The landscape of regional missense mutational intolerance quantified from 125,748 exomes.

Missense variants can have a range of functional impacts depending on factors such as the specific amino acid substitution and location within the gene. To interpret their deleteriousness, studies have sought to identify regions within genes that are specifically intolerant of missense variation 1-12 . Here, we leverage the patterns of rare missense variation in 125,748 individuals in the Genome Aggregation Database (gnomAD) 13 against a null mutational model to identify transcripts that display regional differences in missense constraint. Missense-depleted regions are enriched for ClinVar 14 pathogenic variants, de novo missense variants from individuals with neurodevelopmental disorders (NDDs) 15,16 , and complex trait heritability. Following ClinGen calibration recommendations for the ACMG/AMP guidelines, we establish that regions with less than 20% of their expected missense variation achieve moderate support for pathogenicity. We create a missense deleteriousness metric (MPC) that incorporates regional constraint and outperforms other deleteriousness scores at stratifying case and control de novo missense variation, with a strong enrichment in NDDs. These results provide additional tools to aid in missense variant interpretation.

Binding to nucleosome poises human SIRT6 for histone H3 deacetylation.

Sirtuin 6 (SIRT6) is an NAD+-dependent histone H3 deacetylase that is prominently found associated with chromatin, attenuates transcriptionally active promoters and regulates DNA repair, metabolic homeostasis and lifespan. Unlike other sirtuins, it has low affinity to free histone tails but demonstrates strong binding to nucleosomes. It is poorly understood how SIRT6 docking on nucleosomes stimulates its histone deacetylation activity. Here, we present the structure of human SIRT6 bound to a nucleosome determined by cryogenic electron microscopy. The zinc finger domain of SIRT6 associates tightly with the acidic patch of the nucleosome through multiple arginine anchors. The Rossmann fold domain binds to the terminus of the looser DNA half of the nucleosome, detaching two turns of the DNA from the histone octamer and placing the NAD+ binding pocket close to the DNA exit site. This domain shows flexibility with respect to the fixed zinc finger and moves with, but also relative to, the unwrapped DNA terminus. We apply molecular dynamics simulations of the histone tails in the nucleosome to show that in this mode of interaction, the active site of SIRT6 is perfectly poised to catalyze deacetylation of the H3 histone tail and that the partial unwrapping of the DNA allows even lysines close to the H3 core to reach the enzyme.

The Scalable Variant Call Representation: Enabling Genetic Analysis Beyond One Million Genomes.

The Variant Call Format (VCF) is widely used in genome sequencing but scales poorly. For instance, we estimate a 150,000 genome VCF would occupy 900 TiB, making it both costly and complicated to produce and analyze. The issue stems from VCF's requirement to densely represent both reference-genotypes and allele-indexed arrays. These requirements lead to unnecessary data duplication and, ultimately, very large files. To address these challenges, we introduce the Scalable Variant Call Representation (SVCR). This representation reduces file sizes by ensuring they scale linearly with samples. SVCR achieves this by adopting reference blocks from the Genomic Variant Call Format (GVCF) and employing local allele indices. SVCR is also lossless and mergeable, allowing for N+1 and N+K incremental joint-calling. We present two implementations of SVCR: SVCR-VCF, which encodes SVCR in VCF format, and VDS, which uses Hail's native format. Our experiments confirm the linear scalability of SVCR-VCF and VDS, in contrast to the super-linear growth seen with standard VCF files. We also discuss the VDS Combiner, a scalable, open-source tool for producing a VDS from GVCFs and unique features of VDS which enable rapid data analysis. SVCR, and VDS in particular, ensure the scientific community can generate, analyze, and disseminate genetics datasets with millions of samples.

The structure of the NuA4-Tip60 complex reveals the mechanism and importance of long-range chromatin modification.

Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site. Neither the molecular mechanism of this activity nor its physiological importance are known. Here we report the structure of the Pichia pastoris NuA4 complex, with its core resolved at 3.4-Å resolution. Three subunits, Epl1, Eaf1 and Swc4, intertwine to form a stable platform that coordinates all other modules. The HAT module is firmly anchored into the core while retaining the ability to stretch out over a long distance. We provide structural, biochemical and genetic evidence that an unfolded linker region of the Epl1 subunit is critical for this long-range activity. Specifically, shortening the Epl1 linker causes severe growth defects and reduced H4 acetylation levels over broad chromatin regions in fission yeast. Our work lays the foundations for a mechanistic understanding of NuA4's regulatory role and elucidates how its essential long-range activity is attained.

Bioactivated and PEG-Protected Circa 2 nm Gold Nanoparticles for in Cell Labelling and Cryo-Electron Microscopy.

Advances in cryo-electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2 nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate-, thionitrobenzoate-coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)-containing peptides and 2 kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin-mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non-specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG-coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo-EM of vitreous cell sections demonstrate the presence of 2 nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live-cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo-EM.

3D multiscale analysis of the hierarchical porosity in Coscinodiscus sp. diatoms using a combination of tomographic techniques.

A full 3D analysis of the hierarchical porosity in Coscinodiscus sp. diatom structures was carried out by using a multiscale approach that combines three advanced volumetric imaging techniques with resolutions and fields of view covering all the porous characteristics of such complex architectures: electron tomography, "slice and view" approach that uses a dual-beam microscope (FIB-SEM), and array tomography consisting of serial imaging of ultrathin specimen sections. This multiscale approach allowed the whole porosity network to be quantified and provided an unprecedented structural insight into these natural nanostructured materials with internal organization ranging from micrometer to nanometer. The analysed species is made of several nested layers with different pore sizes, shapes and connectivities and characterized by the presence of interconnected pores structured in various ways. The first evidence of the presence of a nanometric porosity made of ellipsoidal pores in the siliceous diatom frustules is also provided.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Atomic structure of the SAGA complex and it's interaction with TBP.

The transcription of eukaryotic protein genes is controlled by a plethora of proteins which act together in multi-component complexes to facilitate the DNA dependent RNA polymerase II (Pol II) enzyme to bind to the transcription start site and to generate messenger RNA from the gene's coding sequence. The protein that guides the transcription machinery to the exact transcription start site is called TATA-box Binding Protein, or TBP. TBP is part of two large protein complexes involved in Pol II transcription, TFIID and SAGA. The two complexes share several subunits implicated in the interaction with TBP and contain proteins with structural elements highly homologous to nucleosomal histones. Despite the intensive study of transcription initiation, the mode of interaction of TBP with these complexes and its release upon DNA binding was elusive. In this study we demonstrate the quasi-atomic model of SAGA in complex with TBP. The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves a deformed octamer of histone-fold domains at the core of SAGA. This deformed octamer is precisely tuned to establish a peripheral site for TBP binding, where it is protected by steric hindrance against the binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires the general transcription factor TFIIA and whose efficiency correlates with the affinity of DNA to TBP.As the TBP binding machinery is highly similar in TFIID and SAGA, we demonstrated a universal mechanism of how TBP is delivered to gene promoters during transcription initiation.

La transcription des gènes des protéines eucaryotes est contrôlée par une pléthore de protéines agissant de concert sous forme de complexes multi-composants pour faciliter la liaison de l'enzyme ARN polymérase II ADN-dépendante (Pol II) au site d'initiation de la transcription et pour générer un ARN messager à partir de la séquence codante du gène. La protéine qui guide la machinerie de transcription vers le site d'initiation de la transcription est appelée protéine de liaison à la boîte TATA, ou TBP. TBP fait partie de deux complexes protéiques impliqués dans la transcription par la Pol II, TFIID et SAGA. Les deux complexes partagent plusieurs sous-unités impliquées dans l'interaction avec TBP et comportent des protéines présentant des éléments structuraux hautement homologues aux histones nucléosomiques. Malgré l'étude intensive de l'initiation de la transcription, le mode d'interaction de TBP avec ces complexes ainsi que sa libération lors de sa liaison de l'ADN étaient évasifs. Dans cette étude, nous avons déterminé un modèle quasi-atomique de SAGA en complexe avec TBP. La structure révèle le réseau d'interactions qui coordonnent les différents domaines fonctionnels de SAGA et résout un octamère déformé des domaines homologues aux histones au cœur de SAGA. Cet octamère déformé est précisément adapté pour établir un site périphérique de liaison à TBP, où ce dernier est protégé par une inhibition stérique contre la fixation d'un ADN parasite. L'analyse biochimique complémentaire a mis en évidence un mécanisme de libération de TBP de SAGA qui nécessite le facteur de transcription général TFIIA et dont l'efficacité corrèle avec l'affinité de l'ADN pour TBP.Comme le mécanisme de liaison de TBP est très similaire dans TFIID et SAGA, nous avons mis en évidence un mécanisme universel décrivant la manière dont TBP est délivré aux promoteurs de gènes lors de l'initiation de la transcription.

Gold labelling of a green fluorescent protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles.

Advances in microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNPs remains challenging in comparison to the conjugation of 150 kDa IgG to AuNPs. Herein, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP via thiolate exchange. After pegylation, the modified AuNPs are able to non-covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to form immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy.

Architecture of the multi-functional SAGA complex and the molecular mechanism of holding TBP.

In eukaryotes, transcription of protein encoding genes is initiated by the controlled deposition of the TATA-box binding protein TBP onto gene promoters, followed by the ordered assembly of a pre-initiation complex. The SAGA co-activator is a 19-subunit complex that stimulates transcription by the action of two chromatin-modifying enzymatic modules, a transcription activator binding module, and by delivering TBP. Recent cryo electron microscopy structures of yeast SAGA with bound nucleosome or TBP reveal the architecture of the different functional domains of the co-activator. An octamer of histone fold domains is found at the core of SAGA. This octamer, which deviates considerably from the symmetrical analogue forming the nucleosome, establishes a peripheral site for TBP binding where steric hindrance represses interaction with spurious DNA. The structures point to a mechanism for TBP delivery and release from SAGA that requires TFIIA and whose efficiency correlates with the affinity of DNA to TBP. These results provide a structural basis for understanding specific TBP delivery onto gene promoters and the role played by SAGA in regulating gene expression. The properties of the TBP delivery machine harboured by SAGA are compared with the TBP loading device present in the TFIID complex and show multiple similitudes.

Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy.

Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular complexes. The small wavelength of electron and the availability of electron optics enable the direct visualization of macromolecular assemblies in a large range of sizes between 5 and 100 nm. This informs us about the degree of multimerization or aggregation and provides precise information about their general shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional (3D) information can be gained from 2D projections of the sample, leading to a structural description. When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules can be gained, and structure-function relations can be established. However, the way the sample is prepared for its observation within the vacuum of an electron microscope determines the information that can be retrieved from the experiment. We will review two commonly used specimen preparation protocols for subsequent single-particle electron microscopy observation, namely negative staining and vitrification.

Cryo-FIB-SEM as a promising tool for localizing proteins in 3D.

Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is an invaluable tool to visualize the 3D architecture of cell constituents and map cell networks. Recently, amorphous ice embedding techniques have been associated with FIB-SEM to ensure that the biological material remains as close as possible to its native state. Here we have vitrified human HeLa cells and directly imaged them by cryo-FIB-SEM with the secondary electron InLens detector at cryogenic temperature and without any staining. Image stacks were aligned and processed by denoising, removal of ion beam milling artefacts and local charge imbalance. Images were assembled into a 3D volume and the major cell constituents were modelled. The data illustrate the power of the workflow to provide a detailed view of the internal architecture of the fully hydrated, close-to-native, entire HeLa cell. In addition, we have studied the feasibility of combining cryo-FIB-SEM imaging with live-cell protein detection. We demonstrate that internalized gold particles can be visualized by detecting back scattered primary electrons at low kV while simultaneously acquiring signals from the secondary electron detector to image major cell features. Furthermore, gold-conjugated antibodies directed against RNA polymerase II could be observed in the endo-lysosomal pathway while labelling of the enzyme in the nucleus was not detected, a shortcoming likely due to the inadequacy between the size of the gold particles and the voxel size. With further refinements, this method promises to have a variety of applications where the goal is to localize cellular antigens while visualizing the entire native cell in three dimensions.

Structure of SAGA and mechanism of TBP deposition on gene promoters.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.

Over the rainbow: A practical guide for fluorescent protein selection in plant FRET experiments.

Receptor-like kinases (RLK) and receptor-like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo- and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is, however, limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Förster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (a) The emission and excitation spectra of the donor and acceptor, respectively, must overlap; (b) they should not interfere with proper folding, activity, or localization of the fusion proteins; (c) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near-endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET-based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.

A Cellular Insulator against CLE45 Peptide Signaling.

Plants continuously elaborate their bodies through post-embryonic, reiterative organ formation by apical meristems [1]. Meristems harbor stem cells, which produce daughter cells that divide repeatedly before they differentiate. How transitions between stemness, proliferation, and differentiation are precisely coordinated is not well understood, but it is known that phytohormones as well as peptide signals play important roles [2-7]. For example, in Arabidopsis thaliana root meristems, developing protophloem sieve elements (PPSEs) express the secreted CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide and its cognate receptor, the leucine-rich repeat receptor kinase (LRR-RK) BARELY ANY MERISTEM 3 (BAM3). Exogenous CLE45 application or transgenically increased CLE45 dosage impairs protophloem formation, suggesting autocrine inhibition of PPSE differentiation by CLE45 signaling. Since CLE45 and BAM3 are expressed throughout PPSE development, it remains unclear how this inhibition is eventually overcome. The OCTOPUS (OPS) gene is required for proper PPSE differentiation and therefore the formation of continuous protophloem strands. OPS dosage increase can mend the phenotype of other mutants that display protophloem development defects in association with CLE45-BAM3 hyperactivity [8, 9]. Here, we provide evidence that OPS protein promotes differentiation of developing PPSEs by dampening CLE45 perception. This markedly quantitative antagonism is likely mediated through direct physical interference of OPS with CLE45 signaling component interactions. Moreover, hyperactive OPS confers resistance to other CLE peptides, and ectopic OPS overexpression triggers premature differentiation throughout the root. Our results thus reveal a novel mechanism in PPSE transition toward differentiation, wherein OPS acts as an "insulator" to antagonize CLE45 signaling.

Molecular structure of promoter-bound yeast TFIID.

Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 Å, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.

Structural Basis for NusA Stabilized Transcriptional Pausing.

Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, we report single-particle electron cryo-microscopy reconstructions of NusA bound to paused E. coli RNAP elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.

Molecular architecture of potassium chloride co-transporter KCC2.

KCC2 is a neuron specific K+-Cl- co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA.

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.