A proteolytic AAA+ machine poised to unfold protein substrates.

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate.

Automated model-free analysis of cryo-EM volume ensembles with SIREn.

Cryogenic electron microscopy (cryo-EM) has the potential to capture snapshots of proteins in motion and generate hypotheses linking conformational states to biological function. This potential has been increasingly realized by the advent of machine learning models that allow 100s-1,000s of 3D density maps to be generated from a single dataset. How to identify distinct structural states within these volume ensembles and quantify their relative occupancies remain open questions.

NCOA4 initiates ferritinophagy by binding GATE16 using two highly avid short linear interaction motifs.

Cells carefully regulate cytosolic iron, which is a vital enzymatic cofactor, yet is toxic in excess. In mammalian cells, surplus iron is sequestered in ferritin cages that, in iron limiting conditions, are degraded through the selective autophagy pathway ferritinophagy to liberate free iron. Prior work identified the ferritinophagy receptor protein NCOA4, which links ferritin and LC3/GABARAP-family member GATE16, effectively tethering ferritin to the autophagic machinery.

Rapid structural analysis of bacterial ribosomes .

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe the development of a rapid cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures.

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN.

Cryo-electron tomography (cryo-ET) enables observation of macromolecular complexes in their native, spatially contextualized cellular environment. Cryo-ET processing software to visualize such complexes at nanometer resolution via iterative alignment and averaging are well developed but rely upon assumptions of structural homogeneity among the complexes of interest. Recently developed tools allow for some assessment of structural diversity but have limited capacity to represent highly heterogeneous structures, including those undergoing continuous conformational changes.

A proteolytic AAA+ machine poised to unfold a protein substrate.

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 µm wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM's widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation.

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis.

Imaging structurally dynamic ribosomes with cryogenic electron microscopy.

Throughout the history of electron microscopy, ribosomes have served as an ideal subject for imaging and technological development, which in turn has driven our understanding of ribosomal biology. Here, we provide a historical perspective at the intersection of electron microscopy technology development and ribosome biology and reflect on how this technique has shed light on each stage of the life cycle of this dynamic macromolecular machine. With an emphasis on prokaryotic systems, we specifically describe how pairing cryo-EM with clever experimental design, time-resolved techniques, and next-generation heterogeneous structural analysis has afforded insights into the modular nature of assembly, the roles of the many transient biogenesis and translation co-factors, and the subtle variations in structure and function between strains and species.

KsgA facilitates ribosomal small subunit maturation by proofreading a key structural lesion.

Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases, whose precise role is poorly understood. Here, we leverage the power of cryo-EM and machine learning to discover that the E. coli methyltransferase KsgA performs a 'proofreading' function in the assembly of the small ribosomal subunit by recognizing and partially disassembling particles that have matured but are not competent for translation.

Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination.

In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 μm-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EM's widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice.

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN.

Cryo-electron tomography (cryo-ET) allows one to observe macromolecular complexes in their native, spatially contextualized environment. Tools to visualize such complexes at nanometer resolution via iterative alignment and averaging are well-developed but rely on assumptions of structural homogeneity among the complexes under consideration. Recently developed downstream analysis tools allow for some assessment of macromolecular diversity but have limited capacity to represent highly heterogeneous macromolecules, including those undergoing continuous conformational changes.

Uncovering structural ensembles from single-particle cryo-EM data using cryoDRGN.

Single-particle cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique to visualize the structural landscape sampled by a protein complex. However, algorithmic and computational bottlenecks in analyzing heterogeneous cryo-EM datasets have prevented the full realization of this potential. CryoDRGN is a machine learning system for heterogeneous cryo-EM reconstruction of proteins and protein complexes from single-particle cryo-EM data.

Conformational landscape of the yeast SAGA complex as revealed by cryo-EM.

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA.

CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks.

Cryo-electron microscopy (cryo-EM) single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many imaged protein complexes exhibit conformational and compositional heterogeneity that poses a major challenge to existing three-dimensional reconstruction methods. Here, we present cryoDRGN, an algorithm that leverages the representation power of deep neural networks to directly reconstruct continuous distributions of 3D density maps and map per-particle heterogeneity of single-particle cryo-EM datasets.

Role of Era in assembly and homeostasis of the ribosomal small subunit.

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting.

Addressing preferred specimen orientation in single-particle cryo-EM through tilting.

We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.

Structure and dynamics of bacterial ribosome biogenesis.

Bacterial ribosome biogenesis has been an active area of research for more than 30 years and has served as a test-bed for the development of new biochemical, biophysical and structural techniques to understand macromolecular assembly generally. Recent work inspecting the process in vivo has advanced our understanding of the role of ribosome biogenesis factors, the co-transcriptional nature of assembly, the kinetics of the process under sub-optimal conditions, and the rRNA folding and ribosome protein binding pathways. Additionally, new structural work enabled by single-particle electron microscopy has helped to connect in vitro ribosomal protein binding maps to the underlying RNA.

Modular Assembly of the Bacterial Large Ribosomal Subunit.

The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation.

YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit.

YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.