Cryo-EM uniqueness in structure determination of macromolecular complexes: A selected structural anthology

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.

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.

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

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

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

Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

Physical and chemical processes at solid-liquid interfaces play a crucial role in many natural and technological phenomena, including catalysis, solar energy and fuel generation, and electrochemical energy storage. Nanoscale characterization of such interfaces has recently been achieved using cryogenic electron microscopy, thereby providing a new path to advancing our fundamental understanding of interface processes. This contribution provides a practical guide to mapping the structure and chemistry of solid-liquid interfaces in materials and devices using an integrated cryogenic electron microscopy approach. In this approach, we pair cryogenic sample preparation which allows stabilization of solid-liquid interfaces with cryogenic focused ion beam (cryo-FIB) milling to create cross-sections through these complex buried structures. Cryogenic scanning electron microscopy (cryo-SEM) techniques performed in a dual-beam FIB/SEM enable direct imaging as well as chemical mapping at the nanoscale. We discuss practical challenges, strategies to overcome them, as well as protocols for obtaining optimal results. While we focus in our discussion on interfaces in energy storage devices, the methods outlined are broadly applicable to a range of fields where solid-liquid interface play a key role.

While lithium ion batteries with electrodes based on intercalation compounds have dominated the portable energy storage market for decades, the energy density of these materials is fundamentally limited. Today, rapidly growing demand for this type of energy storage is driving research into materials that utilize alternative reaction mechanisms to enable higher energy densities. Transition metal compounds are one such class of materials, with storage enabled by “conversion” reactions, where the material is converted to new compound upon lithiation. MoS2 is one example of this type of material that has generated a large amount of interest recently due to its high theoretical lithium storage capacity compared to graphite. Here, cryogenic scanning transmission electron microscopy techniques are used to reveal the atomic‐scale processes that occur during reaction of a model monolayer MoS2 system by enabling the unaltered atomic structure to be determined at various levels of lithiation. It is revealed that monolayer MoS2 can undergo a conversion reaction even with no substrate, and that the resulting particles are smaller than those that form in bulk MoS2, likely due to the more limited 2D diffusion. Additionally, while bilayer MoS2 undergoes intercalation with a corresponding phase transition before conversion, monolayer MoS2 does not.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.

Optimal application of protein engineering technology will require understanding how the amino acid sequence of a polypeptide chain specifies its spatial conformation. It is now clear that polypeptide chain folding and subunit assembly proceeds through sequential pathways involving defined intermediates. The critical amino acid instructions directing these pathways appear to be dispersed through the sequence. Recent genetic approaches have begun to identify which residues in a chain are important in maintaining the pathway, and avoiding aggregated states. Investigations of the P22 tailspike endorhamnosidase suggest that one general class of mutants, tss mutants, may be particularly informative in identifying sequences determining the conformation of the intermediates. Certain inherited human diseases may represent single amino acid substitutions causing folding defects.

Temperature-sensitive mutations in the coat protein of phage P22 severely restrict formation of infectious particles at restrictive temperature. A set of 25 temperature-sensitive strains, which had been localized to regions of the coat gene (Casjens, S., Eppler, K., Sampson, L., Parr, R., and Wyckoff, E. (1991) Genetics 127, 637-647), define 17 sites of single amino acid substitutions by DNA sequencing. Particles assembled from the mutant proteins at permissive temperature were not thermolabile at restrictive temperature, nor defective in the infectious process. At restrictive temperature, ts mutant polypeptide chains were synthesized at near wild-type rates. These inactive chains were not degraded within the cells. The ts chains failed to interact with scaffolding proteins to form the procapsid precursor shell, and they did not polymerize with each to form aberrant shells. Rather, the mutant coat protein accumulated as insoluble aggregates, with the amorphous morphology of inclusion bodies. The results indicate that the chains fail to reach the conformation needed for subunit-subunit or subunit-scaffolding interaction. These mutations appear to be of the class of temperature-sensitive folding mutations, which destabilize an intermediate in the intracellular folding pathway.

Some globular proteins contain repeated structural patterns within the same polypeptide chain. Several enzymes have a pseudo-symmetric two-lobed architecture: a pair of connected but well separated domains with very similar structures are grouped round an approximate 2-fold symmetry axis close to the active centre. On a smaller scale the same motif may appear inside a single protein domain: the polypeptide chain folds into two successive topologically similar subdomains which interlock symmetrically and form a compact globule. In such a domain the two halves come into close contact round the dyad axis; as if the structural integrity of the domain depended on the interactions between its halves, while one separated subdomain could not exist as an independent folding unit. Many of these paired structures seem to have evolved from dimeric precursors by tandem gene duplication. They contain repeated amino acid sequences or precisely repeated structural elements in which equivalent sets of alpha-carbon atoms can be superimposed with root mean square deviations of the order of 1-2 A. Here it is shown that copper-zinc superoxide dismutase contains two paired subdomains, and the significance of the repeated folding pattern is discussed.

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5′-phosphate-dependent enzyme