Single Particle Image Analysis Research Articles

Binding of N-Terminus Fragments of Cardiac Myosin-Binding C-protein to Actin

Myosin-binding protein C (C-protein) is a sarcomeric protein regularly associated with thick filaments of vertebrate skeletal and heart muscle and is involved in the regulating of muscle contraction. C-protein belongs to the immunoglobulin- fibronectin superfamily of proteins and it consists of 11 tandemly arranged immunoglobulin-like domains. Four Ig-domains at the N-terminus of the cardiac isoform (C0-C1-m-C2 or C0C2) influence the actin -myosin S1 interaction whereas the C-terminal domains (C7-C10) play a structural role in the sarcomere, binding to myosin thick filaments. It has been shown that C0C2 fragment stabilizes F-actin, decorating the filaments in a highly regular arrangement.We used electron microscopy and single particle image analysis to reconstruct complexes of F-actin with cardiac C-protein fragments containing 4 Ig-domains (C0C2) and 2 Ig-domains (C0C1). Both fragments regularly decorated actin filaments and image analysis has revealed substantial rod-shaped density connecting adjacent monomers along the actin filament. The fragments induced dendritic growth of actin filaments in the first several minutes of co-polymerization followed by formation of regular sheets of filaments and ordered bundles. The results will be discussed in the terms of suggested involvement of C-protein in the modulation of contractile cycles of heart muscles.

Amyloid fibril length distribution quantified by atomic force microscopy single-particle image analysis.

Amyloid fibrils are proteinaceous nano-scale linear aggregates. They are of key interest not only because of their association with numerous disorders, such as type II diabetes mellitus, Alzheimer's and Parkinson's diseases, but also because of their potential to become engineered high-performance nano-materials. Methods to characterise the length distribution of nano-scale linear aggregates such as amyloid fibrils are of paramount importance both in understanding the biological impact of these aggregates and in controlling their mechanical properties as potential nano-materials. Here, we present a new quantitative approach to the determination of the length distribution of amyloid fibrils using tapping-mode atomic force microscopy. The method described employs single-particle image analysis corrected for the length-dependent bias that is a common problem associated with surface-based imaging techniques. Applying this method, we provide a detailed characterisation of the length distribution of samples containing long-straight fibrils formed in vitro from β2-microglobulin. The results suggest that the Weibull distribution is a suitable model in describing fibril length distributions, and reveal that fibril fragmentation is an important process even under unagitated conditions. These results demonstrate the significance of quantitative length distribution measurements in providing important new information regarding amyloid assembly.

Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a approximately 17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a approximately 35 A-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.

CD2AP Structure And Progression Of Renal Disease

CD2AP is a scaffolding molecule that was originally cloned as an interaction partner of CD2 in T lymphocytes. In the kidney, CD2AP is strongly expressed in podocytes, a cell type that regulates the filtration barrier. The protein directly interacts with filamentous actin and a variety of cell membrane proteins including the kidney filter protein nephrin. In addition to discrete binding sites for actin and nephrin, CD2AP possesses three SH3 domains and a proline-rich region containing, in turn, binding sites for SH3 domains. CD2AP is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. We have initiated structural studies of recombinant CD2AP protein using electron microscopy and single particle image analysis. Negative stain electron microscopy of revealed uniform particles with a size and morphology suggesting a tetrameric organization, subsequently verified with chemical crosslinking. Single particle image analysis was used to generate a three-dimensional map of the CD2AP tetramer at 21 Å resolution. The electron density map reveals an extended structure allowing the identification of specific subdomains. The tetramer is organized around a central core, including density assigned to the C-terminal coiled-coil domain, surrounded by four loosely attached arms radiating out from the center, which we have assigned to the N-terminal SH3 domains. We have further identified CD2AP as a substrate for cytoplasmic cathepsin L, a protease that is induced in early podocyte damage. Cleavage of CD2AP with cathepsin L results in a C-terminal core domain that is structurally competent but releases the CD2AP binding partner dendrin resulting in translocation of dendrin to the nucleus where it promotes apoptosis. Based on our analysis of the cathepsin L cleavage sites within CD2AP we conclude that cytosolic cathepsin L releases the N-terminal arms producing a structurally competent C-terminal core domain.

Reconstruction of the P2X2 Receptor Reveals a Vase-Shaped Structure with Lateral Tunnels above the Membrane

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.

Smallest Structure of Biological Macromolecular Complex Revealed by Single Particle Image Analysis

Smallest Structure of Biological Macromolecular Complex Revealed by Single Particle Image Analysis

Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.

Ca 2+-Induced Tropomyosin Movement in Scallop Striated Muscle Thin Filaments

Striated muscle contraction in most animals is regulated at least in part by the troponin–tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca 2+ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca 2+, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca 2+ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca 2+, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca 2+ in regulated thin filaments. This Ca 2+-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca 2 + regulated.

Structural Analysis of the Saf Pilus by Electron Microscopy and Image Processing

Bacterial pili are important virulence factors involved in host cell attachment and/or biofilm formation, key steps in establishing and maintaining successful infection. Here we studied Salmonella atypical fimbriae (or Saf pili), formed by the conserved chaperone/usher pathway. In contrast to the well-established quaternary structure of typical/FGS-chaperone assembled, rod-shaped, chaperone/usher pili, little is known about the supramolecular organisation in atypical/FGL-chaperone assembled fimbriae. In our study, we have used negative stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the Salmonella typhimurium Saf pilus. Our results show atypical/FGL-chaperone assembled fimbriae are composed of highly flexible linear multi-subunit fibres that are formed by globular subunits connected to each other by short links giving a “beads on a string”-like appearance. Quantitative fitting of the atomic structure of the SafA pilus subunit into the electron density maps, in combination with linker modelling and energy minimisation, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf pilus. Short intersubunit linker regions provide the molecular basis for flexibility of the Saf pilus by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre.

Single Particle Image Reconstruction of the Human Recombinant Kv2.1 Channel

Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K + currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic β-cells. They are therefore an important drug target for the treatment of Type II diabetes mellitus. We used electron microscopy and single particle image analysis to derive a three-dimensional density map of recombinant human Kv2.1. The tetrameric channel is egg-shaped with a diameter of ∼80 Å and a long axis of ∼120 Å. Comparison to known crystal structures of homologous domains allowed us to infer the location of the cytoplasmic and transmembrane assemblies. There is a very good fit of the Kv1.2 crystal structure to the assigned transmembrane assembly of Kv2.1. In other low-resolution maps of K + channels, the cytoplasmic N-terminal and transmembrane domains form separate rings of density. In contrast, Kv2.1 displays contiguous density that connects the rings, such that there are no large windows between the channel interior and the cytoplasmic space. The crystal structure of KcsA is thought to be in a closed conformation, and the good fit of the KcsA crystal structure to the Kv2.1 map suggests that our preparations of Kv2.1 may also represent a closed conformation. Substantial cytoplasmic density is closely associated with the T1 tetramerization domain and is ascribed to the ∼184 kDa C-terminal regulatory domains within each tetramer.

Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.

Atypical AAA+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104

SummaryHsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose (ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.

A structural investigation of complex I and I + III 2 supercomplex from Zea mays at 11–13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III 2 supercomplex from the C 4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III 2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.

Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.

Rapid purification of active γ‐secretase, an intramembrane protease implicated in Alzheimer’s disease

Gamma-secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-beta precursor protein generates the amyloid-beta protein (Abeta) of Alzheimer's disease, partially inhibiting gamma-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of gamma-secretase, but at low resolution (15 A). The limited amount of purified gamma-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four gamma-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active gamma-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

Tissue specific differences in fibrillin microfibrils analysed using single particle image analysis

Tissue specific differences in fibrillin microfibrils analysed using single particle image analysis

Octomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris

Pyruvate-ferredoxin oxidoreductatse (PFOR) carries out the central step in oxidative decarboxylation of pyruvate to acetyl-CoA. We have purified this enzyme from Desulfovibrio vulgaris Hildenborough ( DvH) as part of a systematic characterization of as many multiprotein complexes as possible for this organism, and the three-dimensional structure of this enzyme has been determined by a combination of electron microscopy (EM), single particle image analysis, homology modeling and computational molecular docking. Our results show that the 1 MDa DvH PFOR complex is a homo-octomer, or more precisely, a tetramer of the dimeric form of the related enzyme found in Desulfovibrio africanus ( Da), with which it shares a sequence identity of 69%. Our homology model of the DvH PFOR dimer is based on the Da PFOR X-ray structure. Docking of this model into our 17 Å resolution EM-reconstruction of negatively stained DvH PFOR octomers strongly suggests that the difference in oligomerization state for the two species is due to the insertion of a single valine residue (Val383) within a surface loop of the DvH enzyme. This study demonstrates that the strategy of intermediate resolution EM reconstruction coupled to homology modeling and docking can be powerful enough to infer the functionality of single amino acid residues.

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ′) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.