ABSTRACT
Gas vesicles are gas-filled nanocompartments that allow a diverse group of bacteria and archaea to control their buoyancy. The molecular basis of their properties and assembly remains unclear. Here, we report the 3.2 Å cryo-EM structure of the gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Two helical half shells connect through a characteristic arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. The fold of GvpA features a corrugated wall structure typical for force-bearing thin-walled cylinders. Small pores enable gas molecules to diffuse across the shell, while the exceptionally hydrophobic interior surface effectively repels water. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and demonstrates molecular features of shell reinforcement by GvpC. Our findings will further research into gas vesicle biology and facilitate molecular engineering of gas vesicles for ultrasound imaging.
Subject(s)
Archaea , Biological Evolution , Cryoelectron Microscopy , Engineering , Reinforcement, PsychologyABSTRACT
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
Subject(s)
Cryoelectron Microscopy , Glycine Hydroxymethyltransferase , Glycine Hydroxymethyltransferase/metabolism , Glycine Hydroxymethyltransferase/genetics , Glycine Hydroxymethyltransferase/chemistry , Humans , RNA/metabolism , RNA/genetics , Serine/metabolism , Allosteric Regulation , Protein Binding , Phylogeny , Models, Molecular , Protein Conformation , Structure-Activity Relationship , Glycine/metabolism , Glycine/chemistry , Binding SitesABSTRACT
Gas vesicles mediate buoyancy-based motility in aquatic bacteria and archaea and are the only protein-based structures known to enclose a gas-filled volume. Their unique physicochemical properties and ingenious architecture rank them among the most intriguing macromolecular assemblies characterised to date. This review covers the 60-year journey in quest for a high-resolution structural model of gas vesicles, first highlighting significant strides made in establishing the detailed ultrastructure of gas vesicles through transmission electron microscopy, X-ray fibre diffraction, atomic force microscopy, and NMR spectroscopy. We then survey the recent progress in cryogenic electron microscopy studies of gas vesicles, which eventually led to a comprehensive atomic model of the mature assembly. Synthesising insight from these structures, we examine possible mechanisms of gas vesicle biogenesis and growth, presenting a testable model to guide future experimental work. We conclude by discussing future directions in the structural biology of gas vesicles, particularly considering advancements in AI-driven structure prediction.
Subject(s)
Bacteria , Proteins , Proteins/chemistry , Archaea , BiologyABSTRACT
RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.
Subject(s)
DNA/chemistry , Protein Subunits/chemistry , RNA Polymerase I/chemistry , RNA/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Gene Expression , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , RNA/genetics , RNA/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/isolation & purification , RNA Polymerase I/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Resolution-dependent loss of contrast in cryo-EM maps may obscure features at high resolution that are critical for map interpretation. Post-processing of cryo-EM maps can improve the interpretability by adjusting the resolution-dependence of structure factor amplitudes through map sharpening. Traditionally this has been done by rescaling the relative contribution of low and high-resolution frequencies globally. More recently, the realisation that molecular motion and heterogeneity cause non-uniformity of resolution throughout the map has inspired the development of techniques that optimise sharpening locally. We previously developed LocScale, a method that utilises the radial structure factor from a refined atomic model as a restraint for local map sharpening. While this method has proved beneficial for the interpretation of cryo-EM maps, the dependence on the availability of (partial) model information limits its general applicability. Here, we review the basic assumptions of resolution-dependent contrast loss in cryo-EM maps and propose a route towards a robust alternative for local map sharpening that utilises information on expected scattering properties of biological macromolecules, but requires no detailed knowledge of the underlying molecular structure. We examine remaining challenges for implementation and discuss possible applications.
Subject(s)
Electrons , Cryoelectron Microscopy/methods , Models, Molecular , Protein ConformationABSTRACT
In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.
Subject(s)
RNA Polymerase I/chemistry , RNA Polymerase I/metabolism , Saccharomyces cerevisiae/enzymology , Transcription, Genetic , Cryoelectron Microscopy , DNA, Fungal/metabolism , Models, Molecular , Protein Conformation , Protein Multimerization , RNA, Ribosomal/biosynthesis , Saccharomyces cerevisiae/geneticsABSTRACT
Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.
Subject(s)
Models, Molecular , RNA Polymerase III/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Cryoelectron Microscopy , Protein Binding , Protein Structure, TertiaryABSTRACT
Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.
Subject(s)
Software , Cryoelectron Microscopy , Macromolecular Substances , Models, Molecular , Protein ConformationABSTRACT
Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.
Subject(s)
Autophagy , Vacuoles/metabolism , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/metabolism , Aminopeptidases/chemistry , Aminopeptidases/metabolism , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Biological Transport , Cytoplasm/metabolism , Membranes/metabolism , Models, Biological , Protein Binding , Protein Conformation , Protein Multimerization , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/geneticsABSTRACT
Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-ß unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.
Subject(s)
Bacterial Proteins/metabolism , Carbon-Carbon Ligases/metabolism , Leucine/metabolism , Multienzyme Complexes/metabolism , Mycobacterium tuberculosis/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Bacterial Proteins/chemistry , Carbon-Carbon Ligases/chemistry , Gene Knockout Techniques , Immunoprecipitation , Mass Spectrometry , Metabolism/physiology , Molecular Sequence Data , Protein Structure, Quaternary , Sequence Homology, Nucleic AcidABSTRACT
With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
Subject(s)
Capsid Proteins/ultrastructure , Tobacco Mosaic Virus/ultrastructure , Cryoelectron Microscopy/instrumentation , Models, Molecular , Protein Structure, QuaternaryABSTRACT
Conformational heterogeneity of biological macromolecules is a challenge in single-particle averaging (SPA). Current standard practice is to employ classification and filtering methods that may allow a discrete number of conformational states to be reconstructed. However, the conformation space accessible to these molecules is continuous and, therefore, explored incompletely by a small number of discrete classes. Recently developed heterogeneous reconstruction algorithms (HRAs) to analyse continuous heterogeneity rely on machine-learning methods that employ low-dimensional latent space representations. The non-linear nature of many of these methods poses a challenge to their validation and interpretation and to identifying functionally relevant conformational trajectories. These methods would benefit from in-depth benchmarking using high-quality synthetic data and concomitant ground truth information. We present a framework for the simulation and subsequent analysis with respect to the ground truth of cryo-EM micrographs containing particles whose conformational heterogeneity is sourced from molecular dynamics simulations. These synthetic data can be processed as if they were experimental data, allowing aspects of standard SPA workflows as well as heterogeneous reconstruction methods to be compared with known ground truth using available utilities. The simulation and analysis of several such datasets are demonstrated and an initial investigation into HRAs is presented.
Subject(s)
Algorithms , Benchmarking , Cryoelectron Microscopy , Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Molecular Dynamics Simulation , Software , Machine Learning , Macromolecular Substances/chemistryABSTRACT
Guanylate-binding proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) mediating host defense against intracellular pathogens. Their antimicrobial activity hinges on their ability to self-associate and coat pathogen-associated compartments or cytosolic bacteria. Coat formation depends on GTPase activity but how nucleotide binding and hydrolysis prime coat formation remains unclear. Here, we report the cryo-electron microscopy structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and describe the molecular ultrastructure of the GBP1 coat on liposomes and bacterial lipopolysaccharide membranes. Conformational changes of the middle and GTPase effector domains expose the isoprenylated C terminus for membrane association. The α-helical middle domains form a parallel, crossover arrangement essential for coat formation and position the extended effector domain for intercalation into the lipopolysaccharide layer of gram-negative membranes. Nucleotide binding and hydrolysis create oligomeric scaffolds with contractile abilities that promote membrane extrusion and fragmentation. Our data offer a structural and mechanistic framework for understanding GBP1 effector functions in intracellular immunity.
ABSTRACT
Rlf is a guanine nucleotide exchange factor for the small G-proteins RalA and RalB and couples Ras- to Ral-signalling. Here the crystal structure of the catalytic module of Rlf consisting of a REM- and a CDC25-homology domain is determined. The structure is distinguished by an extended three stranded ß-sheet called the flagpole. The flagpole is a conserved element in the RalGDS family of guanine nucleotide exchange factors and stabilises the orientation of the REM-domain relative to the CDC25-homology domain. A proline-rich sequence in the flagpole is unique to Rlf and several proteins that interact with this sequence by SH3 domains are identified. Conformational pre-selection results in a gain of affinity and contributes to the establishment of SH3 domain selectivity.
Subject(s)
Transcription Factors/chemistry , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Guanine Nucleotide Exchange Factors , Humans , Hydrogen Bonding , Mice , Models, Molecular , Myosin Type I/chemistry , Peptide Fragments/chemistry , Phospholipase C gamma/chemistry , Protein Binding , Protein Structure, Secondary , src Homology DomainsABSTRACT
Single molecule localization microscopy offers resolution nearly down to the molecular level with specific molecular labelling, and is thereby a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labelling of the structure. This missing information can be completed by merging information from many structurally identical particles in a particle fusion approach similar to cryo-EM single-particle analysis. In this paper, we present a data analysis of particle fusion results of fluorescently labelled Nup96 nucleoporins in the Nuclear Pore Complex to show that Nup96 occurs in a spatial arrangement of two rings of 8 units with two Nup96 copies per unit giving a total of 32 Nup96 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.
Subject(s)
Artificial Intelligence , Nuclear Pore , Nuclear Pore Complex Proteins , Coloring AgentsABSTRACT
A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.
ABSTRACT
Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.
Subject(s)
Microtubules , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Dyneins/metabolism , Kinesins/genetics , Kinesins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Myosins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
Subject(s)
Cryoelectron Microscopy/methods , Microfluidics/instrumentation , Microfluidics/methods , Protein Array Analysis/methods , Specimen Handling/instrumentation , Specimen Handling/methods , Cryoelectron Microscopy/instrumentation , Proteasome Endopeptidase Complex , Water/chemistryABSTRACT
Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.