1. Volume 25, Issue 8, Pages 1264-1274.e3 (August 2017)
Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters 
Alberto Collauto, Smriti Mishra, Aleksei Litvinov, Hassane S. Mchaourab, Daniella Goldfarb 
Structure 
Volume 25, Issue 8, Pages e3 (August 2017)
DOI: /j.str
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2. Structure 2017 25, 1264-1274.e3DOI: (10.1016/j.str.2017.06.014)
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3. Figure 1 Conformational Dynamics of BmrCD and MsbA
(A) Ribbon model of BmrCD NBDs in the dimer state showing the location of spin label pairs monitoring the consensus (440/441) and the degenerate (348/532) NBSs. The distance distributions show distance components characteristic of the HES in the presence of substoichiometric ratio of Mg2+/ATP.
(B) Ribbon diagram of MsbA NBDs (PDB: 3B60) in the dimer state showing the location of spin label pair (350/471). Similar to BmrCD, distance components corresponding to the HES are observed under substoichiometric levels of Mg2+/ATP. See also Figures S1 and S2.
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4. Figure 2
The W-Band 31P-ENDOR Spectra Reveal the Identity of the Coordinated Nucleotide Phosphate
Normalized 31P-ENDOR of (A) MsbA and (B) BmrCD in the ATP-bound state (+Mn2+/AMPPNP), under turnover conditions (+Mn2+/ATP, 1 and 30 min) and the post-hydrolysis state (+Mn2+/ADP). For reference, the normalized 31P-ENDOR spectra of solutions of Mn2+/AMPPNP, Mn2+/ATP, and Mn2+/ADP are presented in (C).
The dashed lines and the arrows denote the distinctive features that are present in the spectrum of ATP-bound Mn2+ and absent in the spectrum of ADP-bound Mn2+.
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5. Figure 3
EDNMR Spectra Demonstrates Distinct Coordination of Nucleotide in the NBSs of MsbA and BmrCD
(A and C) MsbA +Mn2+/AMPPNP and +Mn2+/ADP.
(B and D) BmrCD +Mn2+/AMPPNP and +Mn2+/ADP.
(A and B) The full scans; (C and D) details of the 14N double-quantum region and of the 31P single-quantum region. See also Figures S3 and S4.
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6. Figure 4
EDNMR of Mn2+/15N5-ATP Identifies a BmrCD Nitrogen Atom Coordinated to Mn2+
(A) MsbA + Mn2+/ATP(15N5) and (B) BmrCD + Mn2+/ATP(15N5) for samples frozen after 5 s (red) and 1 hr (black) of incubation at 30°C. In all panels the spectrum of Mn2+/ATP(15N5) in the absence of any protein is presented in (blue), prepared in the corresponding MsbA and BmrCD buffers. Spectra were normalized to the 55Mn signal at 94 MHz. See also Figures S3 and S4.
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7. Figure 5 ATP Turnover Kinetics Followed by 31P-EDNMR Spectroscopy
(A) MsbA +Mn2+/ATP, 30°C.
(B) BmrCD +Mn2+/ATP, 30°C.
The data are fit to a mono-exponential model (see text); fit curves (solid line), 95% confidence intervals (shaded area) and half-life (dashed line) are shown. Filled and empty symbols correspond to two different experiments to illustrate the reproducibility of the method. See also Figure S5.
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8. Figure 6
31P-ENDOR for Mn2+/ATP Turnover in the Presence of Excess Vi Shows Spectral Characteristics of Post-hydrolysis State
Normalized 31P-ENDOR spectra of (A) MsbA and (B) BmrCD.
The dashed lines identify the positions of the characteristic signals for Mn2+/ATP (see Figure 2).
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9. Figure 7
EDNMR in the Presence of ATP and Excess Vi Reveals Asymmetric Trapping of Vi in BmrCD NBSs
W-band EDNMR spectra of (A and C) MsbA-Mn2+/ADP-Vi [MsbA + Mn2+/ATP + Vi].
(B and D) BmrCD-Mn2+/ADP-Vi [BmrCD + Mn2+/ATP + Vi].
(A and B) The full scans; (C and D) details of the 14N double-quantum region, the 51V/23Na single-quantum region and of the 31P single-quantum region. See also Figure S7.
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