Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination.

ZYG11B is a substrate specificity factor for Cullin-RING ubiquitin ligase (CRL2) involved in many biological processes, including Gly/N-degron pathways. Yet how the binding of ZYG11B with CRL2 is coupled to substrate recognition and ubiquitination is unknown. We present the Cryo-EM structures of the CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer.

Dodecamer assembly of a metazoan AAA chaperone couples substrate extraction to refolding.

Ring-forming AAA chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states.

Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity.

Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2.

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before.

High-power near-concentric Fabry-Perot cavity for phase contrast electron microscopy.

Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry-Perot cavity as a phase plate for TEM.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding.

Enhancing the signal-to-noise ratio and generating contrast for cryo-EM images with convolutional neural networks.

In cryogenic electron microscopy (cryo-EM) of radiation-sensitive biological samples, both the signal-to-noise ratio (SNR) and the contrast of images are critically important in the image-processing pipeline. Classic methods improve low-frequency image contrast experimentally, by imaging with high defocus, or computationally, by applying various types of low-pass filter. These contrast improvements typically come at the expense of the high-frequency SNR, which is suppressed by high-defocus imaging and removed by low-pass filtration.

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses.

Allosteric coupling between α-rings of the 20S proteasome.

Proteasomal machinery performs essential regulated protein degradation in eukaryotes. Classic proteasomes are symmetric, with a regulatory ATPase docked at each end of the cylindrical 20S. Asymmetric complexes are also present in cells, either with a single ATPase or with an ATPase and non-ATPase at two opposite ends.

General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM.

Affinity grids have great potential to facilitate rapid preparation of even quite impure samples in single-particle cryo-electron microscopy (EM). Yet despite the promising advances of affinity grids over the past decades, no single strategy has demonstrated general utility. Here we chemically functionalize cryo-EM grids coated with mostly one or two layers of graphene oxide to facilitate affinity capture.

Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring.

Protein nanoribbons template enamel mineralization.

As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date.

Ion transport and regulation in a synaptic vesicle glutamate transporter.

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential.

Exploring long-range cooperativity in the 20S proteasome core particle from using methyl-TROSY-based NMR.

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel.

The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Cells into Quiescence.

The class of Cullin-RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in ).

A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM.

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them.