Single Particle Cryo-Electron Microscopy: From Sample to Structure.

Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8Å), but provides more detailed information (2.65Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

Single Particle Kinetic and Electron Cryo-Microscopy Studies of Flavivirus Membrane Fusion

Cryogenic electron microscopy (cryo-EM) has become in the past 10 years one of the major tools for the structure determination of proteins. Nowadays, the structure prediction field is experiencing the same revolution and, using AlphaFold2, it is possible to have high-confidence atomic models for virtually any polypeptide chain, smaller than 4000 amino acids, in a simple click. Even in a scenario where all polypeptide chain folding were to be known, cryo-EM retains specific characteristics that make it a unique tool for the structure determination of macromolecular complexes. Using cryo-EM, it is possible to obtain near-atomic structures of large and flexible mega-complexes, describe conformational panoramas, and potentially develop a structural proteomic approach from fully ex vivo specimens.

Volta phase plate data collection facilitates image processing and cryo-EM structure determination.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.Teaser:A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

A new maximum likelihood–based algorithm allows cryo-EM structure determination of macromolecular complexes even when conformational heterogeneity is present.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Cryo-EM structure and functional analyses show that human iron exporter ferroportin (Fpn) can bind and transport calcium ions and the transport activity is reduced when the concentration of ferrous ions is high.

In this research the phenomenon of monoclonal antibodies (mAbs) cooperative bactericidal activity and the structure of the N.meningitidis antigen NadA var.3 are investigated using Transmission Electron Microscopy (TEM). The mAbs cooperativity is a mechanism that occurs when mAbs that individually show low or no bactericidal activity become bactericidal when coupled together. Revealing the structural bases of cooperative bactericidal activity is fundamental for a thorough understanding of antibody-based mechanisms of protection. Meningococcal factor H binding protein (fHbp), surface-exposed lipoprotein presents in the vaccine against serogroup B Neisseria meningitidis, Bexsero®. A comparison of the structure of the murine cooperative complex with the human cooperative one has been performed revealing a higher level of flexibility and instability of the first complex. Moreover we have been able to prove a simultaneous binding of factor H (fH) to the immune cooperative complex. The 3D structures of the cooperative complexes demonstrated that the angle formed between fHbp-antibodies is orientated only depending on the epitope location on the antigen. Non-cooperative human complexes show a structural dissimilarities compared with the cooperative couples mainly for the absence of the complex formation due to the partial epitopes overlapping, as proved by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX_MS). The NadA var. 3, the variant included in Bexsero® vaccine, structure was determined by the Cryo-Electron Microscopy (Cryo-EM) technique combined with Single Particle (SP) reconstruction method. The 3D reconstruction of NadA var.3, generated from Cryo-TEM data, shows an elongated and thin stalk decorated by a globular compact head characterized by a three-fold symmetry. Moreover the 3D EM map shows three evident points of interruption in the density of the stalk region presumably correlated with the three interruptions present in the coiled-coil periodicity of NadA var.3 the sequence. This result indicates a possible alternative mechanism of the antigen flexibility.

Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.