DOI: 10.5061/dryad.m905qfvfj
title: Data from: Conformational dynamics and asymmetry in multimodal inhibition of membrane-bound pyrophosphatases

description: Abstract Membrane-bound pyrophosphatases (mPPases) are homodimeric
 proteins that hydrolyse pyrophosphate and pump H+/Na+ across membranes.
 They are crucial for the virulence of protist pathogens, making them
 attractive drug targets. In this study, we investigate the inhibitory
 effects of seven distinct bisphosphonates against Thermotoga maritima
 mPPase to explore their mode of action and assist in future small molecule
 inhibitor development. We solved two structures of mPPase bound to the
 inhibitors in the enzyme active sites and probed the conformational
 dynamics of mPPase under multiple inhibitors and functionally relevant
 conditions by double electron-electron resonance (DEER) spectroscopy. We
 found that mPPase adopts distinct conformational equilibria in solution in
 the presence of different inhibitors, including states consistent with
 asymmetric binding in the active site (closed-open), but a symmetric
 apo-like conformation on the periplasmic side (open-open). Combined with
 solid-supported membrane-based electrophysiology recordings, this revealed
 that during catalysis, one monomer of the dimer remains open, and Na+ can
 only be pumped in a closed state. These results further support
 symmetry-breaking across the membrane, consistent with
 half-of-the-sites-reactivity.
description: # Data from: Conformational dynamics and asymmetry in multimodal
 inhibition of membrane-bound pyrophosphatases Dataset DOI:
 [10.5061/dryad.m905qfvfj](https://doi.org/10.5061/dryad.m905qfvfj) ##
 Description of the data and file structure README: **Conformational
 dynamics and asymmetry in multimodal inhibition of membrane-bound
 pyrophosphatases** Description of the data and file structure These data
 are the source data for TmPPase activity in the presence of various
 compounds, TmPPase electrometric measurement, and primary data of TmPPase
 for all measurement variations. The files are structured according to the
 table below: | Figure | Panel | ID | Metadata | Raw data | |
 :--------------------- | :---- |
 :-------------------------------------------------- |
 :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | 1-figure supplement 1 | - | - | - | Figure 1-Source data 1-(Figure1-figure supplement 1).xlsx | | 1 | C | - | - | Figure 1-Source data 2-(Figure\_1C).xlsx | | 4 | B | red trace; black trace | apo\_525\_bckg.dat, apo\_525\_res.txt | 05\_ DEER\_p33-96d33-895\_50K\_32-32-20-32\_n200\_tauav\_5000d2\_unphased.DSC; | | 4 | B | green trace; black trace | Ca2+\_525\_bckg.dat, Ca2+\_525\_res.txt | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n336\_tauav\_5000d2.DSC; | | 4 | B | blue trace; black trace | IDP\_525\_bckg.dat, IDP\_525\_res.txt | 115Scans\_IDP\_PELDOR\_525R1.DTA; 115Scans\_IDP\_PELDOR\_525R1.DSC; 115Scans\_IDP\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | B | orange trace; black trace | ZLD\_525\_bckg.dat, ZLD\_525\_res.txt | 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DSC; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DTA; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | B | maroon trace; black trace | ETD\_525\_bckg.dat, ETD\_525\_res.txt | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DSC; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DTA; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | B | purple trace; | ETD\_Ca2+\_525\_bckg.dat, ETD\_Ca2+\_525\_res.txt | 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DSC; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DTA; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause\_comaprative\_deer\_analysis.mat | | 4 | B | pink trace; black trace | PAM\_525\_bckg.dat, PAM\_525\_res.txt | JW\_013\_130225\_PELDOR.DSC; JW\_013\_130225\_PELDOR.DTA; JW\_013\_130225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | B | gold trace; black trace  | ALE\_525\_bckg.dat, | JW\_014\_190225\_PELDOR\_16step.DSC; JW\_014\_190225\_PELDOR\_16step.DTA; JW\_014\_190225\_PELDOR\_16step\_comparative\_deer\_analysis.mat | | 4 | C | red trace; black dotted trace | apo\_525\_fit.dat, apo\_525\_res.txt | 05\_ DEER\_p33-96d33-895\_50K\_32-32-20-32\_n200\_tauav\_5000d2\_unphased.DSC; | | 4 | C | green trace; black dotted trace | Ca2+\_525\_fit.dat, Ca2+\_res.txt | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n336\_tauav\_5000d2.DSC; | | 4 | C | blue trace; black dotted trace | IDP\_525\_fit.dat, IDP\_525\_res.txt | 115Scans\_IDP\_PELDOR\_525R1.DTA; 115Scans\_IDP\_PELDOR\_525R1.DSC; 115Scans\_IDP\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | C | orange trace; black dotted trace | ZLD\_525\_fit.dat, ZLD\_525\_res.txt | 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DSC; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DTA; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | C | maroon trace; black dotted trace | ETD\_525\_fit.dat, | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DSC; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DTA; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | C | purple trace; | ETD\_Ca2+\_525\_fit.dat, ETD\_Ca2+\_525\_res.txt | 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DSC; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DTA; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause\_comaprative\_deer\_analysis.mat | | 4 | C | pink trace; black dotted trace | PAM\_525\_fit.dat, PAM\_525\_res.txt | JW\_013\_130225\_PELDOR.DSC; JW\_013\_130225\_PELDOR.DTA; JW\_013\_130225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | C | gold trace; black dotted trace  | ALE\_525\_fit.dat, ALE\_525\_res.txt | JW\_014\_190225\_PELDOR\_16step.DSC; JW\_014\_190225\_PELDOR\_16step.DTA; JW\_014\_190225\_PELDOR\_16step\_comparative\_deer\_analysis.mat | | 4 | D | red trace | apo\_525\_distr.dat, apo\_525\_res2.txt | 05\_ DEER\_p33-96d33-895\_50K\_32-32-20-32\_n200\_tauav\_5000d2\_unphased.DSC; | | 4 | D | green solid trace; green dotted trace | Ca2+\_525\_distr.dat, Ca2+\_525\_res2.txt, In\_silico\_525\_Ca.txt | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n336\_tauav\_5000d2.DSC; | | 4 | D | blue trace; blue dotted trace | IDP\_525\_distr.dat,  IDP\_525\_res2.txt, | 115Scans\_IDP\_PELDOR\_525R1.DTA; 115Scans\_IDP\_PELDOR\_525R1.DSC; 115Scans\_IDP\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | D | orange trace; orange dotted trace   | ZLD\_525\_distr.dat,  ZLD\_525\_res2.txt, | 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DSC; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2.DTA; 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | D | maroon trace | ETD\_525\_distr.dat,  ETD\_525\_res2.txt, | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DSC; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2.DTA; 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2\_comparative\_deer\_analysis.mat | | 4 | D | purple solid trace; purple dotted trace | ETD\_Ca2+\_525\_distr.dat,  ETD\_Ca2+\_525\_res2.txt, | 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DSC; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause.DTA; 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause\_comaprative\_deer\_analysis.mat | | 4 | D | pink trace | PAM\_525\_distr.dat,  PAM\_525\_res2.txt, | JW\_013\_130225\_PELDOR.DSC; JW\_013\_130225\_PELDOR.DTA; JW\_013\_130225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4 | D | gold trace | ALE\_525\_distr.dat,  ALE\_525\_res2.txt, | JW\_014\_190225\_PELDOR\_16step.DSC; JW\_014\_190225\_PELDOR\_16step.DTA; JW\_014\_190225\_PELDOR\_16step\_comparative\_deer\_analysis.mat | | 4 | D | black solid trace | In\_silico\_525\_asymmetry.txt | N/A | | 4 | E | red trace; black trace | apo\_bckg.dat, apo\_res.txt | YM\_013\_300125\_PELDOR\_16step\_pc.DSC, YM\_013\_300125\_PELDOR\_16step\_pc.DTA,  | | 4 | E | green trace; black trace | Ca2+\_bckg.dat, Ca2+\_res.txt | JW\_013\_250126\_PELDOR\_16pc.DSCJW\_013\_250126\_PELDOR\_16pc.DTA | | 4 | E | blue trace; black trace | IDP\_bckg.dat, IDP\_res.txt | YM\_013\_290125\_PELDOR\_16step\_pc.DSC, | | 4 | E | orange trace; black trace | ZLD\_bckg.dat, ZLD\_res.txt | JW\_013\_230225\_PELDOR.DSC, JW\_013\_230225\_PELDOR.DTA, JW\_013\_230225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | E | maroon trace; black trace | ETD\_bckg.dat, ETD\_res.txt | JW\_013\_250225\_PELDOR\_70scans.DSC, JW\_013\_250225\_PELDOR\_70scans.DTA, JW\_013\_250225\_PELDOR\_70scans\_comparative\_deer\_analysis.mat | | 4 | E | purple trace; black trace | ETD\_Ca2+\_bckg.datETD\_Ca2+\_res.txt | JW\_013\_240225\_PELDOR.DSC, JW\_013\_240225\_PELDOR.DTA, JW\_013\_240225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | E | pink trace; black trace | PAM\_bckg.dat, PAM\_res.txt | JW\_013\_220225\_PELDOR.DSC, JW\_013\_220225\_PELDOR.DTA, JW\_013\_220225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | E | gold trace; black trace | ALE\_bckg.dat, ALE\_res.txt | JW\_015\_190225\_PELDOR.DSC, JW\_015\_190225\_PELDOR.DTA, JW\_015\_190225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | F | red trace; black dotted trace | apo\_fit.dat, apo\_res.txt | YM\_013\_300125\_PELDOR\_16step\_pc.DSC, YM\_013\_300125\_PELDOR\_16step\_pc.DTA,  | | 4 | F | green trace; black dotted trace | Ca2+\_fit.dat, Ca2+\_res.txt | JW\_013\_250126\_PELDOR\_16pc.DSCJW\_013\_250126\_PELDOR\_16pc.DTA | | 4 | F | blue trace; black dotted trace | IDP\_fit.dat, IDP\_res.txt | YM\_013\_290125\_PELDOR\_16step\_pc.DSC, | | 4 | F | orange trace; black dotted trace | ZLD\_fit.dat, ZLD\_res.txt | JW\_013\_230225\_PELDOR.DSC, JW\_013\_230225\_PELDOR.DTA, JW\_013\_230225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | F | maroon trace; black dotted trace | ETD\_fit.dat, ETD\_res.txt | JW\_013\_250225\_PELDOR\_70scans.DSC, JW\_013\_250225\_PELDOR\_70scans.DTA, JW\_013\_250225\_PELDOR\_70scans\_comparative\_deer\_analysis.mat | | 4 | F | purple trace; black dotted trace | ETD\_Ca2+\_fit.dat, ETD\_Ca2+\_res.txt | JW\_013\_240225\_PELDOR.DSCJW\_013\_240225\_PELDOR.DTA, JW\_013\_240225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | F | pink trace; black dotted trace | PAM\_fit.dat, PAM\_res.txt | JW\_013\_220225\_PELDOR.DSC, JW\_013\_220225\_PELDOR.DTA, JW\_013\_220225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | F | gold trace; black dotted trace | ALE\_fit.dat, ALE\_res.txt | JW\_015\_190225\_PELDOR.DSCJW\_015\_190225\_PELDOR.DTA, JW\_015\_190225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | G | red trace | apo\_distr.dat, apo\_res2.txt | YM\_013\_300125\_PELDOR\_16step\_pc.DSC, YM\_013\_300125\_PELDOR\_16step\_pc.DTA,  | | 4 | G | green solid trace; green dotted trace | Ca2+\_distr.dat, Ca2+\_res2.txt, In\_silico\_599\_Ca.txt | JW\_013\_250126\_PELDOR\_16pc.DSCJW\_013\_250126\_PELDOR\_16pc.DTA | | 4 | G | blue solid trace; blue dotted trace | IDP\_distr.dat, IDP\_res2.txt, In\_silico\_599\_IDP.txt | YM\_013\_290125\_PELDOR\_16step\_pc.DSC, | | 4 | G | orange solid trace; orange dotted trace | ZLD\_distr.dat, ZLD\_res2.txt, In\_silico\_599\_ZLD.txt | JW\_013\_230225\_PELDOR.DSC, JW\_013\_230225\_PELDOR.DTA, JW\_013\_230225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | G | maroon trace | ETD\_distr.dat, ETD\_res2,txt | JW\_013\_250225\_PELDOR\_70scans.DSC, JW\_013\_250225\_PELDOR\_70scans.DTA, JW\_013\_250225\_PELDOR\_70scans\_comparative\_deer\_analysis.mat | | 4 | G | purple solid trace; purple dotted trace | ETD\_Ca2+\_distr.dat, ETD\_\_Ca2+\_res2.txt, In\_silico\_599\_ETD+Ca.txt | JW\_013\_240225\_PELDOR.DSCJW\_013\_240225\_PELDOR.DTA, JW\_013\_240225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | G | pink trace | PAM\_distr.dat, PAM\_res2.txt | JW\_013\_220225\_PELDOR.DSC JW\_013\_220225\_PELDOR.DTA, JW\_013\_220225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | G | gold trace | ALE\_distr.dat, ALE\_res2.txt | JW\_015\_190225\_PELDOR.DSCJW\_015\_190225\_PELDOR.DTA, JW\_015\_190225\_PELDOR\_comparative\_deer\_analysis.mat | | 4 | G | black trace | In\_silico\_599\_Asymmetry.txt | N/A | | 4\_figure supplement 1 | B | red trace; dotted black trace | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_bckg.dat; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_res.txt | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DTA; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DSC; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | B | green trace; dotted black trace | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_bckg.dat; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_res.txt | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DTA; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DSC; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | B | purple trace; dotted black trace | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_bckg.dat; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_res.txt | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DTA; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DSC; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | B | maroon trace; dotted black trace | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_bckg.dat; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_res.txt | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DTA; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DSC; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | B | orange trace; dotted black trace | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_bckg.dat; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_res.txt | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DTA; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DSC; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | B | cyan trace; dotted black trace | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_bckg.dat; 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_res.txt | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DTA; 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DSC; | | 4\_figure supplement 1 | C | red trace; dotted black trace | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_fit.dat; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_res.txt | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DTA; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DSC; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | C | green trace; dotted black trace | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_fit.dat; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_res.txt | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DTA; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DSC; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | C | purple trace; dotted black trace | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_fit.dat; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_res.txt | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DTA; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DSC; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | C | maroon trace; dotted black trace | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_fit.dat; | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DTA; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DSC; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | C | orange trace; dotted black trace | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_fit.dat; | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DTA; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DSC; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | C | cyan trace; dotted black trace | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_fit.dat; | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DTA; 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DSC; | | 4\_figure supplement 1 | D | red trace | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_distr.dat; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_res2.txt | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DTA; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94.DSC; 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | D | solid green trace; dotted green trace | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_distr.dat, 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_res2.txt; In\_silico\_211\_Ca.txt | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DTA; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69.DSC; 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | D | solid purple trace; dotted purple trace | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_distr.dat; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_res2.txt;In\_silico\_211\_Ca+ETD.txt | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DTA; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381.DSC; 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | D | maroon trace | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_distr.dat; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_res2.txt; | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DTA; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215.DSC; 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_comparative\_deer\_analysis.mat | | 4\_figure supplement 1 | D | orange trace; dotted orange trace | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_distr.dat, 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_res2.txt; In\_silico\_211\_ZLD | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DTA; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24.DSC; 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_comparative\_deer\_analysis.mat; | | 4\_figure supplement 1 | D | cyan trace; dotted cyan trace | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_distr.dat;03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_res2.txt; In\_silico\_211\_IDP.txt | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DTA; 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90.DSC; 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_comparative\_deer-analysis.mat; | | 4\_figure supplement 2 | A | red trace | cw\_211\_apo.txt | 20240524\_130620050\_211R1\_Apo\_DDM\_result.DTA; 20240524\_130620050\_211R1\_Apo\_DDM\_result.DSC; 20231201\_181806852\_231201\_Tmppase\_10mMCa\_DDM\_Anokhi\_25scans\_result.csv | | 4\_figure supplement 2 | A | blue trace | cw\_211\_IDP.txt | 20210729\_132905035\_TmPPase\_T211C\_MTSSL\_5mMIDP\_29\_07\_2021\_result.csv | | 4\_figure supplement 2 | A | maroon trace | cw\_211\_ETD.txt | 20210729\_134728016\_TmPPase\_T211C\_MTSSL\_5mMEtidronate\_29\_07\_2021\_result.csv | | 4\_figure supplement 2 | B | red trace | cw\_525\_apo.txt | 20231201\_144904045\_MTSSL\_RT1\_result.DTA; 20231201\_144904045\_MTSSL\_RT1\_result.DSC; 20231201\_144904045\_MTSSL\_RT1\_result.csv | | 4\_figure supplement 2 | B | green trace | cw\_525\_Ca2+.txt | 20231201\_181806852\_231201\_Tmppase\_10mMCa\_DDM\_Anokhi\_25scans\_result.DSC; 20231201\_181806852\_231201\_Tmppase\_10mMCa\_DDM\_Anokhi\_25scans\_result.DTA, 20231201\_181806852\_231201\_Tmppase\_10mMCa\_DDM\_Anokhi\_25scans\_result.csv | | 4\_figure supplement 2 | B | maroon trace | cw\_525\_ETD.txt | 20231201\_170019018\_231201\_Tmppase\_2mMETD\_DDM\_Anokhi\_35scans\_result.DSC; 20231201\_170019018\_231201\_Tmppase\_2mMETD\_DDM\_Anokhi\_35scans\_result.DTA; 20231201\_170019018\_231201\_Tmppase\_2mMETD\_DDM\_Anokhi\_35scans\_result.csv | | 4\_figure supplement 2 | B | purple trace | cw\_525\_ETD+Ca2+.txt | 20231201\_174410786\_231201\_Tmppase\_2mMETD\_10mMCa\_DDM\_Anokhi\_50scans\_30sSweep\_result.DSC; 20231201\_174410786\_231201\_Tmppase\_2mMETD\_10mMCa\_DDM\_Anokhi\_50scans\_30sSweep\_result.DTA; 20231201\_174410786\_231201\_Tmppase\_2mMETD\_10mMCa\_DDM\_Anokhi\_50scans\_30sSweep\_result.csv | | 4\_figure supplement 2 | C | red trace | cw\_599\_apo.txt | 20250213\_112443451\_599R1 0.05DDM 20250213\_result.DTA; 20250213\_112443451\_599R1 0.05DDM 20250213\_result.DSC; 20250213\_112443451\_599R1 0.05DDM 20250213\_result.csv | | 4\_figure supplement 2 | C | green trace | cw\_599\_Ca2+.txt | 20241211\_175126338\_20241211 mPPase 0.02DDM Ca 30min\_result.DSC; 20241211\_175126338\_20241211 mPPase 0.02DDM Ca 30min\_result.DTA; 20241211\_175126338\_20241211 mPPase 0.02DDM Ca 30min\_result.csv | | 4\_figure supplement 2 | C | blue trace | cw\_599\_IDP.txt | 20241211\_180955909\_20241211\_mPPase\_0.02DDM\_IDP\_30min\_result.DSC; 20241211\_180955909\_20241211\_mPPase\_0.02DDM\_IDP\_30min\_result.DTA; 20241211\_180955909\_20241211\_mPPase\_0.02DDM\_IDP\_30min\_result.csv | | 4\_figure supplement 2 | C | orange trace | cw\_599\_ZLD.txt | 20250219\_153214708\_mPPase 599R1 2mMZLD 20250219\_result.DSC; 20250219\_153214708\_mPPase 599R1 2mMZLD 20250219\_result.DTA; 20250219\_153214708\_mPPase 599R1 2mMZLD 20250219\_result.csv | | 4\_figure supplement 2 | C | maroon trace | cw\_599\_ETD.txt | 20250219\_123315027\_mPPase\_599R1\_2mMETD\_20250219\_result.DSC; 20250219\_123315027\_mPPase\_599R1\_2mMETD\_20250219\_result.DTA; 20250219\_123315027\_mPPase\_599R1\_2mMETD\_20250219\_result.csv | | 4\_figure supplement 2 | C | purple trace | cw\_599\_ETD+Ca2+.txt | 20250219\_135030122\_mPPase\_599R1\_2mMETD\_10mMCa\_20250219\_result.DSC; 20250219\_135030122\_mPPase\_599R1\_2mMETD\_10mMCa\_20250219\_result.DTA; 20250219\_135030122\_mPPase\_599R1\_2mMETD\_10mMCa\_20250219\_result.csv; | | 4\_figure supplement 2 | C | pink trace | cw\_599\_PAM.txt | 20250214\_132807174\_599R1 PAM 20250214 0.05DDM\_result.DSC; 20250214\_132807174\_599R1 PAM 20250214 0.05DDM\_result.DTA; 20250214\_132807174\_599R1 PAM 20250214 0.05DDM\_result.csv | | 4\_figure supplement 2 | C | gold trace | cw\_599\_ALE.txt | 20250214\_150501028\_20250214\_599R1\_ALE\_0.05DDM\_result.DTA; 20250214\_150501028\_20250214\_599R1\_ALE\_0.05DDM\_result.DSC; | | 4\_figure supplement 3 | A | red trace; dotted black trace; solid black trace | 525R1\_apo\_CDA\_time\_domain.dat | 05\_ DEER\_p33-96d33-895\_50K\_32-32-20-32\_n200\_tauav\_5000d2\_unphased\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | green trace; dotted black trace; solid black trace | 525R1\_Ca2+\_CDA\_time\_domain.dat | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n336\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | blue trace; dotted black trace; solid black trace | 525R1\_IDP\_CDA\_time\_domain.dat | 115Scans\_IDP\_PELDOR\_525R1\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | orange trace; dotted black trace; solid black trace | 525R1\_ZLD\_CDA\_time\_domain.dat | 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | maroon trace; dotted black trace; solid black trace | 525R1\_ETD\_CDA\_time\_domain.dat | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | purple trace; dotted black trace; solid black trace | 525R1\_ETD+Ca2+\_CDA\_time\_domain.dat | 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | pink trace; dotted black trace; solid black trace | 525R1\_PAM\_CDA\_time\_domain.dat | JW\_013\_130225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | A | gold trace; dotted black trace; solid black trace | 525R1\_PAM\_CDA\_time\_domain.dat | JW\_014\_190225\_PELDOR\_16step\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | red trace | 525R1\_apo\_CDA\_P(r).dat | 05\_ DEER\_p33-96d33-895\_50K\_32-32-20-32\_n200\_tauav\_5000d2\_unphased\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | green trace; green dotted trace | 525R1\_Ca2+\_CDA\_P(r).dat; In\_silico\_525\_Ca2+.txt | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n336\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | blue trace; blue dotted trace | 525R1\_IDP\_CDA\_P(r).dat; In\_silico\_525\_IDP.txt | 115Scans\_IDP\_PELDOR\_525R1\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | orange trace; orange dotted trace  | 525R1\_ZLD\_CDA\_P(r).dat;  In\_silico\_525\_ZLD.txt | 04a\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n620\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | maroon trace | 525R1\_ETD\_CDA\_P(r).dat | 04\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n287\_tauav\_5000d2\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | purple trace; purple dotted trace | 525R1\_ETD+Ca2+\_CDA\_P(r).dat;  In\_silico\_525\_ETD\_Ca.txt | 06\_DEER\_p33-96d33-895\_50K\_32-32-24-32\_n437\_tauav\_5000d2\_pause\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | pink trace | 525R1\_PAM\_CDA\_P(r).dat | JW\_013\_130225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | gold trace | 525R1\_ALE\_CDA\_P(r).dat | JW\_014\_190225\_PELDOR\_16step\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | B | black solid trace | In\_silico\_525\_asymmetry.txt | N/A | | 4\_figure supplement 3 | C | red trace; dotted black trace; solid black trace | 599R1\_apo\_CDA\_time\_domain.dat | YM\_013\_300125\_PELDOR\_16step\_pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | green trace; dotted black trace; solid black trace | 599R1\_Ca2+\_time\_domain.dat | JW\_013\_250126\_PELDOR\_16pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | blue trace; dotted black trace; solid black trace | 599R1\_IDP\_time\_domain.dat | YM\_013\_290125\_PELDOR\_16step\_pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | orange trace; dotted black trace; solid black trace | 599R1\_ZLD\_time\_domain.dat | JW\_013\_230225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | maroon trace; dotted black trace; solid black trace | 599R1\_ETD\_time\_domain.dat | JW\_013\_250225\_PELDOR\_70scans\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | purple trace; dotted black trace; solid black trace | 599R1\_ETD+Ca2+\_time\_domain.dat | JW\_013\_240225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | pink trace; dotted black trace; solid black trace | 599R1\_PAM\_time\_domain.dat | JW\_013\_220225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | C | gold trace; dotted black trace; solid black trace | 599R1\_ALE\_time\_domain.dat | JW\_015\_190225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | red trace | 599R1\_apo\_CDA\_P(r).dat | YM\_013\_300125\_PELDOR\_16step\_pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | green trace; green dotted trace | 599R1\_Ca2+\_CDA\_P(r).dat; In\_silico\_599\_Ca.txt | JW\_013\_250126\_PELDOR\_16pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | blue trace; blue dotted trace  | 599R1\_IDP\_P(r).dat; In\_silico\_599\_IDP.txt | YM\_013\_290125\_PELDOR\_16step\_pc\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | orange trace; dotted orange trace | 599R1\_ZLD\_P(r).dat;  In\_silico\_599\_ZLD.txt | JW\_013\_230225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | maroon trace | 599R1\_ETD\_P(r).dat | JW\_013\_250225\_PELDOR\_70scans\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | purple trace; purple dotted trace | 599R1\_ETD+Ca2+\_P(r).dat, In\_silico\_599\_ETD+Ca2+.txt | JW\_013\_240225\_PELDOR\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | D | pink trace | 599R1\_PAM\_P(r).dat | JW\_013\_220225\_PELDOR\_comparative\_DEER\_analysis.dat | | 4\_figure supplement 3 | D | gold trace | 599R1\_ALE\_P(r).dat | JW\_015\_190225\_PELDOR\_comparative\_DEER\_analysis.dat | | 4\_figure supplement 3 | D | black solid trace | In\_silico\_599\_asymmetry.txt | N/A | | 4\_figure supplement 3 | E | red trace; dotted black trace; solid black trace | 211R1\_apo\_time\_domain.dat | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | E | green trace; dotted black trace; solid black trace | 211R1\_Ca2+\_time\_domain.dat | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | E | blue trace; dotted black trace; solid black trace | 211R1\_IDP\_time\_domain.dat | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | E | orange trace; dotted black trace; solid black trace | 211R1\_ZLD\_time\_domain.dat | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | E | maroon trace; dotted black trace; solid black trace | 211R1\_ETD\_time\_domain.dat | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | E | purple trace; dotted black trace; solid black trace | 211R1\_ETD+Ca2+\_time\_domain.dat | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_validation\_sets.mat | | 4\_figure supplement 3 | F | red trace | 211R1\_apo\_time\_domain.dat | 03\_DEER\_SAMPLET1\_33.96GHz\_12111G\_n94\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | F | green trace | 211R1\_Ca2+\_time\_domain.dat | 03\_DEER\_T211R1\_CaCl2\_33.895GHz\_n69\_comparative\_DEER\_analysis.dat | | 4\_figure supplement 3 | F | blue trace | 211R1\_IDP\_time\_domain.dat | 03\_DEER\_SAMPLET2\_33.96GHz\_12111G\_n90\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | F | orange trace | 211R1\_ZLD\_time\_domain.dat | 04\_DEER\_T211R1\_ZOL\_33.895GHz\_n24\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | F | maroon trace | 211R1\_ETD\_time\_domain.dat | 03\_DEER\_T211R1\_EDTN\_33.865GHz\_n215\_comparative\_DEER\_analysis.mat | | 4\_figure supplement 3 | F | purple trace | 211R1\_ETD+Ca2+\_time\_domain.dat | 05\_TR211\_APO\_CaETD\_DEER\_32-32-26\_2ktau\_0dB\_48VG\_50K\_n381\_comparative\_DEER\_analysis.mat | | 5 | A | - | - | Firgure 5-Source data 1-(Figure\_5A).xlsx | ### Files and variables #### File: Underpinning_data.zip **Description:** These data are the source data for TmPPase activity in the presence of various compounds, TmPPase electrometric measurement, and primary data of TmPPase for all measurement variations. ## Code/software DEER/PELDOR files can be viewed and analysed using the DeerLab program - [https://jeschkelab.github.io/DeerLab/](https://jeschkelab.github.io/DeerLab/) or the Comparative DeerAnalyzer - [https://ethz.ch/content/dam/ethz/special-interest/chab/imps/epr-dam/documents/software/deer-15-on/CDAv2Installer_web.exe](https://ethz.ch/content/dam/ethz/special-interest/chab/imps/epr-dam/documents/software/deer-15-on/CDAv2Installer_web.exe)