(A) Structures of bMRP1 captured in ligand-free (left), LTC4-bound (middle), and ATP-bound (right) conformations (PDB accession numbers: 5UJ9, 5UJA, and 6BHU). The positions of tag insertions for …
(A) Schematic of the surface immobilization and single-molecule imaging strategy. Labeled bMRP1 molecules were immobilized on a glass surface and imaged at room temperature with …
(A) Contour plots (top) and histograms (bottom) of FRET distributions obtained with WT bMRP1 in the following conditions (from left to right): apo, + LTC4 (10 µM), + ATP (5 mM), + ATP/LTC4 (5 mM/10 …
(A) Evidence lower bound determined by ebFRET, which was used to determine the number of non-zero FRET states and provide initial estimates of model parameters. (B–F) Models with two (B), three (C), …
(A) FRET contour plots (top) and histograms (bottom) obtained at varying concentrations of LTC4. n denotes the number of molecules analyzed for each condition. (B) FRET contour plots and histograms …
(A) Representative single-molecule donor (green) and acceptor (red) fluorescence trajectories, and the corresponding FRET trajectories (blue) obtained at a frame rate of 25 ms. Idealized FRET states …
Idealized FRET states are overlaid in orange.
Dashed lines represent the mean values for the five discrete FRET states.
(A) Representative smFRET trajectories of active molecules from the perturbation experiments obtained at a frame rate of 300 ms. A limiting (10 µM) or saturating (5 mM) concentration of ATP with or …
(A) Representative fluorescence and FRET trajectories of the inactive molecules. These molecules transitioned among distinct IF states but never visited the OF state before photobleaching. (B) …
(A) Contour plots (top) and corresponding transition density plots (bottom) of smFRET trajectories aligned at the appearance of the first OF state from the perturbation experiments in which 10 µM …
(A) Example smFRET trajectories for apo and LTC4-only conditions showing spontaneous transitions into the OF state independent of ATP. (B) Average lifetime of the OF states under different …
The corresponding time constants (τOF) are reported.
(A) The structure of WT bMRP1 in the presence of saturating ATP and LTC4 shown in blue, overlaid with the structure of the non-hydrolyzing bMRP1-E1454Q mutant in the presence of saturating ATP and …
The number of particles used in each step is indicated next to the arrow associated with that step. After initial classification and refinement in RELION, two separate routes were taken to generate …
(A) Cryo-EM density in the degenerate ATPase site from the Frealign map using all particles, displayed in the same way as Figure 5C. (B) Cryo-EM density in the consensus ATPase site from the …
MRP1 is intrinsically dynamic, transitioning between multiple IF conformations both in the absence and presence of ATP. Under physiological conditions, ATP rapidly binds to the IF state, promoting …
Condition | tIF (s) | tOF (s) |
---|---|---|
WT, 5 mM ATP | 16.1 ± 2.2 | 28.8 ± 4.8 |
WT, 5 mM ATP + 10 µM LTC4 | 4.7 ± 0.8 | 30.8 ± 5.2 |
E1454Q, 5 mM ATP + 10 µM LTC4 | 7.7 ± 1.5 | 31.7 ± 5.5 |
Shown are the average lifetimes (mean ± SEM) of the composite IF state and the OF state for WT and E1454Q MRP1 with indicated ATP and substrate concentrations. The effect of dye photobleaching on the apparent IF/OF lifetime has been corrected for.
Data collection | ||
---|---|---|
Microscope | Titan krios (FEI) | |
Voltage (kV) | 300 | |
Detector | K2 Summit (Gatan) | |
Pixel size (Å) | 1.03 | |
Defocus range (μm) | 0.7 to 2.4 | |
Movies | 3604 | |
Frames/movie | 50 | |
Dose rate (electrons/pixel/s) | 8.0 | |
Total dose (electrons/Å2) | 75 | |
Number of particles | 1,143,729 | |
Model composition | ||
Non-hydrogen atoms | 9684 | |
Protein residues | 1210 | |
Lipids/Detergents/Ligands | 3 CHS/1 ATP/1 ADP/2 Mg2+ | |
Refinement | ||
Resolution (Å) | 3.23 | |
Rwork | 0.265 | |
Rfree | 0.276 | |
RMS deviations | ||
Bond lengths (Å) | 0.003 | |
Bond angles (°) | 1.297 | |
Validation | ||
Molprobity score | 1.11 | |
Clashscore, all atoms | 1.07 | |
Favored rotamers (%) | 97.7 | |
Ramachandran plot (%) | ||
Favored | 95.7 | |
Allowed | 4.3 | |
Outliers | 0.0 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Biotinylated 6x-His Tag monoclonal antibody | Invitrogen | Cat# MA1-21315-BTIN | Molar ratio of 2:1 (antibody:bMRP1) |
Cell line | Sf9 | ATCC | CRL-1711 | |
Cell line | HEK293S GnTI- | ATCC | CRL-3022 | |
Recombinant DNA reagent | bovine MRP1 in pUC57 vector | Bio Basic | Codon-optimized | |
Recombinant DNA reagent | bovine MRP1 in a modified pEG BacMam vector | Johnson and Chen, 2017 | Suitable for expression in mammalian cells | |
Recombinant DNA reagent | bovine MRP1 with S6/A1 peptides for site-specific labeling | This paper | ||
Recombinant DNA reagent | bovine MRP1 E1454Q with S6/A1 peptides for site-specific labeling | This paper | ||
Recombinant DNA reagent | Sfp pet29b C-terminal His Tag | (Worthington and Burkart, 2006) | Addgene Plasmid# 75015 | |
Recombinant DNA reagent | pET15b-ACPs (from S. pneumoniae) | Gift from Michael Johnson | Addgene Plasmid# 63687 | |
Chemical compound, drug | Cy3 maleimide mono-reactive dye | GE Healthcare | Cat# PA23031 | |
Chemical compound, drug | LD655 maleimide mono-reactive dye | Lumidyne Technologies | Cat# LD655-MAL | |
Chemical compound, drug | Trolox | Sigma-Aldrich | Cat# 238813 | |
Chemical compound, drug | 4-Nitrobenzyl alcohol (NBA) | Sigma-Aldrich | Cat# N12821 | |
Chemical compound, drug | Cyclooctatetraene (COT) | Sigma-Aldrich | Cat# 138924 | |
Chemical compound, drug | 3,4-Dihydroxybenzoic acid (PCA) | Sigma-Aldrich | Cat# 37580 | |
Chemical compound, drug | Digitonin | Sigma-Aldrich | Cat# D141 | |
Peptide, recombinant protein | Protocatechuate 3,4-Dioxygenase (PCD) | Sigma-Aldrich | Cat# P8279 | |
Peptide, recombinant protein | Leukotriene C4 | Cayman Chemical | Cat# 20210 | |
Commercial assay, kit | NHS-activated Sepharose 4 Fast Flow resin | GE Healthcare | Cat# 17-0430-01 | |
Commercial assay, kit | Superose 6, 10/300 GL | GE Healthcare | Cat# 17-5172-01 | |
Software, algorithm | SPARTAN | (Juette et al., 2016) | https://www.scottcblanchardlab.com/software | |
Software, algorithm | ebFRET | (van de Meent et al., 2014) | http://ebfret.github.io | |
Software, algorithm | MATLAB | MathWorks | https://www.mathworks.com/products/matlab.html | |
Software, algorithm | Origin | OriginLab | https://www.originlab.com | |
Software, algorithm | GraphPad Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
Software, algorithm | RELION 1.4 | (Scheres, 2012) | https://www2.mrc-lmb.cam.ac.uk/relion | |
Software, algorithm | Frealign | (Grigorieff, 2016) | https://grigoriefflab.janelia.org/frealign | |
Software, algorithm | Coot | (Emsley and Cowtan, 2004) | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot | |
Software, algorithm | PHENIX | (Adams et al., 2010) | https://www.phenix-online.org | |
Software, algorithm | REFMAC | (Brown et al., 2015) | https://www.ccp4.ac.uk/html/refmac5.html | |
Software, algorithm | MolProbity | (Chen et al., 2010) | https://molprobity.biochem.duke.edu | |
Software, algorithm | Chimera | (Pettersen et al., 2004) | https://www.cgl.ucsf.edu/chimera | |
Software, algorithm | PyMOL | PyMOL | https://www.pymol.org | |
Software, algorithm | cryoSPARC | (Punjani et al., 2017) | https://cryosparc.com | |
Other | R1.2/1.3 400 mesh Au holey carbon grids | Quantifoil | Cat# 1210627 | 1 µg/mL |