Conformational variability in Escherichia coli 70S ribosome as revealed by 3D cryo-electron microscopy

Abstract Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.

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Cryo-electron Microscopy Structure of the Acinetobacter baumannii 70S Ribosome and Implications for New Antibiotic Development

mBio ◽

10.1128/mbio.03117-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 8

Author(s):

Christopher E. Morgan ◽

Wei Huang ◽

Susan D. Rudin ◽

Derek J. Taylor ◽

James E. Kirby ◽

...

Keyword(s):

Electron Microscopy ◽

Acinetobacter Baumannii ◽

Bacterial Infections ◽

Bound States ◽

Antimicrobial Agents ◽

Gram Negative ◽

Content Type ◽

Antibiotic Development ◽

Cryo Electron Microscopy ◽

70S Ribosome

ABSTRACT Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections. IMPORTANCE Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen.

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Structure and structural variations of the Escherichia coli 30 S ribosomal subunit as revealed by three-dimensional cryo-electron microscopy 1 1Edited by W. Baumeister

Journal of Molecular Biology ◽

10.1006/jmbi.1999.2538 ◽

1999 ◽

Vol 286 (5) ◽

pp. 1285-1291 ◽

Cited By ~ 46

Author(s):

Irene S. Gabashvili ◽

Rajendra K. Agrawal ◽

Robert Grassucci ◽

Joachim Frank

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Structural Variations ◽

Cryo Electron Microscopy

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The Cryo-Electron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod

10.1101/164657 ◽

2017 ◽

Author(s):

Manuela K. Hospenthal ◽

Tiago R. D. Costa ◽

Adam Redzej ◽

James Lillington ◽

Gabriel Waksman

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Urinary Tract ◽

Virulence Factors ◽

Biomechanical Properties ◽

Bacterial Attachment ◽

Host Cells ◽

Hostile Environment ◽

Cryo Electron Microscopy

ABSTRACTChaperone-usher pili are long, polymeric protein fibres displayed on the surface of many bacterial pathogens. These critical virulence factors allow bacteria to specifically attach to host cells during infection. The type 1 and P pili of uropathogenicEscherichia coli(UPEC) play important roles during UPEC’s colonisation of the urinary tract, mediating bacterial attachment to the bladder and kidney, respectively. Also, their biomechanical properties that allow them to reversibly uncoil in response to flow-induced forces are critical for UPEC’s ability to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2 Å resolution cryo-electron microscopy (cryo-EM) structure of the type 1 pilus rod, which together with the previous structure of the P pilus rod, enables us to understand the remarkable “spring-like” properties of chaperone-usher pili in more detail.

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High-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit and validation of nucleotide modifications

10.1101/695429 ◽

2019 ◽

Author(s):

Vanja Stojković ◽

Alexander G. Myasnikov ◽

Iris D. Young ◽

Adam Frost ◽

James S. Fraser ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

High Resolution ◽

Rna Structure ◽

Ribosome Biogenesis ◽

Precise Determination ◽

23S Rrna ◽

Modified Nucleotides ◽

X Ray Crystallography ◽

Cryo Electron Microscopy

ABSTRACTPost-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides’ positions, a task that has previously been accomplished by X-ray crystallography. Here we present a cryo-electron microscopy (cryo-EM) structure of Escherichia coli (E. coli) 50S subunit at an average resolution of 2.2Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for identification of post-transcriptional rRNA modifications using a cryo-EM map. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

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Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer

Communications Biology ◽

10.1038/s42003-021-02916-2 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Wenxin Hu ◽

Hongjin Zheng

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Molecular Basis ◽

Iron Uptake ◽

Structural Features ◽

Biological Processes ◽

Type Ii ◽

Functional Studies ◽

Cryo Electron Microscopy ◽

Translocation Pathway

AbstractAs one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer’s ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

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