Cryo-electron microscopy and single particle analysis

Cryo-EM has emerged as a key technology in modern structural biology illustrated by the Nobel prize award to three of its pioneers - Jacques Dubochet, Joachim Frank and Richard Henderson - in 2017. In cryo-EM, biological samples are embedded in vitrified ice and then imaged under cryo-low-dose conditions upon cooling the sample at liquid nitrogen temperature inside the microscope. Beside the development of modern cyro-electron microscopes and highly automated data acquisition schemes, the introduction of Direct Electron Detector systems (DEDs) provoked a resolution revolution in single particle cryo-EM, now enabling near-atomic resolution (Fig. 1, Ignatiou et al., 2019, Nat Commun.). Starting in 2004, we established a state-of-the-art cryo-electron microscopy (cryo-EM) facility within the Berlin-Brandenburg research consortium “UltraStructure Network” (USN), which was at first initiated in close collaboration with Prof. Christian Spahn (Charité Berlin) and Prof. Roland Beckmann (Charité Berlin, now LMU Munich). Our facility has a strong focus on structure determination of macromolecular protein complexes using cryo-EM in combination with the single particle approach. We provide a technology platform for sample screening, semi-automated sample vitrification and automated data acquisition as well as intense computing resources for image processing. Core instrument of our facility is a 300 kV Tecnai G2 Polara cryo-electron microscope (FEI) equipped with Gatan k2summit direct electron detector.

Figure 1: Cryo-EM analysis of bacterial virus assembly (collaboration with P. Tavares, CNRS, Gif-sur-Yvette, and E. Orlova, Birkbeck, London). (A) The maturation of the bacteriophage SPP1 involves several scaffolding proteins driving the assembly of major capsid proteins. (B) Representative micrographs showing different assembly intermediates. (C) The near-atomic resolution cryo-EM structures of the procapsid I, procapsid II and the mature phage capsid provide a molecular basis for sequential structural rearrangements during phage capsid maturation (from Ignatiou et al., 2019, Nat commun.).

In single particle cryo-EM, a purified protein complex in aqueous solution is applied to a cryo-EM grid and then embedded in a thin layer of vitreous ice by rapid plunge freezing into liquid ethane. 3D information is then derived by averaging over thousands if not hundred thousands of projection images of individual molecules (referred to as particle images), assuming that they are all identical. In practice, however, preparations of protein complexes rather have to be considered as heterogeneous samples due to variable complex assembly (extrinsic heterogeneity) and conformational flexibility (intrinsic heterogeneity), respectively. Moreover, it is now commonly accepted that different conformational states often represent intermediate states within a metastable energy landscape; and tuning this energy landscape by e.g. ligand binding and/or hydrolysis of nucleotides thereby provides the basis for the biological function of these complexes (Munro et al., 2009, Trends Biochem Sci).

Within the collaborative research center SFB740 (project Z1), we established a fully automated pipeline for high-throughput data collection, on-the-fly drift correction and CFT-based quality control of DED super-resolution movie stacks. Recording thousands of high-quality micrographs at a routine level was crucial to implement multiparticle refinement strategies developed in Christian Spahn’s lab (IMBP Charité), which are essential to overcome intrinsic and extrinsic heterogeneity exhibited by most protein complexes. Combining these tools enabled us to study for the very first time ex-vivo derived protein complexes such as actively translating human polysomes derived from HEK cells, which represent heterogeneous samples by nature. Analyzing polysome preparations, we could identify 11 distinct functional states, 10 of which representing intermediates of the ribosomal elongation cycle and refine the highest-occupied state, the 80S POST-complex, to near-atomic resolution (Fig. 2, Behrmann et al., 2015, Cell).

Figure 2: High-resolution cryo-EM map of the human ribosome in the POST state filtered to 3.5 Å (A) and corresponding atomic models of the ribosomal subunits (B). (C–F) Enlarged regions of the cryo-EM map showing well-resolved alpha-helices (C) or beta-strands (D) including individual side-chains, strong p-stacking interactions (E) and individual nucleotides (F) with nearby ions (from Behrmann et al., 2015, Cell).

Automated high-throughput DED data collection in combination with multiparticle refinement are now routinely used to study the assembly of macromolecular machineries e.g. the biogenesis of ribosomes (Nikolay et al., 2018, Molecular Cell) or virus assembly (Fig. 1, Ignatiou et al., 2019, Nat Commun.) at a structural level. Together with Matthew Kraushar (AG Spahn, IMBP, Charité), we exploited multi-particle cryo-EM to study for the first time molecules derived ex vivo from the nervous system to reveal the molecular architecture of ex vivo-derived embryonic and perinatal mouse neocortex ribosomes to near-atomic resolution. We found that Ebp1 maintains neuronal proteostasis by binding with high occupancy to the 60S subunit at the peptide tunnel exit site, a well-known binding site for chaperones that shepherd an emerging nascent chain to functional protein (Kraushar et al., manuscript in review). Multiparticle refinement has proofed essential for deciphering high conformational flexibility of many dynamic biological processes such as e.g. transcription regulation by fine-tuning the activity of RNA polymerases. Together with Markus Wahl (FU Berlin), we studied the molecular mechanism of bacterial ribosomal RNA transcription (Huang et al., manuscript under revision) as well as λN-dependent processive transcription antitermination. λN, an intrinsically unstructured viral protein, modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG in order to reprogram the transcriptional apparatus (Said et al., 2017, Nat Microbiol.; Krupp et al., 2019, Mol. Cell).

The resolution power of DED systems now enables studying molecular mechanisms of enzymatic catalysis even in small enzyme complexes comprising a molecular weight of only 200-400 kDa and even below. For example, cryo-EM analysis of metal-containing formate dehydrogenase (FDH) from Rhodobacter capsulatus (RcFDH) as isolated and in the presence of NADH not only revealed the complex arrangement of Fe-S clusters in the enzyme, it also indicated that NADH reduction leads to charging of electron carrying cofactors of RcFDH (Fig. 3, Radon et al., 2020, Nat Commun.).

Figure 3: Structural analysis of metal-containing formate dehydrogenase from Rhodobacter capsulatus (RcFDH). (a) Schematic representation of RcFDH subunits (FdsA, red; FdsB, blue; FdsG, green; FdsD, yellow) and its metal cofactors. (b) Three-dimensional reconstruction and (c) ribbon representation of the atomic model derived from the EM structure. Cofactors are shown as spheres (from Radon et al., 2020, Nat Commun.).

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