High-resolution cryo-EM Reconstructions Research Articles

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

High-resolution cryo-EM reconstructions in the presence of substantial aberrations.

Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the ‘core factor’ subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.

Throughput and resolution with a next-generation direct electron detector.

Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy (cryo-EM) by facilitating the correction of beam-induced motion and radiation damage, and also by providing high-resolution image capture. A new-generation DED, the DE64, has been developed by Direct Electron that has good performance in both integrating and counting modes. The camera has been characterized in both modes in terms of image quality, throughput and resolution of cryo-EM reconstructions. The modulation transfer function, noise power spectrum and detective quantum efficiency (DQE) were determined for both modes, as well as the number of images per unit time. Although the DQE for counting mode was superior to that for integrating mode, the data-collection throughput for this mode was more than ten times slower. Since throughput and resolution are related in single-particle cryo-EM, data for apoferritin were collected and reconstructed using integrating mode, integrating mode in conjunction with a Volta phase plate (VPP) and counting mode. Only the counting-mode data resulted in a better than 3 Å resolution reconstruction with similar numbers of particles, and this increased performance could not be compensated for by the increased throughput of integrating mode or by the increased low-frequency contrast of integrating mode with the VPP. These data show that the superior image quality provided by counting mode is more important for high-resolution cryo-EM reconstructions than the superior throughput of integrating mode.

Cryo-EM structure of the hibernating Thermus thermophilus 100S ribosome reveals a protein-mediated dimerization mechanism

In response to cellular stresses bacteria conserve energy by dimerization of ribosomes into inactive hibernating 100S ribosome particles. Ribosome dimerization in Thermus thermophilus is facilitated by hibernation-promoting factor (TtHPF). In this study we demonstrate high sensitivity of Tt100S formation to the levels of TtHPF and show that a 1:1 ratio leads to optimal dimerization. We report structures of the T. thermophilus 100S ribosome determined by cryo-electron microscopy to average resolutions of 4.13 Å and 4.57 Å. In addition, we present a 3.28 Å high-resolution cryo-EM reconstruction of a 70S ribosome from a hibernating ribosome dimer and reveal a role for the linker region connecting the TtHPF N- and C-terminal domains in translation inhibition by preventing Shine−Dalgarno duplex formation. Our work demonstrates that species-specific differences in the dimerization interface govern the overall conformation of the 100S ribosome particle and that for Thermus thermophilus no ribosome-ribosome interactions are involved in the interface.

Human β-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability

Cell biological studies have shown that protofilament number, a fundamental feature of microtubules, can correlate with the expression of different tubulin isotypes. However, it is not known if tubulin isotypes directly control this basic microtubule property. Here, we report high-resolution cryo-EM reconstructions (3.5-3.65Å) of purified human α1B/β3 and α1B/β2B microtubules and find thatthe β-tubulin isotype can determine protofilament number. Comparisons of atomic models of 13- and 14-protofilament microtubules reveal howtubulin subunit plasticity, manifested in "accordion-like" distributed structural changes, can accommodate distinct lattice organizations. Furthermore, compared to α1B/β3 microtubules, α1B/β2B filaments are more stable to passive disassembly and against depolymerization by MCAK or chTOG, microtubule-associated proteins with distinct mechanisms of action. Mixing tubulin isotypes in different proportions results in microtubules with protofilament numbers and stabilities intermediate to those of isotypically pure filaments.Together, our findings indicate that microtubule protofilament number and stability can be controlled through β-tubulin isotype composition.

A deep convolutional neural network approach to single-particle recognition in cryo-electron microscopy

BackgroundSingle-particle cryo-electron microscopy (cryo-EM) has become a mainstream tool for the structural determination of biological macromolecular complexes. However, high-resolution cryo-EM reconstruction often requires hundreds of thousands of single-particle images. Particle extraction from experimental micrographs thus can be laborious and presents a major practical bottleneck in cryo-EM structural determination. Existing computational methods for particle picking often use low-resolution templates for particle matching, making them susceptible to reference-dependent bias. It is critical to develop a highly efficient template-free method for the automatic recognition of particle images from cryo-EM micrographs.ResultsWe developed a deep learning-based algorithmic framework, DeepEM, for single-particle recognition from noisy cryo-EM micrographs, enabling automated particle picking, selection and verification in an integrated fashion. The kernel of DeepEM is built upon a convolutional neural network (CNN) composed of eight layers, which can be recursively trained to be highly “knowledgeable”. Our approach exhibits an improved performance and accuracy when tested on the standard KLH dataset. Application of DeepEM to several challenging experimental cryo-EM datasets demonstrated its ability to avoid the selection of un-wanted particles and non-particles even when true particles contain fewer features.ConclusionsThe DeepEM methodology, derived from a deep CNN, allows automated particle extraction from raw cryo-EM micrographs in the absence of a template. It demonstrates an improved performance, objectivity and accuracy. Application of this novel method is expected to free the labor involved in single-particle verification, significantly improving the efficiency of cryo-EM data processing.

Exposing the Molecular Machinery of BK Polyomavirus

BK polyomavirus (BKV) is an opportunistic pathogen that poses a serious threat to organ transplant recipients. In this issue of Structure, Hurdiss and colleagues' (Hurdiss et al., 2016) beautiful new high-resolution cryo-EM reconstruction of BKV provides a structural roadmap for the ongoing development of therapeutic antibodies and vaccines targeting this potentially deadly virus. The study also serves as a platform for exploring the basic biology of virion assembly and infectious entry.

Integrative Modeling Approaches to Interpret High-Resolution cryo-EM Reconstructions

Modeling 3D-EM reconstructions with computational tools currently enables the interpretation at near-atomic resolution of different functional states of macromolecules, thereby deciphering the functional mechanism of biologically relevant complexes. Recent advances in cryo-EM, such as direct electron detectors, specimen preparation, image processing, and data automation, are increasing the number of determined structures, particularly at high (<5 Å) resolutions. Here, several new integrative approaches are presented to retrieve structural information from these accurate reconstructions by incorporating modeling constraints from complementary biophysical techniques (crystallography, SAXS, FRET, etc.) or any other source of structural information (cross-linking, mutagenesis, prediction data, etc.). First, a two-step integrative approach was developed to unravel the topology of helical bundles using cryo-EM maps, distance restraints, and secondary structure predictions. This method unambiguously localized all helices of a key unassigned proteasome helical bundle and provided a topologically correct model that was later confirmed by crystallography. Second, our normal mode based flexible fitting algorithm, iMODFIT, was accelerated and adapted to deal with high resolution cryo-EM maps and other experimental constraints. Third, a fast loop-closure algorithm (RCD) was combined with integrative fitting strategies for modeling loops into unfilled densities. We strongly believe that these tools will facilitate the interpretation of the incoming high-resolution maps.

Tropomyosin Movement on F-actin Analyzed by Energy Landscape Determination

Muscle contraction is regulated by movement of tropomyosin over the surface of actin filaments. At low-Ca2+, tropomyosin blocks myosin-binding on actin, whereas, in a two-step process, Ca2+-saturated troponin and myosin open the myosin-binding site leading to contraction. No obvious steric obstructions or geometrical barriers on actin limit such tropomyosin movement. However, lacking atomic models for these transitions, the pathways taken by tropomyosin during regulatory movements are uncertain. Here, end-points for regulatory transitions were determined by fitting tropomyosin to EM reconstructions. Reconstructions of negatively-stained low-Ca2+ thin filaments yield an atomic model very close to that described for troponin-free actin-tropomyosin by Li et al. (2011), while comparable high-Ca2+ filament maps suggest an azimuthal sliding of tropomyosin parallel to its superhelical path. Additionally, Raunser et al. (2012-Biophys. Soc. abst.) describe an atomic model of tropomyosin based on high-resolution cryo-EM reconstructions of myosin-decorated filaments, showing expected azimuthal movement and pronounced ∼15A axial displacement of tropomyosin toward the pointed-end of the thin filament. To evaluate transitions between these various regulatory positions, we explored the energy landscape between “end-states” over a comprehensive grid of 832 tropomyosin locations relative to F-actin coordinates. The position of tropomyosin was varied azimuthally and axially relative to F-actin, and then the structure energy-minimized. The resulting electrostatic energy landscape shows a wide energy basin with a minimum centered near the blocked-state. The width of this basin indicates that large azimuthal and axial oscillations of tropomyosin are possible. By contrast, in the myosin-induced open-position, tropomyosin is located at an energy peak, representing a region with no obvious complementary electrostatic interactions between tropomyosin and F-actin. Our results therefore suggest that the open-position is reached only because of tropomyosin interaction with myosin, while binding of tropomyosin to the F-actin surface becomes less important.

Bypassing Translation Initiation

A high-resolution cryo-EM reconstruction of a ribosome-bound dicistrovirus IRES (Schüler et al., 2006) and the crystal structure of its ribosome binding domain (Pfingsten et al., 2006) provide new insights into an exceptional eukaryotic translation mechanism.