In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR's oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR's cooperative interactions translate to consequences in development and disease.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6–7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Chemical synapses are the major sites of communication between neurons in the nervous system and mediate either excitatory or inhibitory signaling. At excitatory synapses, glutamate is the primary neurotransmitter and upon release from presynaptic vesicles, is detected by postsynaptic glutamate receptors, which include ionotropic AMPA and NMDA receptors. Here we have developed methods to identify glutamatergic synapses in brain tissue slices, label AMPA receptors with small gold nanoparticles (AuNPs), and prepare lamella for cryo-electron tomography studies. The targeted imaging of glutamatergic synapses in the lamella is facilitated by fluorescent pre- and postsynaptic signatures, and the subsequent tomograms allow for identification of key features of chemical synapses, including synaptic vesicles, the synaptic cleft and AuNP-labeled AMPA receptors. These methods pave the way for imaging natively derived brain regions at high resolution, using unstained, unfixed samples preserved under near-native conditions.

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

Mutations in the retinal protein retinoschisin (RS1) cause progressive loss of vision in young males, a form of macular degeneration called X-linked retinoschisis (XLRS). We previously solved the structure of RS1, a 16-mer composed of paired back-to-back octameric rings. Here, we show by cryo-electron microscopy that RS1 16-mers can assemble into extensive branched networks. We classified the different configurations, finding four types of interaction between the RS1 molecules. The predominant configuration is a linear strand with a wavy appearance. Three less frequent types constitute the branch points of the network. In all cases, the "spikes" around the periphery of the double rings are involved in these interactions. In the linear strand, a loop (usually referred to as spike 1) occurs on both sides of the interface between neighboring molecules. Mutations in this loop suppress secretion, indicating the possibility of intracellular higher-order assembly. These observations suggest that branched networks of RS1 may play a stabilizing role in maintaining the integrity of the retina.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1). TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.