dna rna protein complexes  (Thermo Fisher)

Journal: bioRxiv

Article Title: Fast and Automated Protein-DNA/RNA Macromolecular Complex Modeling from Cryo-EM Maps

doi: 10.1101/2022.09.29.510189

Figure Lengend Snippet: This process illustrates the use of the microscope to analyze a portion of a biological structure, get the area of interest and transform the image data into a 3D density map. Once complete, the map will contain both the protein-DNA/RNA complex.

Article Snippet: However, only 12,166 structural models are available to these maps . shows an overall process of how Cryo-EM data is processed to make a protein-DNA/RNA complex model. DeepTracer is a fully automated deep learning-based method for fast de novo multi-chain protein complex structure modeling from cryo-EM maps .

Techniques: Microscopy

Journal: bioRxiv

Article Title: Fast and Automated Protein-DNA/RNA Macromolecular Complex Modeling from Cryo-EM Maps

doi: 10.1101/2022.09.29.510189

Figure Lengend Snippet: The left figure shows a protein structure and a closeup of three amino acids. The right figure shows the nucleic structure of DNA. Blue represents nitrogen atoms, red represents oxygen atoms, orange represents phosphates, light gray shows the side chain carbons, whereas dark gray shows the backbone carbons. Each side chain can have 20 different conformations for amino acids and 5 different kinds of DNA/RNA nucleotides.

Article Snippet: However, only 12,166 structural models are available to these maps . shows an overall process of how Cryo-EM data is processed to make a protein-DNA/RNA complex model. DeepTracer is a fully automated deep learning-based method for fast de novo multi-chain protein complex structure modeling from cryo-EM maps .

Techniques:

Journal: bioRxiv

Article Title: Fast and Automated Protein-DNA/RNA Macromolecular Complex Modeling from Cryo-EM Maps

doi: 10.1101/2022.09.29.510189

Figure Lengend Snippet: There are three cryo-EM maps that go through each pipeline in order to predict the structure. Each model built by DeepTracer (blue) and Phenix (red) are compared to each other, while the deposited model (purple) shows how the figure should appear. Darker pigments represent nucleotides whereas lighter pigments represent amino acids. (a) Result of DNA model EMD-12900. Both the DNA and amino acids appear more complete in the DeepTracer pipeline. (b) Result of RNA model EMD-6777. Both maps had issues tracking the single stranded nucleotides. Additionally, Phenix also had a few false positive nucleotides. (c) Result of RNA model EMD-31963. Phenix had a better prediction for the double helix RNA structure, but DeepTracer did better with the amino acid structure.

Article Snippet: However, only 12,166 structural models are available to these maps . shows an overall process of how Cryo-EM data is processed to make a protein-DNA/RNA complex model. DeepTracer is a fully automated deep learning-based method for fast de novo multi-chain protein complex structure modeling from cryo-EM maps .

Techniques: Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

doi: 10.1093/nar/gkab797

Figure Lengend Snippet: Design of nucleic acid-protein nano-sandwich composite. ( A ) Architecture of the nano-sandwich composite containing a streptavidin protein tetramer (yellow) that associates with two flanking nucleic acid nanotriangles, each bound through two DNA-conjugated biotin ligands (blue). The rotational arrangement of the nanotriangles in this and the following schematics is arbitrarily shown in an eclipsed configuration. The design of the nano-sandwich components allows for eclipsed or staggered arrangement. ( B ) Nucleic acid hybrid nanotriangles that self-assemble from RNA corner modules and DNA connectors (DNA out ) controlled by a DNA guide strand (DNA in ) with three hybridization sites for the connector units. AFM imaging confirmed the nanotriangle architecture . ( C ) Programmed assembly of modified nucleic acid nanotriangles that carry 0–3 biotin ligands attached at DNA connectors (DNA out ). Increasing number of biotin ligands is revealed by decreasing mobility of modified nanotriangles on native polyacrylamide gel. Lane C is a mobility control sample containing a previously established mixture of unmodified polygonal RNA–DNA hybrid nanoshapes .

Article Snippet: The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies.

Techniques: Hybridization, Imaging, Modification

Journal: Nucleic Acids Research

Article Title: Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

doi: 10.1093/nar/gkab797

Figure Lengend Snippet: Structure characterization of nucleic acid-streptavidin nano-sandwich composite. ( A ) Nano-sandwich composite formation was demonstrated in solution by observing FRET signal from fluorescently labeled streptavidin (Alexa 488 donor dye) and RNA corner modules (Cy5 acceptor dye). Nano-sandwich composite prepared according to synthetic route II (Figure ) produced a robust signal whereas omission of DNA components abolished FRET. ( B ) Purification of nano-sandwich composite by native PAGE yielded a clean sample for structure determination by cryo-EM. ( C ) Representative electron micrograph of nano-sandwich composite sample. ( D ) Two orthogonal views of cryo-EM maps obtained by 3D reconstruction at ∼5.8 Å resolution under superimposed 3D symmetry for the nano-sandwich composite, shown as iso-density surface.

Article Snippet: The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies.

Techniques: Labeling, Produced, Purification, Clear Native PAGE, Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

doi: 10.1093/nar/gkab797

Figure Lengend Snippet: Complex formation of streptavidin with preformed nucleic acid nanotriangles. ( A ) PAGE analysis of mixtures that contained streptavidin tetramer protein (SA) and nucleic acid nanotriangles (T) carrying a defined number of biotin ligands. Nanotriangles with a single biotin modification formed complexes with 1–3 triangles bound to a single streptavidin tetramer (SA:T n , n = 1–3). Nanotriangles conjugated with two or three biotin ligands formed equimolar complexes (SA:T 1 ) in addition to significant aggregation. Compaction of equimolar SA:T 1 assemblies by increasing number of biotin ligands that tether nanotriangles to streptavidin increased complex mobility. Double bands for equimolar SA:T 1 complexes observed with nanotriangles carrying one or three biotin ligands suggest formation of two different complex configurations due the non-equivalence of biotin binding sites on the protein tetramer. Lane 1 is a mobility control sample containing an RNA–DNA hybrid nanotriangle with a single biotin ligand. ( B ) AFM imaging of SA:T 1 complexes assembled from streptavidin and nanotriangles with two biotin ligands (panel A, lane 3). Scale bar, 50 nm. The histogram shows particle height measured by AFM which was consistent with equimolar complexes of a streptavidin tetramer positioned on top of a single RNA–DNA hybrid nanotriangle .

Article Snippet: The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies.

Techniques: Modification, Binding Assay, Imaging

Journal: Nucleic Acids Research

Article Title: Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

doi: 10.1093/nar/gkab797

Figure Lengend Snippet: Synthesis of nucleic acid-streptavidin nano-sandwich composite. ( A ) Route II for kinetic-trapping assembly: II-a), binding of biotin-conjugated DNA out connector oligonucleotides to streptavidin (SA) furnishing the protein–DNA sandwich core complex precursor (SA-DNA); II-b), addition of DNA in guide and unmodified DNA out connector to the core complex precursor; II-c) induction of RNA–DNA hybrid nanotriangle self-assembly by addition of RNA corner modules. ( B ) PAGE analysis of precursors and products in synthetic routes for the nano-sandwich composite. In route I (shown also in Figure , lane 3), preformed RNA–DNA hybrid nanotriangles carrying 2 biotin ligands (lane 2) are mixed with streptavidin which resulted in mostly aggregation (lane 3). Lane C is a mobility control sample containing a previously established mixture of polygonal RNA–DNA hybrid nanoshapes. In route II, a mixture containing preformed complex of biotin-conjugated DNA connectors bound to streptavidin (II-a) in addition to unconjugated DNA connector and DNA guide strand (II-b) was prepared first (lane 5). Subsequent addition of RNA corner modules furnished the nano-sandwich composite as a single predominant product (lane 6). ( C ) AFM imaging of the nano-sandwich composite product from gel lane 6 (scale bar, 50 nm). The histogram shows particle height measured by AFM.

Article Snippet: The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies.

Techniques: Binding Assay, Imaging

Journal: Nucleic Acids Research

Article Title: Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

doi: 10.1093/nar/gkab797

Figure Lengend Snippet: Modular design concepts for supramolecular sandwich architectures from protein, DNA and RNA components based on hybrid nanoshapes. ( A ) Proteins that recognize single- or double-stranded nucleic acid can be introduced by DNA or RNA components that contain protein binding sites in the modular connector modules (see Figure ) . ( B ) Extended materials can be obtained from sandwich composites by connecting nanoshapes containing RNA three-way junction (3WJ) corner motifs as we previously reported .

Article Snippet: The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies.

Techniques: Protein Binding