(A) Closed SecYEG (PDB: 5AWW, Tanaka et al., 2015). SecYEG (light red) is shown embedded in a modelled membrane (grey) with the plug helix highlighted (purple). Also identified are the transmembrane …
(A) Closed state (PDB: 1RHZ) (Van den Berg et al., 2004). Accessible volumes (orange for dye attached to the reference residue and cyan for the plug probe) obtained by modelling the dye positions …
(A) Protease protection assay performed with wild-type WT SecYEG (unlabelled) and SecYMKEG mutant labelled with AlexaFluor dyes. A 233 amino acid N-terminal fragment of proOmpA was used as …
(A) Expected changes in the FRET efficiency as a consequence of plug displacement during translocation. Pre-translocation, high FRET closed state (black dashed line) changes rapidly to a low FRET, …
(A) SecYMKEG alone. All data were collected using the confocal setup and histograms are fitted to a sum (solid magenta) of three Gaussians (dashed lines), of which the two minor contributions …
FRET distributions from confocal experiment were corrected for contribution from the 50% SecYMKEG in opposite orientation by subtracting appropriately scaled FRET distribution of SecYMKEG alone (Figu…
The top panel depicts a time trace of fluorescence counts with one train of recurring bursts belonging to the same vesicle. For detecting transitions within a set time window (e.g. 50 ms), an …
(A) Two-dimensional FRET efficiency contour plots (transition density plots) were obtained from bursts collected for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 0.1 mM ATP using the …
(A) Burst width distribution for diffusing proteoliposomes. (B) Recurrence probability was obtained from data in (A) as described in Materials and methods. The recurrence time of 80 ms, …
A detailed sampling of time resolved transition density plots shown in Figure 3A for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 0.1 mM ATP. Note a wider scale (count contour level …
Opening (blue) and closing (red) transition times as a function of ATP concentration for SecYMKEG:SecA:SecB in the presence of 1 mM ATP (no proOmpA). Neither opening (average 24.3 ± 5.2 ms) nor …
Time evolution of states for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 1 mM AMP-PNP. RASP time window is indicated in each panel. The scale bar on the lower right depicts count …
Arrhenius plot of reciprocal transition times (rate constants) for the plug opening (blue) and closing (red) under saturating 2 mM ATP concentration. Activation energy of 61.2 ± 4.5 kJ/mol for …
(A) SecYMKEG alone. Transition density for delays up to 21 ms were obtained from RASP analysis of 10,000 events. In all panels, the crosshair symbols indicate positions of the open and closed state …
(A) Two-dimensional FRET efficiency histograms for SecYMK,R357EEG alone. Transition density for delays up to 21 ms were obtained from RASP analysis of 10,000 events. In all panels, the crosshair …
BVA can discriminate between static and dynamic heterogeneity. Blue dots show measured standard deviations (SE) for all individual bursts. Due to statistical nature of these estimates, the variance …
(A) Dwell time dependence for open state (EFRET ~0.2) on the substrate length (schematically shown in the inset, SS depicted as orange bar) in the presence (black) or absence (red) of SecB. Ordinary …
Reactions were performed in the presence or absence of SecB. Gel lane: 10% of starting proOmpA loaded without protease treatment (positive and normalization control); control without ATP, …
Ensemble ATPase activity stimulated by proOmpA constructs with different length in the presence (black) or absence (red) of SecB (10 µM). The rates were obtained under saturating ATP/proOmpA …
(A) Without SecB, uncorrected distributions (shown as probability density functions) are shown in green, corrected distribution shown in red. Solid lines represent fitted gamma distribution …
(A) Without SecB. Error bars represent the standard deviation (s.d.) computed from the distribution of the dwell times. (B) As A), but in the presence of SecB (10 μM).
Colour coding: SecYEG – red, SecA – blue, proOmpA/OmpA – thick green line, SS –green rectangle, stationary plug – purple, plug in intermediate or transient state – magenta, lipid bilayer – grey. …
Conditions: SecYMKEG:SecA:SecB:pOA in the presence of 0.1 mM ATP.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Software, algorithm | iSMS software | (Preus et al., 2015) - doi:10.1038/nmeth.3435 | ||
Software, algorithm | LabView | (Lee et al., 2005) - doi: 10.1529/biophysj.104.054114 | ||
Software, algorithm | FRETbursts | (Ingargiola et al., 2016b) - doi: 10.1371/journal.pone.0160716 | ||
Software, algorithm | photon-hdf5 | (Ingargiola et al., 2016a) - doi: 10.1016/j.bpj.2015.11.013 | ||
Software, algorithm | dual-channel burst search | (Nir et al., 2006) - DOI: - 10.1021/jp063483n | ||
Software, algorithm | regularized inverse transform | (Provencher, 1982) - DOI10.1016/0010-4655(82)90173-4 | ||
Software, algorithm | graphics: library Seaborn, based on Matplotlib | (Hunter, 2007) - DOI10.1109/MCSE.2007.55 |
Python script source code.
Primary single molecule fluorescence data file for use with the Python script.