A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein.

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

Chloroplast Sec14-like 1 (CPSFL1) is essential for normal chloroplast development and affects carotenoid accumulation in Chlamydomonas.

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of Chlamydomonas reinhardtii and isolated cpsfl1 The cpsfl1 mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The CPSFL1 gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The cpsfl1 mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and ß-carotene when expressed in Escherichia coli and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

Cryo-EM studies of NAIP-NLRC4 inflammasomes.

The NAIP-NLRC4 family of inflammasomes are components of the innate immune system that sound a molecular alarm in the presence of intracellular pathogens. In this chapter, we provide an in-depth guide to using cryo-electron microscopy (cryo-EM) to investigate these inflammasomes, focusing especially on the techniques we used in our recent structural analysis of the NAIP5-NLRC4 inflammasome. We explain how to circumvent specific obstacles we encountered at each step, from sample preparation through data processing. The methods described here will be useful for further studies of the NAIP5-NLRC4 inflammasome and related supracomplexes involved in innate immune surveillance; they may also be useful for unrelated complexes that present similar issues, such as preferential orientations and compositional heterogeneity.

A unique supramolecular organization of photosystem I in the moss Physcomitrella patens.

The photosynthesis machinery in chloroplast thylakoid membranes is comprised of multiple protein complexes and supercomplexes1,2. Here, we show a novel supramolecular organization of photosystem I (PSI) in the moss Physcomitrella patens by single-particle cryo-electron microscopy. The moss-specific light-harvesting complex (LHC) protein Lhcb9 is involved in this PSI supercomplex, which has been shown to have a molecular density similar to that of the green alga Chlamydomonas reinhardtii3. Our results show that the structural organization is unexpectedly different-two rows of the LHCI belt exist as in C. reinhardtii4, but the outer one is shifted toward the PsaK side. Furthermore, one trimeric LHC protein and one monomeric LHC protein position alongside PsaL/K, filling the gap between these subunits and the outer LHCI belt. We provide evidence showing that Lhcb9 is a key factor, acting as a linkage between the PSI core and the outer LHCI belt to form the unique supramolecular organization of the PSI supercomplex in P. patens.

The structural basis of flagellin detection by NAIP5: A strategy to limit pathogen immune evasion.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form a multiprotein complex called an inflammasome. Here we report the cryo-electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

Enhanced FIB-SEM systems for large-volume 3D imaging.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 106 µm3. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

Architecture of the human XPC DNA repair and stem cell coactivator complex.

The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.

Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor.

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen-deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity.

Atomic force microscopy of photosystem II and its unit cell clustering quantitatively delineate the mesoscale variability in Arabidopsis thylakoids.

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arrays according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets.

Dynamic mechanical responses of Arabidopsis thylakoid membranes during PSII-specific illumination.

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.

Ranking TEM cameras by their response to electron shot noise.

We demonstrate two ways in which the Fourier transforms of images that consist solely of randomly distributed electrons (shot noise) can be used to compare the relative performance of different electronic cameras. The principle is to determine how closely the Fourier transform of a given image does, or does not, approach that of an image produced by an ideal camera, i.e. one for which single-electron events are modeled as Kronecker delta functions located at the same pixels where the electrons were incident on the camera. Experimentally, the average width of the single-electron response is characterized by fitting a single Lorentzian function to the azimuthally averaged amplitude of the Fourier transform. The reciprocal of the spatial frequency at which the Lorentzian function falls to a value of 0.5 provides an estimate of the number of pixels at which the corresponding line-spread function falls to a value of 1/e. In addition, the excess noise due to stochastic variations in the magnitude of the response of the camera (for single-electron events) is characterized by the amount to which the appropriately normalized power spectrum does, or does not, exceed the total number of electrons in the image. These simple measurements provide an easy way to evaluate the relative performance of different cameras. To illustrate this point we present data for three different types of scintillator-coupled camera plus a silicon-pixel (direct detection) camera.

Human TFIID binds to core promoter DNA in a reorganized structural state.

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.

Structural insights into transcriptional repression by noncoding RNAs that bind to human Pol II.

Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 µM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

Modularity of a carbon-fixing protein organelle.

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.