annotation_CSV/PMC4918766.csv

anno_start	anno_end	anno_text	entity_type	sentence	section
44	56	binding-site	site	Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger	TITLE
74	98	sodium-calcium exchanger	protein_type	Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger	TITLE
0	19	Na+/Ca2+ exchangers	protein_type	Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.	ABSTRACT
32	35	Na+	chemical	Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.	ABSTRACT
113	117	Ca2+	chemical	Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.	ABSTRACT
146	150	Ca2+	chemical	Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis.	ABSTRACT
92	111	structural analysis	experimental_method	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
119	128	exchanger	protein_type	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
134	158	Methanococcus jannaschii	species	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
160	166	NCX_Mj	protein	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
168	176	bound to	protein_state	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
177	180	Na+	chemical	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
182	186	Ca2+	chemical	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
190	194	Sr2+	chemical	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
228	231	apo	protein_state	Here, we elucidate their mechanisms of extracellular ion recognition and exchange through a structural analysis of the exchanger from Methanococcus jannaschii (NCX_Mj) bound to Na+, Ca2+ or Sr2+ in various occupancies and in an apo state.	ABSTRACT
130	133	Na+	chemical	This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism.	ABSTRACT
135	139	Ca2+	chemical	This analysis defines the binding mode and relative affinity of these ions, establishes the structural basis for the anticipated 3Na+:1Ca2+ exchange stoichiometry, and reveals the conformational changes at the onset of the alternating-access transport mechanism.	ABSTRACT
44	80	conformational free-energy landscape	evidence	An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.	ABSTRACT
84	90	NCX_Mj	protein	An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.	ABSTRACT
104	117	ion-occupancy	protein_state	An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.	ABSTRACT
135	183	enhanced-sampling molecular-dynamics simulations	experimental_method	An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.	ABSTRACT
207	225	crystal structures	evidence	An independent analysis of the dynamics and conformational free-energy landscape of NCX_Mj in different ion-occupancy states, based on enhanced-sampling molecular-dynamics simulations, demonstrates that the crystal structures reflect mechanistically relevant, interconverting conformations.	ABSTRACT
6	18	calculations	experimental_method	These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.	ABSTRACT
58	65	outward	protein_state	These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.	ABSTRACT
69	75	inward	protein_state	These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.	ABSTRACT
182	185	Na+	chemical	These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.	ABSTRACT
186	190	Ca2+	chemical	These calculations also reveal the mechanism by which the outward-to-inward transition is controlled by the ion-occupancy state, thereby explaining the emergence of strictly-coupled Na+/Ca2+ antiport.	ABSTRACT
0	19	Na+/Ca2+ exchangers	protein_type	Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis.	INTRO
21	24	NCX	protein_type	Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis.	INTRO
66	70	Ca2+	chemical	Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis.	INTRO
0	3	NCX	protein_type	NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.	INTRO
52	56	Ca2+	chemical	NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.	INTRO
138	141	Na+	chemical	NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.	INTRO
166	169	Na+	chemical	NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.	INTRO
175	179	Ca2+	chemical	NCX catalyzes the uphill extrusion of intracellular Ca2+ across the cell membrane, by coupling this process to the downhill permeation of Na+ into the cell, with a 3 Na+ to 1 Ca2+ stoichiometry.	INTRO
17	20	NCX	protein_type	The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport.	INTRO
47	50	NCX	protein_type	The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves.	INTRO
67	93	membrane-spanning segments	structure_element	The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves.	INTRO
121	127	halves	structure_element	The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves.	INTRO
14	20	halves	structure_element	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
32	48	highly conserved	protein_state	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
72	80	α-repeat	structure_element	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
142	152	eukaryotic	taxonomy_domain	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
153	156	NCX	protein_type	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
166	172	halves	structure_element	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
198	229	intracellular regulatory domain	structure_element	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
240	246	absent	protein_state	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
250	259	microbial	taxonomy_domain	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
260	263	NCX	protein_type	Each of these halves contains a highly conserved region, referred to as α-repeat, known to be important for ion binding and translocation; in eukaryotic NCX, the two halves are connected by a large intracellular regulatory domain, which is absent in microbial NCX (Supplementary Fig. 1).	INTRO
91	94	NCX	protein_type	Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information.	INTRO
29	38	structure	evidence	Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.	INTRO
42	48	NCX_Mj	protein	Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.	INTRO
54	78	Methanococcus jannaschii	species	Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.	INTRO
138	141	NCX	protein_type	Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein.	INTRO
5	14	structure	evidence	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
25	34	exchanger	protein_type	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
41	55	outward-facing	protein_state	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
95	112	ion-binding sites	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
126	134	internal	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
136	140	Sint	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
143	151	external	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
153	157	Sext	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
160	172	Ca2+-binding	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
174	177	SCa	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
183	189	middle	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
191	195	Smid	site	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
241	254	occluded from	protein_state	This structure shows the exchanger in an outward-facing conformation and reveals four putative ion-binding sites, denominated internal (Sint), external (Sext), Ca2+-binding (SCa) and middle (Smid), clustered in the center of the protein and occluded from the solvent (Fig. 1a-b).	INTRO
53	63	eukaryotic	taxonomy_domain	With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX.	INTRO
78	84	NCX_Mj	protein	With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX.	INTRO
247	250	NCX	protein_type	With similar ion exchange properties to those of its eukaryotic counterparts, NCX_Mj provides a compelling model system to investigate the structural basis for the specificity, stoichiometry and mechanism of the ion-exchange reaction catalyzed by NCX.	INTRO
43	53	structures	evidence	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
57	71	outward-facing	protein_state	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
72	81	wild-type	protein_state	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
82	88	NCX_Mj	protein	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
89	104	in complex with	protein_state	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
105	108	Na+	chemical	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
110	114	Ca2+	chemical	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
119	123	Sr2+	chemical	In this study, we set out to determine the structures of outward-facing wild-type NCX_Mj in complex with Na+, Ca2+ and Sr2+, at various concentrations.	INTRO
6	16	structures	evidence	These structures reveal the mode of recognition of these ions, their relative affinities, and the mechanism of extracellular ion exchange, for a well-defined, functional conformation in a membrane-like environment.	INTRO
33	63	molecular-dynamics simulations	experimental_method	An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states.	INTRO
86	96	structures	evidence	An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states.	INTRO
6	18	calculations	experimental_method	These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.	INTRO
66	80	outward-facing	protein_state	These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.	INTRO
81	90	exchanger	protein_type	These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.	INTRO
143	156	inward-facing	protein_state	These calculations also reveal how the ion occupancy state of the outward-facing exchanger determines the feasibility of the transition to the inward-facing conformation, thereby addressing a key outstanding question in secondary-active transport, namely how the transported substrates control the alternating-access mechanism.	INTRO
14	17	Na+	chemical	Extracellular Na+ binding	RESULTS
27	48	central binding sites	site	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
87	93	NCX_Mj	protein	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
94	103	structure	evidence	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
141	144	Na+	chemical	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
149	153	Ca2+	chemical	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
169	177	crystals	evidence	The assignment of the four central binding sites identified in the previously reported NCX_Mj structure was hampered by the presence of both Na+ and Ca2+ in the protein crystals.	RESULTS
73	76	Na+	chemical	To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+.	RESULTS
110	114	Ca2+	chemical	To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+.	RESULTS
0	8	Crystals	evidence	Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.	RESULTS
30	34	NaCl	chemical	Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.	RESULTS
45	64	lipidic cubic phase	experimental_method	Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.	RESULTS
66	69	LCP	experimental_method	Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique.	RESULTS
41	44	LCP	experimental_method	The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods).	RESULTS
132	136	NaCl	chemical	The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods).	RESULTS
141	145	EGTA	chemical	The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods).	RESULTS
0	17	X-ray diffraction	experimental_method	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
34	42	crystals	evidence	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
54	57	Na+	chemical	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
85	114	electron-density distribution	evidence	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
124	128	Sext	site	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
130	133	SCa	site	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
138	142	Sint	site	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
157	160	Na+	chemical	X-ray diffraction of these soaked crystals revealed a Na+-dependent variation in the electron-density distribution at sites Sext, SCa and Sint, indicating a Na+ occupancy change (Fig. 1c).	RESULTS
0	20	Occupancy refinement	experimental_method	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
35	38	Na+	chemical	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
52	56	Sint	site	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
61	64	SCa	site	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
72	75	Na+	chemical	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
131	135	Sint	site	Occupancy refinement indicated two Na+ ions bind to Sint and SCa at low Na+ concentrations (Fig. 1c), with a slight preference for Sint (Table 1).	RESULTS
19	22	Na+	chemical	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
26	30	Sext	site	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
70	77	density	evidence	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
106	109	Na+	chemical	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
130	134	Sext	site	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
174	177	Na+	chemical	Binding of a third Na+ to Sext occurs at higher concentrations, as no density was observed there at 10 mM Na+ or lower (Fig. 1c); Sext is however partially occupied at 20 mM Na+, and fully occupied at 150 mM (Fig. 1c).	RESULTS
4	7	Na+	chemical	The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.	RESULTS
22	25	SCa	site	The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.	RESULTS
57	60	Na+	chemical	The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.	RESULTS
62	66	Ca2+	chemical	The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.	RESULTS
121	125	Smid	site	The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated.	RESULTS
41	46	water	chemical	Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.	RESULTS
70	73	Na+	chemical	Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.	RESULTS
87	91	Smid	site	Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.	RESULTS
122	132	simulation	experimental_method	Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study.	RESULTS
11	27	electron density	evidence	First, the electron density at Smid does not depend significantly on the Na+ concentration.	RESULTS
31	35	Smid	site	First, the electron density at Smid does not depend significantly on the Na+ concentration.	RESULTS
73	76	Na+	chemical	First, the electron density at Smid does not depend significantly on the Na+ concentration.	RESULTS
45	49	Smid	site	Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d).	RESULTS
76	79	Na+	chemical	Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d).	RESULTS
4	9	water	chemical	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
22	26	Smid	site	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
33	47	hydrogen-bonds	bond_interaction	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
57	73	highly conserved	protein_state	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
74	79	Glu54	residue_name_number	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
84	90	Glu213	residue_name_number	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
158	168	coordinate	bond_interaction	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
178	181	Na+	chemical	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
190	194	Sext	site	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
196	199	SCa	site	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
204	208	Sint	site	The water molecule at Smid forms hydrogen-bonds with the highly conserved Glu54 and Glu213 (Supplementary Fig. 1d), stabilizing their orientation to properly coordinate multiple Na+ ions at Sext, SCa and Sint.	RESULTS
45	50	Glu54	residue_name_number	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
55	61	Glu213	residue_name_number	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
81	87	Asp240	residue_name_number	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
102	106	Smid	site	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
127	130	Asn	residue_name	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
134	144	eukaryotic	taxonomy_domain	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
145	148	NCX	protein_type	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
159	169	protonated	protein_state	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
206	216	simulation	experimental_method	It can be inferred from this assignment that Glu54 and Glu213 are ionized, while Asp240, which flanks Smid (and is replaced by Asn in eukaryotic NCX) would be protonated, as indicated by the abovementioned simulation study.	RESULTS
0	3	Na+	chemical	Na+-dependent conformational change	RESULTS
4	10	NCX_Mj	protein	The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).	RESULTS
11	21	structures	evidence	The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).	RESULTS
33	36	Na+	chemical	The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).	RESULTS
69	72	Na+	chemical	The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).	RESULTS
84	88	Sext	site	The NCX_Mj structures in various Na+ concentrations also reveal that Na+ binding to Sext is coupled to a subtle but important conformational change (Fig. 2).	RESULTS
5	8	Na+	chemical	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
18	22	Sext	site	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
26	30	high	protein_state	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
51	66	N-terminal half	structure_element	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
70	73	TM7	structure_element	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
91	104	short helices	structure_element	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
106	110	TM7a	structure_element	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
115	119	TM7b	structure_element	When Na+ binds to Sext at high concentrations, the N-terminal half of TM7 is bent into two short helices, TM7a and TM7b (Fig. 2a).	RESULTS
0	4	TM7b	structure_element	TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).	RESULTS
23	44	central binding sites	site	TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).	RESULTS
103	109	Ala206	residue_name_number	TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).	RESULTS
110	122	coordinating	bond_interaction	TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).	RESULTS
127	130	Na+	chemical	TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d).	RESULTS
14	18	Sext	site	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
27	32	empty	protein_state	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
36	39	low	protein_state	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
40	43	Na+	chemical	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
60	64	TM7a	structure_element	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
69	73	TM7b	structure_element	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
103	108	helix	structure_element	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
146	152	Ala206	residue_name_number	However, when Sext becomes empty at low Na+ concentrations, TM7a and TM7b become a continuous straight helix (Fig. 2a), and the carbonyl group of Ala206 retracts away (Fig. 2b-d).	RESULTS
0	4	TM7a	structure_element	TM7a also forms hydrophobic contacts with the C-terminal half of TM6.	RESULTS
16	36	hydrophobic contacts	bond_interaction	TM7a also forms hydrophobic contacts with the C-terminal half of TM6.	RESULTS
46	61	C-terminal half	structure_element	TM7a also forms hydrophobic contacts with the C-terminal half of TM6.	RESULTS
65	68	TM6	structure_element	TM7a also forms hydrophobic contacts with the C-terminal half of TM6.	RESULTS
33	42	structure	evidence	These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).	RESULTS
48	51	Na+	chemical	These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).	RESULTS
55	59	Sext	site	These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).	RESULTS
107	114	helices	structure_element	These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b).	RESULTS
38	41	TM6	structure_element	This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.	RESULTS
46	49	TM1	structure_element	This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.	RESULTS
138	149	transporter	protein_type	This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.	RESULTS
166	179	inward-facing	protein_state	This difference is noteworthy because TM6 and TM1 are believed to undergo a sliding motion, relative to the rest of the protein, when the transporter switches to the inward-facing conformation.	RESULTS
21	26	TM7ab	structure_element	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
84	88	Sext	site	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
93	97	Smid	site	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
105	108	SCa	site	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
113	117	Sint	site	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
125	133	occluded	protein_state	The straightening of TM7ab also opens up a passageway from the external solution to Sext and Smid, while SCa and Sint remain occluded (Fig. 2d).	RESULTS
10	20	structures	evidence	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
24	28	high	protein_state	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
33	36	low	protein_state	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
37	40	Na+	chemical	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
70	84	outward-facing	protein_state	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
85	93	occluded	protein_state	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
98	112	partially open	protein_state	Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively.	RESULTS
47	50	Na+	chemical	This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.	RESULTS
64	68	Sext	site	This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.	RESULTS
85	88	Na+	chemical	This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.	RESULTS
106	110	Sint	site	This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.	RESULTS
115	118	SCa	site	This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa.	RESULTS
4	41	crystallographic titration experiment	experimental_method	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
61	65	K1/2	evidence	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
74	77	Na+	chemical	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
145	149	Sext	site	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
153	171	partially occupied	protein_state	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
180	186	NCX_Mj	protein	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
187	194	crystal	evidence	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
220	228	occluded	protein_state	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
233	247	partially open	protein_state	Our crystallographic titration experiment indicates that the K1/2 of this Na+-driven conformational transition is ~20 mM. At this concentration, Sext is partially occupied and the NCX_Mj crystal is a mixture of both the occluded and partially open conformations.	RESULTS
26	38	Na+ affinity	evidence	This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.	RESULTS
69	72	Na+	chemical	This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.	RESULTS
100	103	NCX	protein_type	This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.	RESULTS
118	128	eukaryotes	taxonomy_domain	This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes.	RESULTS
21	24	Na+	chemical	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
105	116	transporter	protein_type	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
150	166	Hill coefficient	evidence	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
174	177	Na+	chemical	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
210	220	eukaryotic	taxonomy_domain	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
221	224	NCX	protein_type	The finding that the Na+ occupancy change from 2 to 3 ions coincides with a conformational change of the transporter also provides a rationale to the Hill coefficient of the Na+-dependent activation process in eukaryotic NCX.	RESULTS
14	18	Ca2+	chemical	Extracellular Ca2+ and Sr2+ binding and their competition with Na+	RESULTS
23	27	Sr2+	chemical	Extracellular Ca2+ and Sr2+ binding and their competition with Na+	RESULTS
63	66	Na+	chemical	Extracellular Ca2+ and Sr2+ binding and their competition with Na+	RESULTS
17	21	Ca2+	chemical	To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).	RESULTS
31	37	NCX_Mj	protein	To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).	RESULTS
56	59	Na+	chemical	To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).	RESULTS
70	91	titrated the crystals	experimental_method	To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).	RESULTS
97	101	Sr2+	chemical	To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods).	RESULTS
0	4	Sr2+	chemical	Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.	RESULTS
23	26	NCX	protein_type	Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.	RESULTS
40	44	Ca2+	chemical	Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.	RESULTS
75	78	Na+	chemical	Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.	RESULTS
94	120	electron-density intensity	evidence	Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity.	RESULTS
0	23	Protein crystals soaked	experimental_method	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
35	39	Sr2+	chemical	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
51	54	Na+	chemical	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
73	94	electron-density peak	evidence	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
103	106	SCa	site	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
139	143	Sr2+	chemical	Protein crystals soaked with 10 mM Sr2+ and 2.5 mM Na+ revealed a strong electron-density peak at site SCa, indicating binding of a single Sr2+ ion (Fig. 3a).	RESULTS
4	15	Sr2+-loaded	protein_state	The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.	RESULTS
16	22	NCX_Mj	protein	The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.	RESULTS
23	32	structure	evidence	The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.	RESULTS
44	58	partially open	protein_state	The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.	RESULTS
88	91	Na+	chemical	The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations.	RESULTS
11	15	Sr2+	chemical	Binding of Sr2+, however, excludes Na+ entirely.	RESULTS
35	38	Na+	chemical	Binding of Sr2+, however, excludes Na+ entirely.	RESULTS
0	18	Crystal titrations	experimental_method	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
24	34	decreasing	experimental_method	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
35	39	Sr2+	chemical	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
43	53	increasing	experimental_method	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
54	57	Na+	chemical	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
76	80	Sr2+	chemical	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
94	108	outward-facing	protein_state	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
109	115	NCX_Mj	protein	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
170	173	Na+	chemical	Crystal titrations with decreasing Sr2+ or increasing Na+ demonstrated that Sr2+ binds to the outward-facing NCX_Mj with low affinity, and that it can be out-competed by Na+ even at low concentrations (Supplementary Note 1 and Supplementary Fig. 2a-b).	RESULTS
16	19	Na+	chemical	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
30	34	Sr2+	chemical	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
36	39	Na+	chemical	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
60	64	Sr2+	chemical	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
88	94	NCX_Mj	protein	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
102	112	Na+-loaded	protein_state	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
114	128	fully occluded	protein_state	Thus, in 100 mM Na+ and 10 mM Sr2+, Na+ completely replaced Sr2+ (Fig. 3a) and reverted NCX_Mj to the Na+-loaded, fully occluded state.	RESULTS
8	29	titration experiments	experimental_method	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
42	46	Ca2+	chemical	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
51	55	Sr2+	chemical	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
67	73	NCX_Mj	protein	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
100	129	electron density distribution	evidence	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
135	153	crystals soaked in	experimental_method	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
154	158	high	protein_state	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
159	163	Ca2+	chemical	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
168	171	low	protein_state	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
172	175	Na+	chemical	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
192	196	Ca2+	chemical	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
209	213	Smid	site	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
225	228	SCa	site	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
252	255	SCa	site	Similar titration experiments showed that Ca2+ and Sr2+ binding to NCX_Mj are not exactly alike The electron density distribution from crystals soaked in high Ca2+ and low Na+, indicates that Ca2+ can bind to Smid as well as SCa, with a preference for SCa (Fig. 3b).	RESULTS
11	15	Ca2+	chemical	Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b).	RESULTS
113	118	water	chemical	Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b).	RESULTS
145	157	coordinating	bond_interaction	Binding of Ca2+ to both sites simultaneously is highly improbable due to their close proximity, and at least one water molecule can be discerned coordinating the ion (Fig. 3b).	RESULTS
4	11	partial	protein_state	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
12	16	Ca2+	chemical	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
17	26	occupancy	protein_state	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
30	34	Smid	site	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
55	61	Asp240	residue_name_number	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
107	117	coordinate	bond_interaction	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
118	122	Ca2+	chemical	The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+.	RESULTS
9	45	functional and computational studies	experimental_method	Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport.	RESULTS
65	71	Asp240	residue_name_number	Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport.	RESULTS
80	90	protonated	protein_state	Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport.	RESULTS
16	19	NCX	protein_type	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
29	35	Asp240	residue_name_number	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
39	50	substituted	experimental_method	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
54	57	Asn	residue_name	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
97	101	Ca2+	chemical	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
113	117	Smid	site	Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid.	RESULTS
0	3	SCa	site	SCa is therefore the functional Ca2+ site.	RESULTS
32	41	Ca2+ site	site	SCa is therefore the functional Ca2+ site.	RESULTS
13	17	Sr2+	chemical	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
19	23	Ca2+	chemical	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
39	47	affinity	evidence	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
51	65	outward-facing	protein_state	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
66	72	NCX_Mj	protein	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
105	108	Na+	chemical	Similarly to Sr2+, Ca2+ binds with low affinity to outward-facing NCX_Mj and can be readily displaced by Na+ (Supplementary Note 1 and Supplementary Fig. 2c).	RESULTS
32	66	physiological and biochemical data	evidence	This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.	RESULTS
76	86	eukaryotic	taxonomy_domain	This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.	RESULTS
87	90	NCX	protein_type	This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.	RESULTS
95	101	NCX_Mj	protein	This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.	RESULTS
131	144	Ca2+ affinity	evidence	This finding is consistent with physiological and biochemical data for both eukaryotic NCX and NCX_Mj indicating that the apparent Ca2+ affinity is much lower on the extracellular than the cytoplasmic side.	RESULTS
18	50	crystallographic titration assay	experimental_method	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
61	65	Ca2+	chemical	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
92	100	affinity	evidence	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
147	162	Ca2+ affinities	evidence	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
196	199	NCX	protein_type	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
201	203	Km	evidence	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
219	225	NCX_Mj	protein	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
227	229	Km	evidence	Specifically, our crystallographic titration assay indicates Ca2+ binds with sub-millimolar affinity, in good agreement with the external apparent Ca2+ affinities deduced functionally for cardiac NCX (Km ~ 0.32 mM) and NCX_Mj (Km ~ 0.175 mM).	RESULTS
22	51	crystal titration experiments	experimental_method	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
78	91	binding sites	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
95	109	outward-facing	protein_state	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
110	116	NCX_Mj	protein	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
148	152	Sint	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
157	161	Sext	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
166	169	Na+	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
187	190	SCa	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
222	226	Ca2+	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
251	254	Na+	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
279	289	simulation	experimental_method	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
308	312	Sr2+	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
314	318	Smid	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
352	356	Ca2+	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
378	382	Smid	site	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
410	415	water	chemical	Taken together, these crystal titration experiments demonstrate that the four binding sites in outward-facing NCX_Mj exhibit different specificity: Sint and Sext are Na+ specific whereas SCa, previously hypothesized to be Ca2+ specific, can also bind Na+, confirming our earlier simulation study, as well as Sr2+; Smid can also transiently accommodate Ca2+ but during transport Smid is most likely occupied by water.	RESULTS
4	21	ion-binding sites	site	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
25	31	NCX_Mj	protein	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
70	73	Na+	chemical	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
122	125	Na+	chemical	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
130	134	Ca2+	chemical	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
139	143	Sr2+	chemical	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
188	198	eukaryotic	taxonomy_domain	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
199	209	exchangers	protein_type	The ion-binding sites in NCX_Mj can therefore accommodate up to three Na+ ions or a single divalent ion, and occupancy by Na+ and Ca2+ (or Sr2+) are mutually exclusive, as was deduced for eukaryotic exchangers.	RESULTS
2	11	structure	evidence	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
15	21	NCX_Mj	protein	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
22	29	without	protein_state	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
30	33	Na+	chemical	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
37	41	Ca2+	chemical	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
42	47	bound	protein_state	A structure of NCX_Mj without Na+ or Ca2+ bound	RESULTS
3	6	apo	protein_state	An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).	RESULTS
16	30	outward-facing	protein_state	An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).	RESULTS
31	37	NCX_Mj	protein	An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).	RESULTS
140	143	Na+	chemical	An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).	RESULTS
158	162	Ca2+	chemical	An apo state of outward-facing NCX_Mj is likely to exist transiently in physiological conditions, despite the high amounts of extracellular Na+ (~150 mM) and Ca2+ (~2 mM).	RESULTS
29	32	apo	protein_state	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
39	48	structure	evidence	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
52	58	NCX_Mj	protein	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
63	76	crystallizing	experimental_method	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
92	100	lower pH	protein_state	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
112	122	absence of	protein_state	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
123	126	Na+	chemical	We were able to determine an apo-state structure of NCX_Mj, by crystallizing the protein at lower pH and in the absence of Na+ (Methods).	RESULTS
5	14	structure	evidence	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
33	47	partially open	protein_state	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
48	57	structure	evidence	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
67	70	Na+	chemical	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
85	89	Ca2+	chemical	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
97	101	Sr2+	chemical	This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences.	RESULTS
7	12	TM7ab	structure_element	First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.	RESULTS
28	46	extracellular half	structure_element	First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.	RESULTS
54	57	TM6	structure_element	First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.	RESULTS
62	65	TM1	structure_element	First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.	RESULTS
168	181	binding sites	site	First, TM7ab along with the extracellular half of the TM6 and TM1 swing further away from the protein core (Fig. 3c), resulting in a slightly wider passageway into the binding sites.	RESULTS
8	13	Glu54	residue_name_number	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
18	24	Glu213	residue_name_number	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
58	71	binding sites	site	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
91	105	hydrogen-bonds	bond_interaction	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
132	148	ion coordination	bond_interaction	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
156	172	fully Na+-loaded	protein_state	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
173	182	structure	evidence	Second, Glu54 and Glu213 side chains rotate away from the binding sites and appear to form hydrogen-bonds with residues involved in ion coordination in the fully Na+-loaded structure (Fig. 3d).	RESULTS
13	26	binding sites	site	Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules.	RESULTS
36	52	fully accessible	protein_state	Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules.	RESULTS
101	117	electron density	evidence	Although the binding sites are thus fully accessible to the external solution (Fig. 3e), the lack of electron density therein indicates no ions or ordered solvent molecules.	RESULTS
5	8	apo	protein_state	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
9	18	structure	evidence	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
49	57	unloaded	protein_state	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
59	63	open	protein_state	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
73	87	outward-facing	protein_state	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
88	94	NCX_Mj	protein	This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj.	RESULTS
20	29	structure	evidence	Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.	RESULTS
46	62	fully protonated	protein_state	Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.	RESULTS
76	87	transporter	protein_type	Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.	RESULTS
98	101	Na+	chemical	Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.	RESULTS
106	110	Ca2+	chemical	Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind.	RESULTS
49	69	computer simulations	experimental_method	Such interpretation would be consistent with the computer simulations reported below.	RESULTS
8	24	transport assays	experimental_method	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
28	34	NCX_Mj	protein	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
63	74	presence of	protein_state	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
75	78	Na+	chemical	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
82	86	Ca2+	chemical	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
88	94	low pH	protein_state	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
95	106	inactivates	protein_state	Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle.	RESULTS
54	60	NCX_Mj	protein	Ion occupancy determines the free-energy landscape of NCX_Mj	RESULTS
5	34	secondary-active transporters	protein_type	That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states.	RESULTS
214	222	outward-	protein_state	That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states.	RESULTS
227	238	inward-open	protein_state	That secondary-active transporters are able to harness an electrochemical gradient of one substrate to power the uphill transport of another relies on a seemingly simple principle: they must not transition between outward- and inward-open conformations unless in two precise substrate occupancy states.	RESULTS
0	3	NCX	protein_type	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
33	36	Na+	chemical	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
42	46	Ca2+	chemical	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
78	88	antiporter	protein_type	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
90	100	symporters	protein_type	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
217	222	bound	protein_state	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
234	237	apo	protein_state	NCX must be loaded either with 3 Na+ or 1 Ca2+, and therefore functions as an antiporter; symporters, by contrast, undergo the alternating-access transition only when all substrates and coupling ions are concurrently bound, or in the apo state.	RESULTS
64	100	conformational free-energy landscape	evidence	To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.	RESULTS
104	110	NCX_Mj	protein	To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.	RESULTS
175	193	molecular dynamics	experimental_method	To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.	RESULTS
195	197	MD	experimental_method	To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.	RESULTS
199	210	simulations	experimental_method	To examine this central question, we sought to characterize the conformational free-energy landscape of NCX_Mj and to examine its dependence on the ion-occupancy state, using molecular dynamics (MD) simulations.	RESULTS
62	71	structure	evidence	This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above.	RESULTS
75	81	NCX_Mj	protein	This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above.	RESULTS
104	128	crystallographic studies	experimental_method	This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above.	RESULTS
44	54	structures	evidence	As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state.	RESULTS
136	142	NCX_Mj	protein	As it happens, the results confirm that the structures now available are representing interconverting states of the functional cycle of NCX_Mj, while revealing how the alternating-access mechanism is controlled by the ion-occupancy state.	RESULTS
24	38	MD simulations	experimental_method	A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.	RESULTS
97	103	NCX_Mj	protein	A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.	RESULTS
104	113	structure	evidence	A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.	RESULTS
134	151	ion-binding sites	site	A series of exploratory MD simulations was initially carried out to examine what features of the NCX_Mj structure might depend on the ion-binding sites occupancy.	RESULTS
23	32	simulated	experimental_method	Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).	RESULTS
37	53	outward-occluded	protein_state	Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).	RESULTS
131	139	high-Na+	protein_state	Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).	RESULTS
140	157	crystal structure	evidence	Specifically, we first simulated the outward-occluded form, in the ion configuration we previously predicted, now confirmed by the high-Na+ crystal structure described above (Fig. 1b).	RESULTS
9	12	Na+	chemical	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
25	29	Sext	site	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
31	34	SCa	site	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
40	44	Sint	site	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
52	56	D240	residue_name_number	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
60	70	protonated	protein_state	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
77	82	water	chemical	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
101	105	Smid	site	That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid.	RESULTS
4	7	Na+	chemical	The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time.	RESULTS
15	19	Sext	site	The Na+ ion at Sext was then relocated from the site to the bulk solution (Methods), and this system was then allowed to evolve freely in time.	RESULTS
4	7	Na+	chemical	The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.	RESULTS
16	19	SCa	site	The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.	RESULTS
24	28	Sint	site	The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.	RESULTS
75	85	simulation	experimental_method	The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out.	RESULTS
14	25	simulations	experimental_method	These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures.	RESULTS
61	72	transporter	protein_type	These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures.	RESULTS
116	134	crystal structures	evidence	These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures.	RESULTS
45	48	Na+	chemical	The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a).	RESULTS
54	58	Sext	site	The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a).	RESULTS
84	89	TM7ab	structure_element	The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a).	RESULTS
7	10	Na+	chemical	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
20	25	bound	protein_state	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
27	32	TM7ab	structure_element	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
80	99	α-helical fragments	structure_element	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
150	164	hydrogen-bonds	bond_interaction	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
173	177	F202	residue_name_number	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
182	186	A206	residue_name_number	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
192	196	T203	residue_name_number	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
201	205	F207	residue_name_number	When 3 Na+ ions are bound, TM7ab primarily folds as two distinct, non-collinear α-helical fragments, owing to the loss of the backbone carbonyl-amide hydrogen-bonds between F202 and A206, and T203 and F207 (Fig. 4b).	RESULTS
25	29	Sext	site	This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g).	RESULTS
97	100	Na+	chemical	This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g).	RESULTS
147	151	A206	residue_name_number	This distortion occludes Sext from the exterior (Fig. 4d, 4h-i) and appears to be induced by the Na+ ion itself, which pulls the carbonyl group of A206 into its coordination sphere (Fig. 4g).	RESULTS
5	9	Sext	site	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
10	15	empty	protein_state	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
26	31	TM7ab	structure_element	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
50	57	α-helix	structure_element	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
111	114	TM3	structure_element	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
119	122	TM7	structure_element	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
145	150	water	chemical	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
202	206	Sext	site	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
233	244	transporter	protein_type	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
248	266	no longer occluded	protein_state	With Sext empty, however, TM7ab forms a canonical α-helix (Fig. 4a-b, 4g), thereby creating an opening between TM3 and TM7, which in turn allows water molecules from the external solution to reach into Sext (Fig. 4e, 4h-i), i.e. the transporter is no longer occluded.	RESULTS
16	19	Na+	chemical	Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c).	RESULTS
25	28	SCa	site	Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c).	RESULTS
33	37	Sint	site	Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c).	RESULTS
55	58	TM7	structure_element	The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).	RESULTS
63	66	TM2	structure_element	The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).	RESULTS
151	154	Na+	chemical	The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).	RESULTS
162	165	SCa	site	The most noticeable is an increased separation between TM7 and TM2 (Fig. 4f), previously brought together by concurrent backbone interactions with the Na+ ion at SCa (Fig. 4d-e).	RESULTS
0	3	TM1	structure_element	TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c).	RESULTS
8	11	TM6	structure_element	TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c).	RESULTS
76	92	outward-occluded	protein_state	TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c).	RESULTS
38	53	aqueous channel	site	Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i).	RESULTS
76	79	SCa	site	Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i).	RESULTS
84	88	Sint	site	Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i).	RESULTS
4	15	transporter	protein_type	The transporter thus becomes fully outward-open.	RESULTS
29	47	fully outward-open	protein_state	The transporter thus becomes fully outward-open.	RESULTS
111	120	exchanger	protein_type	To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).	RESULTS
167	182	MD calculations	experimental_method	To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).	RESULTS
242	258	outward-occluded	protein_state	To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).	RESULTS
263	281	fully outward-open	protein_state	To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).	RESULTS
312	333	free-energy landscape	evidence	To more rigorously characterize the influence of the ion-occupancy state on the conformational dynamics of the exchanger, we carried out a series of enhanced-sampling MD calculations designed to reversibly simulate the transition between the outward-occluded and fully outward-open states, and thus quantify the free-energy landscape encompassing these states (Methods).	RESULTS
68	71	Na+	chemical	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
75	79	Sext	site	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
81	84	SCa	site	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
89	93	Sint	site	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
100	103	Na+	chemical	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
112	115	SCa	site	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
120	124	Sint	site	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
130	137	without	protein_state	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
138	141	Na+	chemical	As above, we initially examined three occupancy states, namely with Na+ in Sext, SCa and Sint, with Na+ only at SCa and Sint, and without Na+.	RESULTS
6	18	calculations	experimental_method	These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.	RESULTS
40	43	Na+	chemical	These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.	RESULTS
67	78	transporter	protein_type	These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.	RESULTS
108	144	conformational free-energy landscape	evidence	These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape.	RESULTS
9	18	Na+ sites	site	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
44	63	free-energy minimum	evidence	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
168	173	TM7ab	structure_element	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
205	208	TM2	structure_element	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
213	216	TM3	structure_element	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
229	242	binding sites	site	When all Na+ sites are occupied, the global free-energy minimum corresponds to a conformation in which the ions are maximally coordinated by the protein (Fig. 5a, 5c); TM7ab is bent and packs closely with TM2 and TM3, and so the binding sites are occluded from the solvent (Fig. 5b).	RESULTS
40	51	transporter	protein_type	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
64	74	metastable	protein_state	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
76	85	half-open	protein_state	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
109	114	TM7ab	structure_element	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
142	146	Sext	site	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
150	154	open	protein_state	At a small energetic cost, however, the transporter can adopt a metastable ‘half-open’ conformation in which TM7ab is completely straight and Sext is open to the exterior (Fig. 5a, 5b).	RESULTS
4	7	Na+	chemical	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
15	19	Sext	site	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
28	45	fully coordinated	protein_state	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
62	67	water	chemical	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
111	115	A206	residue_name_number	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
147	151	F202	residue_name_number	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
154	158	A206	residue_name_number	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
161	174	hydrogen-bond	bond_interaction	The Na+ ion at Sext remains fully coordinated, but an ordered water molecule now mediates its interaction with A206:O, relieving the strain on the F202:O–A206:N hydrogen-bond (Fig. 5c).	RESULTS
5	14	semi-open	protein_state	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
91	94	Na+	chemical	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
109	112	SCa	site	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
117	121	Sint	site	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
127	130	Na+	chemical	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
185	188	Na+	chemical	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
192	196	Sext	site	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
212	222	metastable	protein_state	This semi-open conformation is nearly identical to that found to be the most probable when Na+ occupies only SCa and Sint (2 × Na+, Fig. 5a), demonstrating that binding (or release) of Na+ to Sext occurs in this metastable conformation.	RESULTS
92	107	aqueous channel	site	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
141	144	SCa	site	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
153	156	TM7	structure_element	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
161	164	TM2	structure_element	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
178	192	gating helices	structure_element	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
193	196	TM1	structure_element	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
201	204	TM6	structure_element	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
219	223	open	protein_state	Interestingly, this doubly occupied state can also access conformations in which the second aqueous channel mentioned above, i.e. leading to SCa between TM7 and TM2 and over the gating helices TM1 and TM6, also becomes open (Fig. 5b-c).	RESULTS
23	44	free-energy landscape	evidence	Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a).	RESULTS
54	72	partially occupied	protein_state	Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a).	RESULTS
101	109	occluded	protein_state	Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a).	RESULTS
34	37	Na+	chemical	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
48	51	SCa	site	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
56	60	Sint	site	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
82	103	free-energy landscape	evidence	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
111	122	transporter	protein_type	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
170	180	fully open	protein_state	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
205	221	aqueous channels	site	Displacement of the two remaining Na+ ions from SCa and Sint further reshapes the free-energy landscape of the transporter (No ions, Fig. 5a), which now can only adopt a fully open state featuring the two aqueous channels (Fig. 5b-c).	RESULTS
22	30	occluded	protein_state	The transition to the occluded state in this apo state is again energetically unfeasible.	RESULTS
45	48	apo	protein_state	The transition to the occluded state in this apo state is again energetically unfeasible.	RESULTS
67	71	open	protein_state	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
76	85	semi-open	protein_state	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
132	135	Na+	chemical	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
175	178	Na+	chemical	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
287	308	free-energy landscape	evidence	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
316	327	transporter	protein_type	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
346	354	occluded	protein_state	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
392	417	Na+ occupancy is complete	protein_state	From a mechanistic standpoint, it is satisfying to observe how the open and semi-open states are each compatible with two different Na+ occupancies, explaining how sequential Na+ binding to energetically accessible conformations (prior to those binding events) progressively reshape the free-energy landscape of the transporter; by contrast, the occluded conformation is forbidden unless the Na+ occupancy is complete.	RESULTS
41	44	Na+	chemical	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
93	103	Ca2+-bound	protein_state	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
144	155	transporter	protein_type	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
223	235	outward-open	protein_state	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
276	280	Ca2+	chemical	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
295	303	occluded	protein_state	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
331	340	semi-open	protein_state	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
385	396	inward-open	protein_state	This processivity is logical since three Na+ ions are involved, but also implies that in the Ca2+-bound state, which includes a single ion, the transporter ought to be able to access all three major conformations, i.e. the outward-open state, in order to release (or re-bind) Ca2+, but also the occluded conformation, and thus the semi-open intermediate, in order to transition to the inward-open state.	RESULTS
26	28	H+	chemical	By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion.	RESULTS
97	106	semi-open	protein_state	By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion.	RESULTS
133	143	fully open	protein_state	By contrast, occupancy by H+, which as mentioned are not transported, might be compatible with a semi-open state as well as with the fully open conformation, but should not be conducive to occlusion.	RESULTS
42	71	enhanced-sampling simulations	experimental_method	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
80	84	Ca2+	protein_state	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
89	97	H+-bound	protein_state	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
108	122	outward-facing	protein_state	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
123	129	NCX_Mj	protein	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
169	172	Na+	chemical	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
249	259	structures	evidence	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
267	277	Ca2+-bound	protein_state	To assess this hypothesis, we carried out enhanced-sampling simulations for the Ca2+ and H+-bound states of outward-facing NCX_Mj analogous to those described above for Na+ (see Supplementary Note 2 and Supplementary Fig. 3-4 for details on how the structures of the Ca2+-bound state was predicted).	RESULTS
4	14	calculated	experimental_method	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
15	36	free-energy landscape	evidence	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
41	51	Ca2+-bound	protein_state	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
52	58	NCX_Mj	protein	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
103	107	Ca2+	chemical	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
149	155	NCX_Mj	protein	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
176	180	Ca2+	chemical	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
186	194	occluded	protein_state	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
196	206	dehydrated	protein_state	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
274	283	exchanger	protein_type	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
303	312	semi-open	protein_state	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
317	321	open	protein_state	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
356	360	Ca2+	chemical	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
373	376	Na+	chemical	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
402	425	aqueous access channels	site	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
439	443	Sext	site	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
448	451	SCa	site	The calculated free-energy landscape for Ca2+-bound NCX_Mj confirms the hypothesis outlined above (1 × Ca2+, Fig. 6a): consistent with the fact that NCX_Mj transports a single Ca2+, the occluded, dehydrated conformation is one of the major energetic minima, but clearly the exchanger can also adopt the semi-open and open states that would be required for Ca2+ release and Na+ entry, via either of the aqueous access channels that lead to Sext and SCa (Fig. 6b-c).	RESULTS
13	24	protonation	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
28	33	Glu54	residue_name_number	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
38	44	Glu213	residue_name_number	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
55	63	occluded	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
133	139	NCX_Mj	protein	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
159	166	protons	chemical	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
176	184	H+-bound	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
204	213	exchanger	protein_type	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
228	237	semi-open	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
267	273	low pH	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
275	278	apo	protein_state	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
279	296	crystal structure	evidence	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
302	304	H+	chemical	By contrast, protonation of Glu54 and Glu213 makes the occluded conformation energetically unfeasible, consistent with the fact that NCX_Mj does not transport protons; in this H+-bound state, though, the exchanger can adopt the semi-open conformation captured in the low pH, apo crystal structure (2 × H+, Fig. 6a-c).	RESULTS
21	54	systematic computational analysis	experimental_method	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
58	72	outward-facing	protein_state	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
73	79	NCX_Mj	protein	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
168	186	Na+/Ca2+ exchanger	protein_type	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
253	274	free-energy landscape	evidence	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
335	343	occluded	protein_state	Taken together, this systematic computational analysis of outward-facing NCX_Mj clearly demonstrates that the alternating-access and ion-recognition mechanisms in this Na+/Ca2+ exchanger are coupled through the influence that the bound ions have on the free-energy landscape of the protein, which in turn determines whether or not the occluded conformation is energetically feasible.	RESULTS
5	13	occluded	protein_state	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
74	81	outward	protein_state	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
86	97	inward-open	protein_state	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
137	148	dehydration	protein_state	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
189	207	complete occupancy	protein_state	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
215	228	binding sites	site	This occluded conformation, which is a necessary intermediate between the outward and inward-open states, and which entails the internal dehydration of the protein, is only attainable upon complete occupancy of the binding sites.	RESULTS
78	85	outward	protein_state	The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.	DISCUSS
91	102	inward-open	protein_state	The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.	DISCUSS
128	144	secondary-active	protein_state	The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.	DISCUSS
145	156	transporter	protein_type	The alternating-access hypothesis implicitly dictates that the switch between outward- and inward-open conformations of a given secondary-active transporter must not occur unless the appropriate type and number of substrates are recognized.	DISCUSS
32	43	antiporters	protein_type	It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles.	DISCUSS
150	169	membrane symporters	protein_type	It is however also non-trivial: antiporters, for example, do not undergo the alternating-access transition without a cargo, but this is precisely how membrane symporters reset their transport cycles.	DISCUSS
35	45	antiporter	protein_type	Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number.	DISCUSS
67	77	antiporter	protein_type	Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch.	DISCUSS
83	92	symporter	protein_type	Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch.	DISCUSS
143	168	alternating-access switch	site	Yet, when multiple species are to be co-translocated, by either an antiporter or a symporter, partial occupancies must not be conducive to the alternating-access switch.	DISCUSS
103	121	structural studies	experimental_method	Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger.	DISCUSS
126	160	quantitative molecular simulations	experimental_method	Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger.	DISCUSS
166	184	Na+/Ca2+ exchanger	protein_type	Here, we have provided novel insights into this intriguing mechanism of conformational control through structural studies and quantitative molecular simulations of a Na+/Ca2+ exchanger.	DISCUSS
29	35	NCX_Mj	protein	Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7).	DISCUSS
25	39	outward-facing	protein_state	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
40	46	NCX_Mj	protein	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
55	68	inward-facing	protein_state	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
69	87	crystal structures	evidence	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
99	117	Ca2+/H+ exchangers	protein_type	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
168	171	NCX	protein_type	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
209	212	TM1	structure_element	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
217	220	TM6	structure_element	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
249	260	transporter	protein_type	The internal symmetry of outward-facing NCX_Mj and the inward-facing crystal structures of several Ca2+/H+ exchangers indicate that the alternating-access mechanism of NCX proteins entails a sliding motion of TM1 and TM6 relative to the rest of the transporter.	DISCUSS
56	76	extracellular region	structure_element	Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release.	DISCUSS
84	91	TM2-TM3	structure_element	Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release.	DISCUSS
96	110	TM7-TM8 bundle	structure_element	Here, we demonstrate that conformational changes in the extracellular region of the TM2-TM3 and TM7-TM8 bundle precede and are necessary for the transition, and are associated with ion recognition and/or release.	DISCUSS
48	63	N-terminal half	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
67	70	TM7	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
72	77	TM7ab	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
123	126	TM2	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
131	134	TM3	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
151	156	TM7ab	structure_element	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
213	229	aqueous channels	site	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
247	264	ion-binding sites	site	The most apparent of these changes involves the N-terminal half of TM7 (TM7ab); together with more subtle displacements in TM2 and TM3, this change in TM7ab correlates with the opening and closing of two distinct aqueous channels leading into the ion-binding sites from the extracellular solution.	DISCUSS
30	33	TM7	structure_element	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
71	88	ion-binding sites	site	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
123	126	TM6	structure_element	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
145	148	TM6	structure_element	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
153	156	TM1	structure_element	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
180	193	inward-facing	protein_state	Interestingly, the bending of TM7 associated with the occlusion of the ion-binding sites also unlocks its interaction with TM6, and thus enables TM6 and TM1 to freely slide to the inward-facing conformation.	DISCUSS
4	22	crystal structures	evidence	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
26	32	NCX_Mj	protein	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
60	64	Na+,	chemical	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
65	70	Ca2+,	chemical	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
71	75	Sr2+	chemical	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
79	81	H+	chemical	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
82	87	bound	protein_state	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
101	110	exchanger	protein_type	The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states.	DISCUSS
115	126	transporter	protein_type	These states can only represent a subset among all possible, but they ought to reflect inherent preferences of the transporter, modulated by the experimental conditions.	DISCUSS
20	27	crystal	evidence	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
31	37	NCX_Mj	protein	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
41	44	LCP	experimental_method	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
50	68	extracellular half	structure_element	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
76	90	gating helices	structure_element	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
92	95	TM6	structure_element	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
100	103	TM1	structure_element	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
190	207	ion-binding sites	site	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
235	238	apo	protein_state	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
240	246	low pH	protein_state	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
247	256	structure	evidence	For example, in the crystal of NCX_Mj in LCP, the extracellular half of the gating helices (TM6 and TM1) form a lattice contact, which might ultimately restrict the degree of opening of the ion-binding sites in some cases (e.g. in the apo, low pH structure).	DISCUSS
17	50	calculated free-energy landscapes	evidence	Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states.	DISCUSS
134	157	crystallized structures	evidence	Nonetheless, the calculated free-energy landscapes, derived without knowledge of the experimental data, reassuringly confirm that the crystallized structures correspond to mechanistically relevant, interconverting states.	DISCUSS
4	15	simulations	experimental_method	The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event.	DISCUSS
58	66	occluded	protein_state	Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.	DISCUSS
130	136	NCX_Mj	protein	Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.	DISCUSS
146	149	Na+	chemical	Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.	DISCUSS
151	155	Ca2+	chemical	Indeed, we show that it is the presence or absence of the occluded state in this landscape that explains the antiport function of NCX_Mj and its 3Na+:1Ca2+ stoichiometry.	DISCUSS
89	101	transporters	protein_type	We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.	DISCUSS
140	150	symporters	protein_type	We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.	DISCUSS
155	166	antiporters	protein_type	We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.	DISCUSS
197	205	occluded	protein_state	We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.	DISCUSS
228	272	protein conformational free-energy landscape	evidence	We posit that a similar principle might govern the alternating-access mechanism in other transporters; that is, we anticipate that for both symporters and antiporters, it is the feasibility of the occluded state, encoded in the protein conformational free-energy landscape and its dependence on substrate binding, that ultimately explains their specific coupling mechanisms.	DISCUSS
122	128	NCX_Mj	protein	In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues.	DISCUSS
137	147	eukaryotic	taxonomy_domain	In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues.	DISCUSS
48	70	ion-binding affinities	evidence	The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.	DISCUSS
89	116	crystallographic titrations	experimental_method	The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.	DISCUSS
125	127	Km	evidence	The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.	DISCUSS
132	143	K1/2 values	evidence	The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.	DISCUSS
168	185	functional assays	experimental_method	The striking quantitative agreement between the ion-binding affinities inferred from our crystallographic titrations and the Km and K1/2 values previously deduced from functional assays has been discussed above.	DISCUSS
113	119	NCX_Mj	protein	Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).	DISCUSS
132	135	NCX	protein_type	Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).	DISCUSS
193	199	NCX_Mj	protein	Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).	DISCUSS
200	210	structures	evidence	Consistent with that finding, mutations that have been shown to inactivate or diminish the transport activity of NCX_Mj and cardiac NCX perfectly map to the first ion-coordination shell in our NCX_Mj structures (Supplementary Fig. 4c-d).	DISCUSS
4	25	crystallographic data	evidence	The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.	DISCUSS
149	159	eukaryotic	taxonomy_domain	The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.	DISCUSS
160	163	NCX	protein_type	The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.	DISCUSS
212	218	NCX_Mj	protein	The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.	DISCUSS
237	255	Na+/Ca2+ exchanger	protein_type	The crystallographic data also provides the long-sought structural basis for the ‘two-site’ model proposed to describe competitive cation binding in eukaryotic NCX, underscoring the relevance of these studies of NCX_Mj as a prototypical Na+/Ca2+ exchanger.	DISCUSS
18	36	crystal titrations	experimental_method	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
66	69	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
70	74	Ca2+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
91	95	Sint	site	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
100	103	SCa	site	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
111	115	Ca2+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
120	123	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
162	165	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
211	214	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
239	242	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
261	266	water	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
280	284	Smid	site	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
313	317	Ca2+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
334	350	Hill coefficient	evidence	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
361	364	Na+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
389	393	Ca2+	chemical	Specifically, our crystal titrations suggest that, during forward Na+/Ca2+ exchange, sites Sint and SCa, which Ca2+ and Na+ compete for, can be grouped into one; Na+ binding to these sites does not require high Na+ concentrations, and two Na+ ions along with a water molecule (at Smid) are sufficient to displace Ca2+, explaining the Hill coefficient of ~2 for Na+-dependent inhibition of Ca2+ fluxes.	DISCUSS
4	8	Sext	site	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
51	66	activation site	site	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
78	81	Na+	chemical	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
127	130	Na+	chemical	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
149	152	Na+	chemical	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
199	207	occluded	protein_state	The Sext site, by contrast, might be thought as an activation site for inward Na+ translocation, since this is where the third Na+ ion binds at high Na+ concentration, enabling the transition to the occluded state.	DISCUSS
26	30	Ca2+	chemical	Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle.	DISCUSS
34	38	Smid	site	Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle.	DISCUSS
8	18	structures	evidence	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
22	28	NCX_Mj	protein	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
29	37	bound to	protein_state	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
38	42	Cd2+	chemical	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
46	50	Mn2+	chemical	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
104	108	Smid	site	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
123	127	Sr2+	chemical	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
142	145	SCa	site	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
182	185	NCX	protein_type	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
199	206	calcium	chemical	Indeed, structures of NCX_Mj bound to Cd2+ or Mn2+, both of which inhibit transport, show these ions at Smid; by contrast, Sr2+ binds only to SCa, and accordingly, is transported by NCX similarly to calcium.	DISCUSS
37	43	NCX_Mj	protein	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
72	76	Ca2+	chemical	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
80	83	Na+	chemical	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
147	174	hydrogen-deuterium exchange	experimental_method	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
176	179	HDX	experimental_method	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
184	190	NCX_Mj	protein	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
199	207	presence	protein_state	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
211	221	absence of	protein_state	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
259	273	outward-facing	protein_state	Lastly, our theory that occlusion of NCX_Mj is selectively induced upon Ca2+ or Na+ recognition is consonant with a recent analysis of the rate of hydrogen-deuterium exchange (HDX) in NCX_Mj, in the presence or absence of these ions, in conditions that favor outward-facing conformations.	DISCUSS
36	40	Ca2+	chemical	Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices.	DISCUSS
44	47	Na+	chemical	Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices.	DISCUSS
89	97	HDX rate	evidence	Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices.	DISCUSS
132	148	α-repeat helices	structure_element	Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices.	DISCUSS
231	248	ion-binding sites	site	We interpret these observations as reflecting that the solvent accessibility of the protein interior is diminished upon ion recognition, consistent with our finding that opening and closing of extracellular aqueous pathways to the ion-binding sites depend on ion occupancy state.	DISCUSS
80	88	occluded	protein_state	In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate.	DISCUSS
194	202	HDX rate	evidence	In addition, the increased compactness of the protein tertiary structure in the occluded state would also slow down the dynamics of the secondary-structure elements, and thus further reduce the HDX rate.	DISCUSS
70	78	HDX rate	evidence	Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations.	DISCUSS
97	100	Na+	chemical	Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations.	DISCUSS
105	109	Ca2+	chemical	Our data would also explain the observation that the reduction in the HDX rate is comparable for Na+ and Ca2+, as well as the finding that the degree of deuterium incorporation remains non-negligible even under saturating ion concentrations.	DISCUSS
7	40	calculated free-energy landscapes	evidence	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
47	50	Na+	chemical	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
55	59	Ca2+	chemical	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
88	99	transporter	protein_type	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
136	145	ion-bound	protein_state	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
219	229	fully open	protein_state	As the calculated free-energy landscapes show, Na+ and Ca2+ induce the occlusion of the transporter in a comparable manner, and yet the ion-bound states retain the ability to explore conformations that are partially or fully open to the extracellular solution, precisely so as to be able to unload and re-load the substrates.	DISCUSS
0	3	Na+	chemical	Na+ binding to outward-facing NCX_Mj.	FIG
15	29	outward-facing	protein_state	Na+ binding to outward-facing NCX_Mj.	FIG
30	36	NCX_Mj	protein	Na+ binding to outward-facing NCX_Mj.	FIG
12	21	structure	evidence	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
25	31	native	protein_state	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
32	46	outward-facing	protein_state	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
47	53	NCX_Mj	protein	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
59	73	crystals grown	experimental_method	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
84	87	Na+	chemical	(a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+.	FIG
36	39	Na+	chemical	Colored spheres represent the bound Na+ (green) and water (red).	FIG
52	57	water	chemical	Colored spheres represent the bound Na+ (green) and water (red).	FIG
50	71	central binding sites	site	(b) Structural details and definition of the four central binding sites.	FIG
4	20	electron density	evidence	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
39	57	Fo-Fc ion omit map	evidence	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
78	82	Smid	site	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
98	103	water	chemical	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
130	134	Sext	site	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
136	139	SCa	site	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
144	148	Sint	site	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
152	155	Na+	chemical	The electron density (grey mesh, 1.9 Å Fo-Fc ion omit map contoured at 4σ) at Smid was modeled as water (red sphere) and those at Sext, SCa and Sint as Na+ ions (green spheres).	FIG
89	92	Na+	chemical	Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1).	FIG
4	25	Fo – Fc ion-omit maps	evidence	All Fo – Fc ion-omit maps are calculated to 2.4 Å and contoured at 3σ for comparison.	FIG
20	24	A206	residue_name_number	The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).	FIG
39	42	Na+	chemical	The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).	FIG
85	99	partially open	protein_state	The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).	FIG
107	115	occluded	protein_state	The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).	FIG
148	151	Na+	chemical	The displacement of A206 reflects the [Na+]-dependent conformational change from the partially open to the occluded state (observed at low and high Na+ concentrations, respectively).	FIG
9	12	Na+	chemical	At 20 mM Na+, both conformations co-exist.	FIG
75	80	water	chemical	No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.	FIG
101	104	SCa	site	No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.	FIG
106	110	Sint	site	No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.	FIG
115	119	Smid	site	No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites.	FIG
0	3	Na+	chemical	Na+-occupancy dependent conformational change in NCX_Mj.	FIG
49	55	NCX_Mj	protein	Na+-occupancy dependent conformational change in NCX_Mj.	FIG
4	19	Superimposition	experimental_method	(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.	FIG
27	33	NCX_Mj	protein	(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.	FIG
34	52	crystal structures	evidence	(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.	FIG
128	131	Na+	chemical	(a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations.	FIG
25	34	interface	site	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
43	46	TM6	structure_element	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
51	56	TM7ab	structure_element	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
64	70	NCX_Mj	protein	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
71	81	structures	evidence	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
107	110	Na+	chemical	(b) Close-up view of the interface between TM6 and TM7ab in the NCX_Mj structures obtained at high and low Na+ concentrations (top and lower panels, respectively).	FIG
47	56	structure	evidence	Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail.	FIG
60	63	low	protein_state	Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail.	FIG
64	67	Na+	chemical	Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail.	FIG
25	42	Na+-binding sites	site	(c) Close-up view of the Na+-binding sites.	FIG
11	15	Sext	site	The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.	FIG
28	37	structure	evidence	The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.	FIG
41	44	low	protein_state	The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.	FIG
45	48	Na+	chemical	The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere.	FIG
56	60	A206	residue_name_number	Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.	FIG
78	89	coordinates	bond_interaction	Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.	FIG
90	93	Na+	chemical	Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.	FIG
97	101	Sext	site	Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen.	FIG
47	64	ion binding sites	site	(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].	FIG
72	82	structures	evidence	(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].	FIG
86	90	high	protein_state	(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].	FIG
95	98	low	protein_state	(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].	FIG
100	103	Na+	chemical	(d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+].	FIG
9	25	solvent channels	site	Putative solvent channels are represented as light-purple surfaces.	FIG
28	31	apo	protein_state	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
32	41	structure	evidence	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
45	51	NCX_Mj	protein	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
66	70	Sr2+	chemical	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
99	102	SCa	site	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
106	123	crystals titrated	experimental_method	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
135	139	Sr2+	chemical	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
151	154	Na+	chemical	Divalent cation binding and apo structure of NCX_Mj. (a) A single Sr2+ (dark blue sphere) binds at SCa in crystals titrated with 10 mM Sr2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
21	25	Sr2+	chemical	Residues involved in Sr2+ coordination are labeled.	FIG
98	107	Na+-bound	protein_state	There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state.	FIG
0	3	T50	residue_name_number	T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.	FIG
8	12	T209	residue_name_number	T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.	FIG
30	40	coordinate	bond_interaction	T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.	FIG
41	45	Sr2+	chemical	T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms.	FIG
5	8	Na+	chemical	High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).	FIG
53	57	Sr2+	chemical	High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).	FIG
70	76	NCX_Mj	protein	High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).	FIG
84	92	occluded	protein_state	High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel).	FIG
25	41	Fo – Fc omit map	evidence	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
49	58	structure	evidence	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
67	70	Na+	chemical	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
128	135	density	evidence	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
141	144	Na+	chemical	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
154	157	H2O	chemical	The contour level of the Fo – Fc omit map of the structure at high Na+ concentration was lowered (to 4σ) so as to visualize the density from Na+ ions and H2O.	FIG
4	8	Ca2+	chemical	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
42	45	SCa	site	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
49	53	Smid	site	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
57	74	crystals titrated	experimental_method	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
86	90	Ca2+	chemical	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
102	105	Na+	chemical	(b) Ca2+ (tanned spheres) binds either to SCa or Smid in crystals titrated with 10 mM Ca2+ and 2.5 mM Na+ (see also Supplementary Fig. 2).	FIG
60	75	Superimposition	experimental_method	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
79	85	NCX_Mj	protein	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
86	96	structures	evidence	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
113	116	Na+	chemical	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
169	179	absence of	protein_state	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
180	183	Na+	chemical	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
188	192	pH 4	protein_state	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
223	226	apo	protein_state	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
259	276	ion-binding sites	site	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
284	287	apo	protein_state	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
292	299	high H+	protein_state	The relative occupancies are 55% and 45%, respectively. (c) Superimposition of NCX_Mj structures obtained at low Na+ concentration (10 mM) and pH 6.5 (brown) and in the absence of Na+ and pH 4 (light green), referred to as apo state. (d) Close-up view of the ion-binding sites in the apo (or high H+) state.	FIG
19	22	E54	residue_name_number	The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.	FIG
27	31	E213	residue_name_number	The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.	FIG
41	48	low Na+	protein_state	The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.	FIG
49	58	structure	evidence	The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison.	FIG
36	40	Sint	site	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
42	46	Smid	site	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
47	50	SCa	site	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
99	116	ion-binding sites	site	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
120	123	apo	protein_state	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
124	130	NCX_Mj	protein	White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj.	FIG
27	36	structure	evidence	Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.	FIG
40	56	outward-occluded	protein_state	Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.	FIG
58	76	fully Na+-occupied	protein_state	Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.	FIG
77	83	NCX_Mj	protein	Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.	FIG
132	135	Na+	chemical	Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+.	FIG
19	29	simulation	experimental_method	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
43	49	NCX_Mj	protein	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
65	68	Na+	chemical	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
69	77	bound at	protein_state	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
78	82	Sext	site	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
84	87	SCa	site	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
92	96	Sint	site	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
139	142	Na+	chemical	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
143	156	bound only at	protein_state	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
157	160	SCa	site	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
165	169	Sint	site	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
240	255	N-terminal half	structure_element	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
259	262	TM7	structure_element	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
264	269	TM7ab	structure_element	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
284	287	Na+	chemical	(a) Representative simulation snapshots of NCX_Mj (Methods) with Na+ bound at Sext, SCa and Sint (orange cartoons, green spheres) and with Na+ bound only at SCa and Sint (marine cartoons, yellow spheres) (b) Close-up of the backbone of the N-terminal half of TM7 (TM7ab), in the same Na+ occupancy states depicted in (a).	FIG
19	39	simulation snapshots	evidence	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
61	64	Na+	chemical	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
65	73	bound at	protein_state	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
74	77	SCa	site	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
82	86	Sint	site	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
125	132	without	protein_state	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
137	140	Na+	chemical	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
141	146	bound	protein_state	(c) Representative simulation snapshots (same as above) with Na+ bound at SCa and Sint (marine cartoons, yellow spheres) and without any Na+ bound (grey cartoons).	FIG
20	38	ion-binding region	site	(d) Close-up of the ion-binding region in the fully Na+-occupied state.	FIG
46	64	fully Na+-occupied	protein_state	(d) Close-up of the ion-binding region in the fully Na+-occupied state.	FIG
30	33	TM2	structure_element	Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state.	FIG
35	38	TM3	structure_element	Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state.	FIG
43	46	TM7	structure_element	Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state.	FIG
87	105	ion-binding region	site	Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state.	FIG
113	135	partially Na+-occupied	protein_state	Approximate distances between TM2, TM3 and TM7 are indicated in Å. (e) Close-up of the ion-binding region in the partially Na+-occupied state.	FIG
20	38	ion-binding region	site	(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).	FIG
46	54	Na+-free	protein_state	(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).	FIG
142	153	simulations	experimental_method	(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).	FIG
162	175	Na+-occupancy	protein_state	(f) Close-up of the ion-binding region in the Na+-free state. (g-i) Analytical descriptors of the changes just described, calculated from the simulations of each Na+-occupancy state depicted in panels (a-f).	FIG
63	101	Bias-Exchange Metadynamics simulations	experimental_method	These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods).	FIG
4	29	Probability distributions	evidence	(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).	FIG
74	90	hydrogen-bonding	bond_interaction	(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).	FIG
102	107	TM7ab	structure_element	(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).	FIG
223	227	Sext	site	(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).	FIG
348	351	SCa	site	(g) Probability distributions of an analytical descriptor of the backbone hydrogen-bonding pattern in TM7ab (Eq. 2). (h) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the Sext site (Eq. 1). (i) Mean value (with standard deviation) of a quantitative descriptor of the solvent accessibility of the SCa site (Eq. 1).	FIG
108	111	NCX	protein_type	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
117	165	Calculated conformational free-energy landscapes	evidence	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
170	184	outward-facing	protein_state	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
185	191	NCX_Mj	protein	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
211	214	Na+	chemical	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
254	267	no ions bound	protein_state	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated conformational free-energy landscapes for outward-facing NCX_Mj, for two different Na+-occupancy states, and for a state with no ions bound.	FIG
4	15	free energy	evidence	The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).	FIG
106	122	aqueous channels	site	The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).	FIG
138	142	Sext	site	The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).	FIG
147	150	SCa	site	The free energy is plotted as a function of two coordinates, each describing the degree of opening of the aqueous channels leading to the Sext and SCa sites, respectively (see Methods).	FIG
22	38	X-ray structures	evidence	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
42	48	NCX_Mj	protein	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
61	65	high	protein_state	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
70	73	low	protein_state	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
74	77	Na+	chemical	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
112	118	low pH	protein_state	Black circles map the X-ray structures of NCX_Mj obtained at high and low Na+ concentration, as well as that at low pH, reported in this study.	FIG
4	23	Density isosurfaces	evidence	(b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.	FIG
28	33	water	chemical	(b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.	FIG
63	81	ion-binding region	site	(b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.	FIG
120	153	conformational free-energy minima	evidence	(b) Density isosurfaces for water molecules within 12 Å of the ion-binding region (grey volumes), for each of the major conformational free-energy minima in each ion-occupancy state.	FIG
0	3	Na+	chemical	Na+ ions are shown as green spheres.	FIG
8	33	inverted-topology repeats	structure_element	The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).	FIG
41	52	transporter	protein_type	The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).	FIG
53	62	structure	evidence	The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).	FIG
111	116	TM1-5	structure_element	The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).	FIG
126	132	TM6-10	structure_element	The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine).	FIG
26	44	ion-binding region	site	(c) Close-up views of the ion-binding region in the same conformational free-energy minima.	FIG
57	90	conformational free-energy minima	evidence	(c) Close-up views of the ion-binding region in the same conformational free-energy minima.	FIG
25	28	Na+	chemical	Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines).	FIG
33	38	water	chemical	Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines).	FIG
4	22	water-density maps	evidence	The water-density maps in (b) is shown here as a grey mesh.	FIG
5	9	D240	residue_name_number	Note D240 is protonated, while E54 and E213 are ionized.	FIG
31	34	E54	residue_name_number	Note D240 is protonated, while E54 and E213 are ionized.	FIG
39	43	E213	residue_name_number	Note D240 is protonated, while E54 and E213 are ionized.	FIG
108	111	NCX	protein_type	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
117	150	Calculated free-energy landscapes	evidence	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
155	169	outward-facing	protein_state	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
170	176	NCX_Mj	protein	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
186	190	Ca2+	chemical	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
199	215	fully protonated	protein_state	Thermodynamic basis for the proposed mechanism of substrate control of the alternating-access transition of NCX. (a) Calculated free-energy landscapes for outward-facing NCX_Mj, for the Ca2+ and the fully protonated state.	FIG
4	15	free energy	evidence	The free energy is plotted as in Fig. 5.	FIG
4	8	Ca2+	chemical	For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6).	FIG
12	15	map	evidence	For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6).	FIG
144	147	map	evidence	For Ca2+, a map is shown in which a correction for the charge-transfer between the ion and the protein is introduced, alongside the uncorrected map (see Supplementary Notes 3-4 and Supplementary Fig. 5-6).	FIG
16	19	map	evidence	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
39	43	open	protein_state	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
66	75	semi-open	protein_state	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
80	88	occluded	protein_state	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
166	174	bound to	protein_state	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
218	221	Na+	chemical	The uncorrected map overstabilizes the open state relative to the semi-open and occluded because it also overestimates the cost of dehydration of the ion, once it is bound to the protein (this effect is negligible for Na+).	FIG
22	40	crystal structures	evidence	Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.	FIG
58	62	Ca2+	chemical	Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.	FIG
84	90	low pH	protein_state	Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.	FIG
95	102	high H+	protein_state	Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study.	FIG
4	29	Water-density isosurfaces	evidence	(b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps.	FIG
106	124	free-energy minima	evidence	(b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps.	FIG
132	148	free-energy maps	evidence	(b) Water-density isosurfaces analogous to those in Fig. 5 are shown for each of the major conformational free-energy minima in the free-energy maps.	FIG
4	8	Ca2+	chemical	The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima.	FIG
100	118	ion-binding region	site	The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima.	FIG
131	164	conformational free-energy minima	evidence	The Ca2+ ion is shown as a red sphere; the protein is shown as in Fig. 5. (c) Close-up views of the ion-binding region in the same conformational free-energy minima.	FIG
25	29	Ca2+	chemical	Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines).	FIG
34	39	water	chemical	Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines).	FIG
4	22	water-density maps	evidence	The water-density maps in (b) are shown here as a grey mesh.	FIG
7	15	occluded	protein_state	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
27	31	Ca2+	chemical	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
32	37	bound	protein_state	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
39	44	helix	structure_element	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
45	50	TM7ab	structure_element	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
83	97	fully occupied	protein_state	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
98	101	Na+	chemical	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
128	134	Ala206	residue_name_number	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
143	171	hydrogen-bonding interaction	bond_interaction	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
177	183	Ser210	residue_name_number	In the occluded state with Ca2+ bound, helix TM7ab bends in the same way as in the fully occupied Na+ state, as the carbonyl of Ala206 forms a hydrogen-bonding interaction with Ser210.	FIG
62	65	NCX	protein_type	Structural mechanism of extracellular forward ion exchange in NCX.	FIG
23	28	Ala47	residue_name_number	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
33	37	TM2b	structure_element	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
43	49	Ala206	residue_name_number	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
54	58	TM7b	structure_element	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
84	89	Glu54	residue_name_number	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
94	98	TM2c	structure_element	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
104	110	Glu213	residue_name_number	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
115	119	TM7c	structure_element	The carbonyl groups of Ala47 (on TM2b) and Ala206 (on TM7b), and the side chains of Glu54 (on TM2c) and Glu213 (on TM7c) are highlighted; these are four of the key residues for ion chelation and conformational changes.	FIG
39	53	gating helices	structure_element	The green open cylinders represent the gating helices TM1 and TM6.	FIG
54	57	TM1	structure_element	The green open cylinders represent the gating helices TM1 and TM6.	FIG
62	65	TM6	structure_element	The green open cylinders represent the gating helices TM1 and TM6.	FIG
32	50	crystal structures	evidence	Asterisks mark the states whose crystal structures have been determined in this study.	FIG
65	98	calculated free-energy landscapes	evidence	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG
120	131	Ca2+-loaded	protein_state	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG
132	146	outward-facing	protein_state	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG
147	155	occluded	protein_state	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG
170	178	unloaded	protein_state	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG
180	190	fully open	protein_state	These states and their connectivity can also be deduced from the calculated free-energy landscapes, which also reveal a Ca2+-loaded outward-facing occluded state, and an unloaded, fully open state.	FIG