Time-resolved Cryogenic Electron Microscopy Research Articles

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-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.

Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species.

Disease-specific tau filaments assemble via polymorphic intermediates

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297–391) into paired helical filaments of Alzheimer’s disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302–316. Nuclear magnetic resonance indicates that the same residues adopt rigid, β-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading.

SummaryRibosomes accurately decode mRNA by proofreading each aminoacyl-tRNA delivered by elongation factor EF-Tu1. Understanding the molecular mechanism of proofreading requires visualizing GTP-catalyzed elongation, which has remained a challenge2–4. Here, time-resolved cryo-EM revealed 33 states following aminoacyl-tRNA delivery by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding center (DC) dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis allows EF-Tu’s GTPase domain to extend away, releasing EF-Tu from tRNA. Then, the 30S subunit locks cognate tRNA in the DC, and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase center. By contrast, the DC fails to lock near-cognate tRNA, allowing dissociation of near-cognate tRNA during both initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between initial selection5,6 and the previously unseen proofreading, which together govern efficient rejection of incorrect tRNA.

The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Late steps in bacterial translation initiation visualized using time-resolved cryo-EM.

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.

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

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 A away. Surprisingly, free RF2 is compact, with only 20 A between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 A resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms. Translation termination is under strong selection pressure for high speed and accuracy. Here the authors provide a 3D view of the dynamics of a translating bacterial ribosome as it recruits a class-1 release factor (RF1 or RF2) upon encountering a stop codon, and propose a structure-based kinetic model for the early steps in bacterial translation termination.

Caught on Camera: Intermediates of Ribosome Recycling

Following termination of protein synthesis, bacterial ribosomes are split into subunits by the joint action of elongation factor G and ribosome recycling factor in the process called ribosome recycling. In this issue ofStructure, Fu etal. (2016) describe visualization of transient intermediates of ribosome recycling using time-resolved cryogenic electron microscopy.

Ribosome Subunit Joining Frozen in Time

In this issue of Structure, Chen etal. (2015) report the use of a mixing-spraying method of time-resolved cryogenic electron microscopy, which allowed the progression of ribosomal subunit association to be visualized on the millisecond timescale.

Structural Dynamics of Ribosome Subunit Association Studied by Mixing-Spraying Time-Resolved Cryogenic Electron Microscopy

Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60ms and 140ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method.

Time-Resolved cryo-EM Study of Ribosome Subunit Association by Mixing-Spraying

Time-resolved cryogenic electron microscopy (cryo-EM) is a technique for visualizing transient structures in a biological specimen in a pre-equilibrium system. Capturing reactions in the sub-second range has been a practical challenge, due to the requirement of depositing the specimen rapidly on the grid without blotting. To capture faster reactions in the sub-second range, Lu and coworkers [1] developed a mixing-spraying method, which allows a reaction involving two macromolecular components to proceed for tens to hundreds of milliseconds. The first study of ribosome subunit association using time-resolved cryo-EM, by Shaikh et al. [2], were performed at 9.4 ms and 43 ms. In present work, we improved the mixing-spraying method, by designing an environmental chamber and optimizing the EM data yield, and applied the method to the study of ribosome subunit association. We captured the subunit association reaction in a pre-equilibrium state, by mixing the subunits and reacting for 60 ms and 140 ms. Our results showed that at 60 ms and 140 ms time points, 33% and 42% of the large subunits have associated into 70S ribosomes, respectively, compared with 85% in a 15-min incubation control experiment. Three distinct conformations of the 70S ribosome were found: non-rotated, non-rotated with 30S head swiveled and rotated. Our results demonstrate the capability of the mixing-spraying method of time-resolve cryo-EM to visualize multiple states of macromolecules in a reaction within a sub-second time frame. In the future, the mixing-spraying method will be applied to study translation initiation, to gain insights on the role of mRNA, initiator tRNA and initiation factors. Supported by HHMI and NIH R01 GM29169 and GM55440 (to J.F.) [1] Lu, Z., et al. (2009). J. Struct. Biol. 168, 388-395. [2] Shaikh, T.R., et al. (2014). Proc. Natl. Acad. Sci. USA 111, 9822-9827.

A Time-Resolved CRYO-EM Study of Ribosome Subunit Association by Mixing-Spraying

The emergence of a high-throughput data processing pipeline and efficient classification algorithms has revived the idea of time-resolved cryogenic electron microscopy (cryo-EM), i.e., capturing time-dependent structures in a biological specimen using cryo-EM. The blotting method now routinely used to prepare cryo-EM specimen is sufficient only to study a reaction in the second to minute range. Capturing faster reactions, in the sub-second range, has been a practical challenge, due to the required step of depositing the specimen on the grid. To address this challenge, Lu et al. (2009) have developed a novel method to prepare time-resolved cryo-EM specimens, by using a nano-fabricated mixing-spraying chip. The mixing-spraying chip allows a two-component reaction to proceed inside the chip for tens to hundreds of milliseconds (ms), which is then stopped by fast freezing. Here we used the improved mixing-spraying method to study ribosome subunit association, and particularly to capture the conformational changes of the ribosome in this reaction. Ribosome subunit association is a pivotal step in translation initiation. Previous ensemble kinetic studies suggested that the ribosome subunit association is a multi-step process, with the ribosomal inter-subunit bridges sequentially formed. Using time-resolved cryo-EM, we were able to capture the association reaction in a pre-equilibrium state by mixing the two ribosomal subunits and reacting for 140ms. However, the fraction population of the associated ribosomes in each conformation was stable from 140ms to 75min, indicating that the ribosome undergoes conformational changes faster than the 140ms time frame upon association of the subunits. Thus, we have demonstrated that the mixing-spraying method of time-resolved cryo-EM is able to visualize the states of macromolecules in a reaction within a sub-second time frame.