5-HT2B receptor bound to LSD in complex with beta-arrestin1 obtained by cryo-electron microscopy (cryoEM)
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ABSTRACT:
SUBMITTER: Ximena Barros-Alvarez
PROVIDER: EMPIAR-11493 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
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Project description:Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry. The catalytic PRMT5 subunits form a core tetramer and the MEP50 subunits are arranged peripherally in complex with the PRMT5 N-terminal domain. The cryo-EM reconstruction shows good side chain definition and shows a well-resolved peak for a bound dehydrosinefungin inhibitor molecule. These results demonstrate the applicability of cryo-EM in determining structures of human protein complexes of biomedical significance and suggests cryo-EM could be further utilized to understand PRMT5 interactions with other biologically important binding proteins and ligands.
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Project description:ObjectiveThe ?IIb?3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain ?. Previous mutagenesis studies suggested that abciximab binds to the ?3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent ?1-?1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the ?IIb?3-binding pocket for fibrinogen or the ?3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the ?IIb?3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the ?3 SDL and neighboring residues, the ?1-?1 helix, and ?3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with ?IIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the ?IIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL.ConclusionsWe deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.
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