Late steps in bacterial translation initiation visualized using time-resolved cryo-EM

Abstract

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNA fMet )-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factors (IFs), which must dissociate before the resulting 70S EC can begin translation elongation 1 . Although comparison of 30S 2-5 and 70S 4,6-8 IC structures have revealed that the ribosome, IFs, and fMet-tRNA fMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a 70S EC structure obtained directly from a 70S IC formed via an IF-catalyzed initiation reaction has precluded an understanding of ribosome, IF, and fMet-tRNA fMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM) 9 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNA fMet positioning during 70S EC formation. Our results demonstrate the power of TR cryo-EM to determine how a time-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

“…Through the years, a toolbox of biophysical methods by which such insights can be obtained has been assembled. These methods include X-ray crystallography [5,6], cryo-electron microscopy (cryoEM) [7,8], nuclear magnetic resonance spectroscopy (NMR) [9,10], electron paramagnetic resonance (EPR) [11,12], Förster resonance energy transfer (FRET) [13,14], small-angle X-ray scattering (SAXS) [15], and molecular dynamics simulation (MD) [16]. Out of this toolbox, EPR is a method that provides such information but requires the presence of unpaired electrons.…”

Ultracompact 3D microfluidics for time-resolved structural biology

“…We achieved~21 ms mixing, corresponding to a 25-65-fold improvement over the previous Tjunction-based mix-and-diffuse-SFX 28 or coaxial hydrodynamic flow-focusing 31 at equivalent flow rates. All nozzles and mixers were validated 51 and refined through numerous X-ray beamtime experiments and can be readily adapted for other time-resolved methods such as in spectroscopy 58 in combination of liquid sheets 54 , small-and wide-angle X-ray scattering 59 , hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) 60 , as well as cryoEM 61 . Our modular design approach will simplify cross-validation of biomolecular dynamics through combining two or more of these different imaging modalities, which may prove essential to make recording of molecular movies through time-resolved mixing-based reaction initiation a routine method in the future.…”

Serial protein crystallography in an electron microscope

“…Augmenting parallel-beam crystallography with coherent scanning diffraction techniques such as convergent-beam diffraction or low-dose ptychography might be a viable way to obtain Bragg reflection phase information 51,52 . Finally, integrating the serial acquisition approach with emerging methods of in situ and time-resolved EM [53][54][55] may open up avenues for roomtemperature structures or structural dynamics studies on beamsensitive systems. All of this makes STEM-based SerialED a versatile, highly efficient and low-cost alternative to canonical structure determination approaches for proteins and beyond.…”