scholarly journals Quantitative Mining of Compositional Heterogeneity in Cryo-EM Datasets of Ribosome Assembly Intermediates

2021 ◽  
Author(s):  
Jessica N Rabuck-Gibbons ◽  
Dmitry Lyumkis ◽  
James R Williamson
Keyword(s):  
Structural Heterogeneity ◽  
Ribosome Assembly ◽  
Compositional Heterogeneity ◽  
Assembly Process ◽  
Branch Points ◽  
Macromolecular Complexes ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Assembly Factor ◽  
Structural Configurations

Macromolecular complexes are dynamic entities whose function is often intertwined with their many structural configurations. Single particle cryo-electron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct populations into different groups through computational classification. However, current workflows are limited, and 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 a set of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which is characterized by significant structural heterogeneity. We identified new branch points in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

scholarly journals Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

2019 ◽  
Author(s):  
Aida Razi ◽  
Joseph H. Davis ◽  
Yumeng Hao ◽  
Dushyant Jahagirdar ◽  
Brett Thurlow ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Small Subunit ◽  
Folding Pathway ◽  
Ribosome Assembly ◽  
Combined Approach ◽  
Quantitative Mass Spectrometry ◽  
Cryo Electron Microscopy ◽  
30S Subunit ◽  
Assembly Factor

SUMMARYTo reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era’s role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

scholarly journals Structural insights into assembly of the ribosomal nascent polypeptide exit tunnel

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel M. Wilson ◽  
Yu Li ◽  
Amber LaPeruta ◽  
Michael Gamalinda ◽  
Ning Gao ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Assembly ◽  
Ribosomal Subunits ◽  
Nascent Polypeptide ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Associated Proteins ◽  
Assembly Factor ◽  
Exit Tunnel ◽  
Structural Insights

Abstract The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

scholarly journals Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 830
Author(s):  
Prasangi Rajapaksha ◽  
Isoiza Ojo ◽  
Ling Yang ◽  
Ankit Pandeya ◽  
Thilini Abeywansha ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Assembly Process ◽  
Multidrug Efflux ◽  
E Coli ◽  
Multidrug Efflux Pumps ◽  
Negative Effect ◽  
Dynamic Assembly ◽  
Pump Assembly

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

scholarly journals Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiwei Wang ◽  
Yan Gao ◽  
Yanting Tang ◽  
Xiaoting Zhou ◽  
Yuezheng Lai ◽  
...  
Keyword(s):  
Rational Design ◽  
Pathogenic Bacteria ◽  
Structural Topology ◽  
Antimicrobial Drugs ◽  
E Coli ◽  
Cytochrome Bd ◽  
Cryo Electron Microscopy ◽  
Functional Mechanisms ◽  
The Relationship ◽  
Potential Oxygen

AbstractCytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

scholarly journals Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA-protein interactions during small ribosomal subunit biogenesis

2013 ◽  
Vol 110 (38) ◽  
pp. 15253-15258 ◽  
Author(s):  
U. A. Hellmich ◽  
B. L. Weis ◽  
A. Lioutikov ◽  
J. P. Wurm ◽  
M. Kaiser ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Assembly Factor

Fluorescence studies on the 30 S ribosome assembly process

FEBS Letters ◽  
1970 ◽  
Vol 11 (1) ◽  
pp. 49-54 ◽  
Author(s):  
A. Bollen ◽  
A. Herzog ◽  
A. Favre ◽  
J. Hibault ◽  
F. Gros
Keyword(s):  
Ribosome Assembly ◽  
Assembly Process ◽  
Fluorescence Studies
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