The Structural Basis for Release-Factor Activation During Translation Termination Revealed by Time-Resolved Cryogenic Electron Microscopy

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factors (IFs), which must dissociate before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2–5 and 70S4,6–8 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a 70S EC structure obtained directly from a 70S IC formed via an IF-catalyzed initiation reaction has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)9 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

We use time-resolved cryogenic transmission electron microscopy (TR-cryo-TEM) on a supersaturated solution of calcium sulfate hemihydrate to examine the early stages of particle formation during the hydration of the hemihydrate. As hydration proceeds, we observe nanoscale amorphous clusters that evolve to amorphous particles and then reorganize to crystalline gypsum within tens of seconds. Our results indicate that a multistep particle formation model, where an amorphous phase forms first, followed by the transformation into a crystalline product, is applicable even at time scales of the order of tens of seconds for this system. The addition of a small amount of citric acid significantly delays the reorganization to gypsum crystals. We hypothesize that available calcium ions form complexes with the acid by binding to the carboxylic groups. Their incorporation into a growing particle produces disorder and extends the time over which the amorphous phase exists. We see evidence of patches of "trapped" amorphous phase within the growing gypsum crystals at time scales of the order of 24 h. This is confirmed by complementary X-ray diffraction experiments. Direct imaging of nanoscale samples by TR-cryo-TEM is a powerful technique for a fundamental understanding of crystallization and many other evolving systems.

All well-established experimental methods for investigating the structure of biological macromolecules have spatial and/or temporal limitations inherent to the technique that limit their applicability to studying macromolecular dynamics. The ability to measure macromolecular dynamics is key to gaining predictive mastery of biological systems. This work demonstrates that sufficient data can be acquired from low-Z soft materials using dynamic transmission electron microscopy (DTEM) to potentially allow measurements of macromolecular motion. While a dynamic measurement of macromolecular motion or structural change requires further effort, it is evident from this work that this is feasible under specific experimental conditions that can be achieved using low-dose, time-resolved transmission electron microscopy.

Atomic-scale contact and noncontact scannings on gold surfaces were performed by gold tips in time-resolved high-resolution transmission electron microscopy (HRTEM) using a piezodriven specimen holder. The structural variations of atomic arrangements in the scanning process were observed in situ with a time resolution of 1/60 s. Possibility of combination-type microscopy of HRTEM and scanning-probe microscopy was shown. Mechanical processing at one atomic-layer scale was demonstrated.

This chapter utilizes a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. The ribosome subunit association reaction was performed using the mixing-spraying device as described with some alterations. The chapter demonstrates the capability of the mixing spraying method to capture the states of macromolecules in a reaction within a sub-second time frame. We designed an environmental chamber to monitor and maintain the temperature and humidity during the time the time-resolved cryo-EM grid is sprayed and plunged. The bridges are important for ribosome dynamics and function. In the future, it will be interesting to study translation initiation using this method of time-resolved cryo-EM to shed light on the role of mRNA, initiator tRNA, and initiation factors on the structural dynamics of the subunit association reaction. The mixing-spraying method can also be applied to a wide variety of reactions involving ribosomes, including aminoacyl-tRNA selection, translocation, termination, and ribosome recycling.

Endocytic budding implies the remodeling of a plasma membrane portion from a flat sheet to a closed vesicle. Clathrin- and actin-mediated endocytosis in yeast has proven a very powerful model to study this process, with more than 60 evolutionarily conserved proteins involved in fashioning primary endocytic vesicles. Major progress in the field has been made during the last decades by defining the sequential recruitment of the endocytic machinery at the cell cortex using live-cell fluorescence microscopy. Higher spatial resolution has been recently achieved by developing time-resolved electron microscopy methods, allowing for the first time the visualization of changes in the plasma membrane shape, coupled to the dynamics of the endocytic machinery. Here, we highlight these advances and review recent findings from yeast and mammals that have increased our understanding of where and how endocytic proteins may apply force to remodel the plasma membrane during different stages of the process.

The first prototype of RF gun based relativistic-energy electron microscopy has been constructed at Osaka University to study ultrafast structural dynamic processes in matter. The RF gun driven by a femtosecond laser has generated a 100-fs-pulse MeV electron beam with emittance of 0.1 mm-mrad and energy spread of 10^-4. Both the electron diffraction and image measurements have been succeeded in the prototype using the femtosecond electron beam. In the diffraction measurement, an excellent quality of diffraction pattern was acquired with electron number of 10^6. The singleshot measurement is available in the prototype. In the image measurement, the TEM image was acquired with a total electron number of 10^8. The magnification was 3,000 times. In the next step, we will reduce further the emittance to increase the beam brightness on the sample, and then improve the spatial resolution to <10 nm.

Characterization of Dynamics in Materials Using Time-Resolved Electron Microscopy Incorporating Electron Counting and Electron Correlation Spectroscopy

Protein crystals are expected to be useful not only for their molecular structure analysis but also as functional materials due to their unique properties. Although the generation and the propagation of defects during crystallization play critical roles in the final properties of protein crystals, the dynamics of these processes are poorly understood. By time-resolved liquid-cell transmission electron microscopy, we observed that nanosized crystal defects are surprisingly mobile during the early stages of the crystallization of a lysozyme as a model protein. This highly dynamic behavior of defects reveals that the lattice molecules are mobile throughout the crystal structure. Moreover, the disappearance of the defects indicated that intermolecular bonds can break and reform rapidly with little energetic cost, as reported in theoretical studies. All these findings are in marked contrast to the generally accepted notion that crystal lattices are rigid with very limited mobility of individual lattice molecules.

Electroporation has been used to permeabilize the skin to enhance the transdermal delivery of molecules. The mechanism is generally believed to be based on the temporary breakdown and permeabilization of the stratum corneum by the applied pulsed electric field. The kinetics of recovery are believed to be in the millisecond range. However, no structural changes within this time scale have been observed. We studied the morphological changes to heat stripped porcine stratum corneum following an electroporating pulse by time-resolved freeze fracture electron microscopy. Pulses at a supraelectroporation threshold of 80 volts and 300 μsec were applied across the stratum corneum with a pair of copper plate electrodes, which also served as cooling contacts. The pulse timing was accomplished through the time-of-flight of a trigger bar attached to the plunging rod holding the sample.. The trigger bar passes through two optical switches. As the sample was propelled towards the liquid propane coolant container, the first switch triggered the pulse generator and the second marked the precise time at which the sample was frozen. The sample was pulsed at set times of 0.5 ms, 12ms, 5 sec, 60 sec, and 300 sec before freezing.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

We have explored the exchange of Cs for interlayer Na in birnessite using several techniques, including transmission electron microscopy (TEM) and time-resolved synchrotron X-ray diffraction (XRD). Our goal was to test which of two possible exchange mechanisms is operative during the reaction: (1) diffusion of cations in and out of the interlayer or (2) dissolution of Na-birnessite and reprecipitation of Cs-birnessite. The appearance of distinct XRD peaks for Na- and Cs-rich phases in partially exchanged samples offered support for a simple diffusion model, but it was inconsistent with the compositional and crystallographic homogeneity of (Na,Cs)-birnessite platelets from core to rim as ascertained by TEM. Time-resolved XRD revealed systematic changes in the structure of the emergent Cs-rich birnessite phase during exchange, in conflict with a dissolution and reprecipitation model. Instead, we propose that exchange occurred by sequential delamination of Mn oxide octahedral sheets. Exfoliation of a given interlayer region allowed for wholesale replacement of Na by Cs and was rapidly followed by reassembly. This model accounts for the rapidity of metal exchange in birnessite, the co-existence of distinct Na- and Cs-birnessite phases during the process of exchange, and the uniformly mixed Na- and Cs-compositions ascertained from point analyses by selected area electron diffraction and energy dispersive spectroscopy of partially exchanged grains.

Nanoscale materials show a variety of interesting physical and chemical properties. Since the properties are mostly determined by mesoscopic- and atomicscale areas of the materials, characterization of nanoscale materials at the atomic level is crucial for understanding the origin of their properties as well as for tailoring them to industrial needs. High-resolution electron microscopy (HREM) is an effective method to observe atomic structures in nanophase materials. The “structure-imaging technique,” originated by Ueda et al., and Cowley and Iijima, contributed much to the development of the method. Innovations are anticipated in (1) three-dimensional analysis of nanophase materials in real space, (2) observation of dynamic phenomena, and (3) extraction of compositional and physical information from nanometer-sized areas. Using the structure-imaging technique, one can observe directly nanometer-scale particles and defect structures such as voids and line and plane defects in crystals, including surfaces and interfaces. There were previously some limitations on the study of the dynamic process on the atomic level since images were captured on films. The recent development of high-sensitivity silicon-integrated-target-television (SIT-TV) cameras has initiated a new stage of HREM.Our time-resolved-high-resolution-electron-microscopy (TRHREM) system is composed of a 200-kV HREM (JEOL: JEM-2010) equipped with a high-sensitivity SIT-TV camera, various types of dedicated sample holders, an ultrahigh-vacuum (UHV) sample-preparation chamber, and a digital videotape recorder, as partly illustrated in Figure 5. The HREM images at 0.8–1.0-million magnification are recorded on the videotape recorder. In the U.S.-Japan TV system, one “frame image” of 1/30-s time resolution is composed of two interlaced “field images” of 1/60-s time resolution. The digital videotape recorder enables us to output the two “field images” separately from two frame memories.

Structural Fluctuation in Co-Pt Nanoparticles Revealed by Time-resolved High-voltage Electron Microscopy