857 research outputs found
Effects of cryo-EM cooling on structural ensembles
No full textStructure determination by cryo electron microscopy (cryo-EM) provides information on structural heterogeneity and ensembles at atomic resolution. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to cryogenic temperatures. To what extent the structural ensemble is perturbed during cooling is currently unknown. Here, to quantify the effects of cooling, we combined continuum model calculations of the temperature drop, molecular dynamics simulations of a ribosome complex before and during cooling with kinetic models. Our results suggest that three effects markedly contribute to the narrowing of the structural ensembles: thermal contraction, reduced thermal motion within local potential wells, and the equilibration into lower free-energy conformations by overcoming separating free-energy barriers. During cooling, barrier heights below 10 kJ/mol were found to be overcome, which is expected to reduce B-factors in ensembles imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures
Chemomechanical Regulation of Snare Proteins Studied with Molecular Dynamics Simulations
No full textAbstractSNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress
tRNA dissociation from EF-Tu after GTP hydrolysis: Primary steps and antibiotic inhibition.
No full textIn each round of ribosomal translation, the translational GTPase elongation factor Tu (EF-Tu) delivers a transfer RNA (tRNA) to the ribosome. After successful decoding, EF-Tu hydrolyzes GTP, which triggers a conformational change that ultimately results in the release of the tRNA from EF-Tu. To identify the primary steps of these conformational changes and how they are prevented by the antibiotic kirromycin, we employed all-atom explicit-solvent molecular dynamics simulations of the full ribosome-EF-Tu complex. Our results suggest that after GTP hydrolysis and Pi release, the loss of interactions between the nucleotide and the switch 1 loop of EF-Tu allows domain D1 of EF-Tu to rotate relative to domains D2 and D3 and leads to an increased flexibility of the switch 1 loop. This rotation induces a closing of the D1-D3 interface and an opening of the D1-D2 interface. We propose that the opening of the D1-D2 interface, which binds the CCA tail of the tRNA, weakens the crucial EF-Tu-tRNA interactions, which lowers tRNA binding affinity, representing the first step of tRNA release. Kirromycin binds within the D1-D3 interface, sterically blocking its closure, but does not prevent hydrolysis. The resulting increased flexibility of switch 1 explains why it is not resolved in kirromycin-bound structures
Dynamics and energetics of elongation factor SelB in the ternary complex and the ribosome.
No full textMolecular simulations of the ribosome and associated translation factors
No full textThe ribosome is a macromolecular complex which is responsible for protein synthesis in all living cells according to their transcribed genetic information. Using X-ray crystallography and, more recently, cryo-electron microscopy (cryo-EM), the structure of the ribosome was resolved at atomic resolution in many functional and conformational states. Molecular dynamics simulations have added information on dynamics and energetics to the available structural information, thereby have bridged the gap to the kinetics obtained from single-molecule and bulk experiments. Here, we review recent computational studies that brought notable insights into ribosomal structure and function
Single-particle Cryo-EM and molecular dynamics simulations: A perfect match
No full textFunder:
http://dx.doi.org/10.13039/501100001659
Deutsche Forschungsgemeinschaf
Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation.
No full textDuring ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation
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