Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system.

Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates.

Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity.

SrmB Rescues Trapped Ribosome Assembly Intermediates.

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action.

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain.

The kinase-inducible domain (KIX) of the transcriptional coactivator CBP binds multiple transcriptional regulators through two allosterically connected sites. Establishing a method for observing activator-specific KIX conformations would facilitate the discovery of drug-like molecules that capture specific conformations and further elucidate how distinct activator-KIX complexes produce differential transcriptional effects. However, the transient and low to moderate affinity interactions between activators and KIX are difficult to capture using traditional biophysical assays.

CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions.

Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase.