Towards high-throughput in situ structural biology using electron cryotomography

Böhning, Jan; Bharat, Tanmay A. M.

doi:10.1016/j.pbiomolbio.2020.05.010

Cited by 37 publications

(31 citation statements)

References 80 publications

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“…This study highlights the utility of FIB milling and cryo-ET to deliver high-resolution insights into tissue-like multicellular specimens, which have traditionally been considered beyond the reach of structural cell biology. With increasing throughput and automation of these methods ( 29 , 30 ), structure determination of molecules in cell–cell junctions may become practical and a possible extension of this work in the future.…”

Section: Discussionmentioning

confidence: 99%

Architecture of cell–cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Melia

Bolla

Katharios-Lanwermeyer

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell–cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam–milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell–cell junctions. Combining our in situ observations at cell–cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell–cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell–cell junctions to prevent or treat problematic, chronic bacterial infections.

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Section: Discussionmentioning

confidence: 99%

Architecture of cell–cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Melia

Bolla

Katharios-Lanwermeyer

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…There has recently been considerable progress in the field of in situ cryo-ET [ 98 ]. Analysing the structure of proteins in their native state in situ is one of the key goals in structural biology.…”

Section: Discussionmentioning

confidence: 99%

Current data processing strategies for cryo-electron tomography and subtomogram averaging

Pyle

Zanetti

2021

Biochemical Journal

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Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

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“…This study highlights the utility of FIB milling and cryo-ET to deliver high-resolution insights into tissue-like multicellular specimens, which have traditionally been considered beyond the reach of structural cell biology. With increasing throughput and automation of these methods (28, 29), structure determination of molecules in cell-cell junctions may become practical, and a possible extension of this work in the future.…”

Section: Discussionmentioning

confidence: 99%

“…It is made The copyright holder for this preprint this version posted February 8, 2021. ; https://doi.org/10.1101/2021.02.08.430230 doi: bioRxiv preprint 10 been considered beyond the reach of structural cell biology. With increasing throughput and automation of these methods (28,29), structure determination of molecules in cell-cell junctions may become practical, and a possible extension of this work in the future.…”

mentioning

confidence: 99%

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Melia

Bolla

Katharios-Lanwermeyer³

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, where cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused-ion beam milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt pre-existing biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

show abstract

Towards high-throughput in situ structural biology using electron cryotomography

Cited by 37 publications

References 80 publications

Architecture of cell–cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Architecture of cell–cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

Current data processing strategies for cryo-electron tomography and subtomogram averaging

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition

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