative helical reconstruction was applied and yielded densities with reliable 8 Å resolution. Following MD simulations were able to resolve functional differences of myosin between the rigor and the strong binding ADP state

Author response

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath beta'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant

Direct visualization of degradation microcompartments at the ER membrane

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within similar to 200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 265 proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control

Statistical analysis and modeling for biomolecular structures

Most of the recent studies on biomolecules address their three dimensional structure since it is closely related to their functions in a biological system. Determination of structure of biomolecules can be done by using various methods, which rely on data from various experimental instruments or on computational approaches to previously obtained data or datasets. Single particle reconstruction using electron microscopic images of macromolecules has proven resource-wise to be useful and affordable for determining their molecular structure in increasing details. The main goal of this thesis is to contribute to the single particle reconstruction methodology, by adding a process of denoising in the analysis of the cryo-electron microscopic images. First, the denoising methods are briefly surveyed and their efficiencies for filtering cryo-electron microscopic images are evaluated. In this thesis, the focus has been set to information theoretic minimum description length (MDL) principle for coding efficiently the essential part of the signal. This approach can also be applied to reduce noise in signals and here it is used to develop a novel denoising method for cryo-electron microscopic images. An existing denoising method has been modified to suit the given problem in single particle reconstruction. In addition, a more general denoising method has been developed, discovering a novel way to find model class by using the MDL principle. This method was then thoroughly tested and compared with co-existing methods in order to evaluate the utility of denoising in single particle reconstruction. A secondary goal in the research for this thesis deals with studying protein oligomerisation, using computational approaches. The focus has been to recognize interacting residues in proteins for oligomerization and to model the interaction site for hantavirus N-protein. In order to unravel the interaction structure, the approach has been to understand the phenomenon of protein folding towards quaternary structure.reviewe

The structure of the COPI coat determined within the cell

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant