Cryo-electron microscopy and single particle analysis

Cryo-EM in combination with the single particle approach 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. The introduction of Direct Electron Detector systems (DEDs) provoked a resolution revolution in single particle cryo-EM, now enabling near-atomic resolution almost routine (Figure 1, Ignatiou et al., 2019, Nat Commun). Starting already 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 provides 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). — **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).

**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, 3D information is derived by averaging over thousands if not hundred thousand of projection images of individual molecules (referred to as particle images), assuming that they are all identical. In practice, however, biological protein complexes typically show variable complex assembly and intrinsic conformational flexibility which often represent different biological relevant functional states. Within the collaborative research center SFB740 (project Z1), we established a fully automated pipeline for high-throughput data collection including on-the-fly drift correction and CFT-based quality control of DED super-resolution movie stacks which was essential for introducing multi-particle refinement strategies developed in Christian Spahn’s lab (IMBP Charité). 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 (Figure 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). — **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).

**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).

Once established, multi-particle refinement was applied to study phage assembly intermediates (Ignatiou et al., 2019, Nat Commun), transcription regulation (Huang et al., 2020, Mol Cell; Said et al., 2021, Science), ribosome assembly (Nikolay et al., 2018, Mol Cell; Nikolay et al., 2021, Mol Cell; Quin et al, unpublished data), and translational control in embryonic brain development (Kraushar et al., 2021, Mol Cell). Exploiting the resolution power of DED systems, single particle cryo-EM now also allows structural analysis of small enzyme complexes comprising a molecular weight of only 200-400 kDa and even below such as e.g. the metal-containing formate dehydrogenase (FDH) from Rhodobacter capsulatus (RcFDH), demonstrating that NADH reduction leads to charging of electron carrying cofactors (Figure 3, Radon et al., 2020, Nat Commun).