Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis
- School of Biochemistry, University of Bristol, United Kingdom
- Biological Mass Spectrometry and Proteomics, MRC Laboratory of Molecular Biology, United Kingdom
- Department of Life Sciences, Imperial College London, United Kingdom
- Living Systems Institute, University of Exeter, United Kingdom
- College of Life and Environmental Sciences, University of Exeter, United Kingdom
Figures
Figure 1 with 1 supplement
Identification of interactions between HTL and BAM.
(a) Schematic representation of sucrose gradient centrifugation tube for fractionation of E. coli total membranes. Numbers 1–6 indicate the fractions taken for SDS-PAGE and immunoblotting shown in (b…
Figure 1—figure supplement 1
Raw western blots of co-immunoprecipitations and affinity pull-downs.
(a) Coomassie-stained SDS-PAGE of sucrose gradient fractions of E. coli C43 total membranes from cells over-producing SecYEG or HTL. Left and right gels show untreated and heat denatured (+∆) …
Figure 2 with 5 supplements
3D characterisation of HTL-BAM by negative stain-EM and cryo-EM in detergent solution, and XL-MS analysis.
(a) Silver-stained SDS-PAGE gels of fractions from glycerol centrifugation gradients, with increasingly large complexes appearing in fractions of higher percentage glycerol, (from left to right). …
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Figure 2—source data 1
Parameters of EM analysis of HTL and HTL-BAM structures.
- https://cdn.elifesciences.org/articles/60669/elife-60669-fig2-data1-v2.odt
Figure 2—figure supplement 1
Glycerol centrifugation gradients of HTL and BAM components.
(a, b, c) Silver-stained SDS-PAGE gels of fractions from the glycerol centrifugation gradients are shown, with increasingly large complexes appearing in fractions of higher percentage glycerol (from …
Figure 2—figure supplement 2
Negative-stained EM micrographs of the HTL-BAM complex.
(a, b, c) Electron micrographs of HTL-BAM complexes in different conditions. Bottom, reference-free (RF) class averages of the largest populations found in the micrographs (top). Micrograph scale …
Figure 2—figure supplement 3
3D characterisation and subunit assignment of HTL-BAM by negative stain-EM in detergent solution.
(a) Silver-stained SDS-PAGE analysis of HTL-BAM fractionated by glycerol density gradient centrifugation (left). Experiment is the same as that shown in Figure 2a (bottom panel), but with GraFix …
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Figure 2—figure supplement 3—source data 1
Mass spectrometry (MS) analysis of the GraFix fractions for image processing.
Analysis of the protein composition of the HTL-BAM preparations in detergent solution by MS. Only proteins of interest are shown in this table. The SDS-PAGE bands corresponding to the GraFix fractions used for negative stain-EM processing (e.g. Figure 2—figure supplement 3a, middle and right, asterisk) were cut from the gel and prepared for protein digestion, extraction, and MS.
- https://cdn.elifesciences.org/articles/60669/elife-60669-fig2-figsupp3-data1-v2.xls
Figure 2—figure supplement 4
Image processing and classification strategy for the cryo-EM data of the HTL-BAM complex.
Relion 2D classification was used to clean the two preliminary datasets and classify the images. Of Set I, 82% of the particles were used for extensive 3D classifications and auto-refinements. Of …
Figure 2—figure supplement 5
Sample preparation and XL-MS analysis of HTL-BAM.
(a) Top, gel filtration chromatography elution profile (Superose 6) of the HTL-BAM complex for cryo-EM and XL-MS analysis. Bottom, silver-stained SDS-PAGE gel of the fractions eluted from the gel …
Figure 3 with 1 supplement
EM structures of HTL in ‘compact’ and ‘open’ states.
Structure and docking of a previously published cryo-EM structure of HTL in the compact state (i) (Botte et al., 2016), the HTL-BAM complex (ii), HTL in the ‘compact’ state (iii) and HTL in the …
Figure 3—figure supplement 1
EM field of wild type HTL in different conditions.
Bottom, reference-free (RF) class averages of the ‘compact’ (comp) and ‘open’ populations found in the micrographs. Percentages of the populations are indicated on the right of the RF images. …
Figure 4 with 1 supplement
Effect of increasing periplasmic distance on the HTL-BAM interaction.
(a) Negative-stain EM model of HTL-BAM (from Figure 2a), annotated with membranes at the experimentally determined distances between the inner- and outer- membranes of E. coli strains containing …
Figure 4—figure supplement 1
Raw western blots of IPs investigating how periplasmic width effects the HTL-BAM interaction.
Legend is the same as described for Figure 1—figure supplement 1a, but the cell strains used here were either E. coli containing WT lpp or the mutant lpp+21. All IPs were conducted in the presence …
Figure 5 with 1 supplement
Effects of SecD depletion upon cell growth, OmpA transport across the inner-membrane, and maturation.
(a) Western blot illustrating depletion of SecD in E. coli JP325 whole cells when grown in the presence of arabinose or glucose. t = 0 represents the time at which an overnight culture (grown in …
Figure 5—figure supplement 1
Raw western blots and control quantifications accompanying SecD depletion experiments from Figure 5.
(a) Western blots of whole cells visualised for SecD. Cell strains used were E. coli JP325 containing either empty pTrc99a, pTrc99a-SecDF, or pTrc99a-SecDD519NF. A preculture of JP325 was grown in …
Figure 6 with 1 supplement
Structural comparison of HTL and HTL519.
Comparison of negative stain-EM structures of ‘compact’ (structures i and iv) and ‘open’ (structures ii and v) conformations of HTL versus the counterpart containing SecDD519NF (HTL519), both in the …
Figure 6—figure supplement 1
Negative-stain EM of the native HTL and the version containing SecDD519N (HTL519).
Electron micrographs of wild type (wt) HTL and HTL containing SecDD519NF (HTL519) in the same conditions (with cardiolipin, and without GraFix). Bottom, reference-free (RF) class averages of the …
Figure 7
Schematic representation of the HTL-BAM machinery.
Model of OMP transfer through the bacterial envelope, facilitated by HTL-BAM and periplasmic chaperones, such as SurA, Skp, PpiD, and YfgM. From left to right: OMP precursors with an N-terminal …
