High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells

Herdman, M.; Kügelgen, Andriko von; Kureisaite-Ciziene, Danguole; Duman, Ramona; Omari, Kamel El; Garman, Elspeth F.; Kjær, Andreas; Kolokouris, Dimitrios; Löwe, Jan; Wagner, Armin; Stansfeld, Phillip J.; Bharat, Tanmay A. M.

doi:10.1016/j.str.2021.10.012

Cited by 17 publications

(23 citation statements)

References 95 publications

(184 reference statements)

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“…The 3.5 Å map is in excellent agreement with the single particle structure of the in vitro reconstituted complex, including the LPS binding site [23]. Furthermore, divalent metal ions, known to be tightly bound to the inner S-layer [10], are resolved (Figure 2b). Surprisingly, at lower isosurface contour levels, we also observed a second LPS binding site (Figure 2c-d).…”

Section: Caulobacter Crescentus S-layersupporting

confidence: 66%

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

Zivanov

Otón

et al. 2022

Preprint

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We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and CTF refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for two cases that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.

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Section: Caulobacter Crescentus S-layersupporting

confidence: 66%

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

Zivanov

Otón

et al. 2022

Preprint

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“…Residues 272 to 377 form the most extensive, ordered insertion that lines the cavity of the pore. This insertion appears to be stabilized by a canonical bacterial SLP metal ion–binding site ( 33 ) coordinated by residues D274, D276, and D310 ( SI Appendix , Fig. S4 ).…”

Section: Resultsmentioning

confidence: 99%

A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria

Kügelgen

Dorst

Alva

et al. 2022

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

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Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM–PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans . Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

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“…Residues 272-377 form the most extensive, ordered insertion that lines the cavity of the pore. This insertion appears to be stabilized by a canonical bacterial SLP metal-ion binding site (32) coordinated by residues D274, D276, and D310 (Figure S4). Likewise, large insertions blocking the pore, with putative metal-ion binding sites, are found between residues 429-471 and 686-753 (Figures 2B and S4).…”

Section: Resultsmentioning

confidence: 99%

“…Deinococcus radiodurans [32] ALTDVPAGHWAKDAIDRLVSR-GVILGYPDGTFRGTQNLTRYEAAIIIARLLDQMRDGET Deinococcus wulumuqiensis [32] ALTDVPAGHWAKDAIDRLVSR-GIILGYPDGTFRGTQNLTRYEAAIIIARLLDQMRDGET Deinococcus wulumuqiensis [32] ALTDVPAGHWAKDAIDRLVSR-GIILGYPDGTFRGTQNLTRYEAAIIIARLLDQMRDGET Deinococcus ficus [33] ALTDVPAGHWAKDAIDKLVSR-GIILGYPDGTFRGTQNLTRYEAAVIIARLLDQMRSGEV Deinobacterium chartae [37] ALSDVPAGHWAKDAVDQLVAK-GIITGFPDGTFRGNEGLTRYQAALIIARVLEQVAAGSV Marinithermus hydrothermalis [24] QFSDVPAGHWAKEAVEKLADE-GIILGFPDGTFRGNEGLTRYQAALIIFRVLETIREEQL Oceanithermus desulfurans [23] QFSDVPAGHWAKEAVEKIAAE-GIILGFPDGTFRGNENLTRYQAAMIIYRLLQKLEPGQM Meiothermus ruber [24] QFSDVPAGHWAKEAVERIAAC-GLITGFPDGTFRGNTNLTRYQAALIFQRLLNEIQQGGE Thermus thermophilus [24] QFSDVPAGHWAKEAVEALAAK-GIILGFPDGTFRGNENLTRYQAALLIYRLLQQIEEELK Thermus thermophilus [24] QFSDVPAGHWAKEAVEALAAK-GIILGFPDGTFRGNENLTRYQAALLIYRLLQQIEEELK…”

Section: Slpamentioning

confidence: 99%

A multi-domain connector links the outer membrane and cell wall in deep-branching bacteria

Kuegelgen

Dorst

Alva

et al. 2022

Preprint

Self Cite

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Deinococcus radiodurans is a deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report an electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Using the power of structure prediction and sequence-based bioinformatics, we further show that SlpA-like proteins are widespread in deep-branching Gram-negative bacteria, plausibly constituting an ancestral superfamily of OM-PG connectors, important for organizing the cell envelopes of many bacteria. Finally, combining our atomic structures with tomography of cell envelopes, we report a model for the cell surface of D. radiodurans, with implications on understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria. Furthermore, the widespread occurrence of SlpA-like OM-PG connectors in deep-branching bacteria will help in understanding the evolutionary transition between Gram-negative and Gram-positive bacteria.

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High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells

Cited by 17 publications

References 95 publications

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria

A multi-domain connector links the outer membrane and cell wall in deep-branching bacteria

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