Structural basis of early translocation events on the ribosome

Rundlet, E.J.; Holm, Mikael; Schacherl, M.; Natchiar, K.S.; Altman, Roger B.; Spahn, C.M.T.; Myasnikov, Alexander G.; Blanchard, Scott C.

doi:10.1016/j.bpj.2021.11.2545

Cited by 12 publications

(34 citation statements)

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“…In addition to achieving high resolution, the recent revolution in cryo-EM facilitated the determination of ensembles of structures in different states 18 – 21 , 72 . These structural ensembles are obtained from EM images of samples that were rapidly cooled down to cryogenic temperatures by plunge-freezing 13 .…”

Section: Discussionmentioning

confidence: 99%

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Effects of cryo-EM cooling on structural ensembles

Bock¹,

Grubmüller²

2022

Nat Commun

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Structure 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.

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Section: Discussionmentioning

confidence: 99%

“…The rapid cooling during plunge-freezing preserves part of the structural heterogeneity generated by room-temperature structural fluctuations and, hence, contains information on functional motions 17 – 21 . However, cooling of the sample is expected to perturb the structural ensemble of biomolecules.…”

Section: Introductionmentioning

confidence: 99%

Effects of cryo-EM cooling on structural ensembles

Bock¹,

Grubmüller²

2022

Nat Commun

View full text Add to dashboard Cite

show abstract

“…In addition to achieving high resolution, the recent revolution in cryo-EM facilitated the determination of ensembles of structures in different states 18;72;19;20;21 . These structural ensembles are obtained from EM images of samples that were rapidly cooled down to cryogenic temperatures by plunge-freezing 13 .…”

Section: Discussionmentioning

confidence: 99%

“…The rapid cooling during plunge-freezing preserves part of the structural heterogeneity generated by room-temperature structural fluctuations and, hence, contains information on functional motions 17;18;19;20;21 . However, cooling of the sample is expected to perturb the structural ensemble of biomolecules.…”

Section: Introductionmentioning

confidence: 99%

Effects of cryo-EM cooling on structural ensembles

Bock¹,

Grubmüller²

2021

Preprint

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Structure 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 by the 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 resulting in reduction of B-factors in the ensemble imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures.

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“…A) Transcription by RNA polymerase (pdb:1i6h) [120]. B) Translation by the ribosome [121]. C) Chaperone action by Hsp90 [110].…”

Section: Biological Use Casesmentioning

confidence: 99%

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Vermeer

Schmid

2022

Preprint

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Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ″DyeCycling″, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

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Structural basis of early translocation events on the ribosome

Cited by 12 publications

References 0 publications

Effects of cryo-EM cooling on structural ensembles

Effects of cryo-EM cooling on structural ensembles

Effects of cryo-EM cooling on structural ensembles

Can DyeCycling break the photobleaching limit in single-molecule FRET?

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