A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex.

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

Cryo-electron Microscopy Imaging of Alzheimer's Amyloid-beta 42 Oligomer Displayed on a Functionally and Structurally Relevant Scaffold.

Amyloid-ß peptide (Aß) oligomers are pathogenic species of amyloid aggregates in Alzheimer's disease. Like certain protein toxins, Aß oligomers permeabilize cellular membranes, presumably through a pore formation mechanism. Owing to their structural and stoichiometric heterogeneity, the structure of these pores remains to be characterized. We studied a functional Aß42-pore equivalent, created by fusing Aß42 to the oligomerizing, soluble domain of the α-hemolysin (αHL) toxin. Our data reveal Aß42-αHL oligomers to share major structural, functional, and biological properties with wild-type Aß42-pores. Single-particle cryo-EM analysis of Aß42-αHL oligomers (with an overall 3.3 Šresolution) reveals the Aß42-pore region to be intrinsically flexible. The Aß42-αHL oligomers will allow many of the features of the wild-type amyloid oligomers to be studied that cannot be otherwise, and may be a highly specific antigen for the development of immuno-base diagnostics and therapies.

Brønsted acid catalyzed intramolecular benzylic cyclizations of alkylpyridines.

Aldehyde and ketone electrophiles incorporated into the side chains of 2- and 4-alkylpyridines participate in intramolecular aldol-like condensations with pyridine benzylic carbons in the presence of Brønsted acid catalysts. Pyridines featuring ß-ketoamide side chains undergo cyclization in the presence of 10 mol% TfOH to afford pyridyl-substituted hydroxy lactams in good yield. These products were found to be resistant to further dehydration under a variety of conditions, however treatment with thionyl chloride elicited an unusual dehydration/oxidation reaction sequence. In contrast, acid-catalyzed cyclization of pyridines tethered to aliphatic aldehydes with amine linkers gives pyridyl-substituted dehydro-piperidine products. Similarly, intramolecular condensation of salicylaldehyde- and salicylketone-substituted pyridines affords pyridyl-substituted benzofurans.

Vascular adaptations following an eight-week sprint interval training intervention in healthy men.

BACKGROUND: Sprint interval training (SIT) has been shown to improve many indices of cardiovascular risk. The effect of SIT on emerging indicators of cardiovascular health, including arterial stiffness and carotid intima media thickness, remains unclear. Thus, the purpose of this study was to assess changes in augmentation index at 75 beats per minute (AIx@75), pulse wave velocity (cfPWV), and carotid intima media thickness (CIMT) at three time points of an 8-week SIT intervention. METHODS: Eighteen sedentary men (age: 24.7±5.1 years, BMI: 26.7±5.8 kg/m2) participated in the study. Subjects trained 3 times a week for 8 weeks. Training consisted of 3-6 consecutive 30-second bouts of maximal intensity cycling, with 4.5 minutes of active recovery between bouts. Baseline, 4-week, and 8-week vascular assessments were performed. Training effects were analyzed using repeated measures ANOVA. Pearson correlations were used to determine the relationship between baseline values and the change scores (baseline to 8 weeks) of each vascular measure. RESULTS: AIx@75 (BL: -3.6±10.9%, 4W: -5.6±8.3%, 8W: -3.2±9.5%), cfPWV (BL: 5.6±1.0 m/s, 4W: 5.8±0.9 m/s, 8W: 5.6±0.7 m/s), and CIMT (BL: 0.51±0.08 mm, 4W: 0.52±0.08 mm, 8W: 0.51±0.08 mm) did not significantly change (all P>0.05). Baseline cfPWV and AIx@75 were negatively correlated to their change from baseline to 8 weeks (P<0.05). CONCLUSIONS: These findings demonstrate that 8 weeks of SIT is an insufficient stimulus to reduce cfPWV, AIx@75, or CIMT in a group of young, healthy men. Baseline arterial stiffness may modulate vascular adaptations to SIT.

Structural and functional insight into regulation of kinesin-1 by microtubule-associated protein MAP7.

Microtubule (MT)-associated protein 7 (MAP7) is a required cofactor for kinesin-1-driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.

Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation.

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii.

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM.

Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 Šresolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.

Polymerization in the actin ATPase clan regulates hexokinase activity in yeast.

The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.

The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes.

BRG1 and BRM, central components of the BAF (mSWI/SNF) chromatin remodelling complex, are critical in chromatin structure regulation. Here, we show that the human BRM (hBRM) bromodomain (BRD) has moderate specificity for H3K14ac. Surprisingly, we also find that both BRG1 and hBRM BRDs have DNA-binding activity. We demonstrate that the BRDs associate with DNA through a surface basic patch and that the BRD and an adjacent AT-hook make multivalent contacts with DNA, leading to robust affinity and moderate specificity for AT-rich elements. Although we show that the BRDs can bind to both DNA and H3K14ac simultaneously, the histone-binding activity does not contribute substantially to nucleosome targeting in vitro. In addition, we find that neither BRD histone nor DNA binding contribute to the global chromatin affinity of BRG1 in mouse embryonic stem cells. Together, our results suggest that association of the BRG1/hBRM BRD with nucleosomes plays a regulatory rather than targeting role in BAF activity.