PMC 20201223 pmc.key 4918766 NO-CC CODE no 0 0 10.1038/nsmb.3230 NIHMS779827 4918766 27183196 590 6 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: 599 surname:Liao;given-names:Jun surname:Marinelli;given-names:Fabrizio surname:Lee;given-names:ChangKeun surname:Huang;given-names:Yihe surname:Faraldo-Gómez;given-names:José D. surname:Jiang;given-names:Youxing TITLE front 23 2016 0 Mechanism of extracellular ion exchange and binding-site occlusion in the sodium-calcium exchanger 0.99964213 site cleaner0 2023-07-12T19:32:26Z SO: binding-site 0.9995489 protein_type cleaner0 2023-07-12T16:55:52Z MESH: sodium-calcium exchanger ABSTRACT abstract 99 Na+/Ca2+ exchangers utilize the Na+ electrochemical gradient across the plasma membrane to extrude intracellular Ca2+, and play a central role in Ca2+ homeostasis. 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. 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. 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. 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. 0.97095776 protein_type cleaner0 2023-07-12T15:31:56Z MESH: Na+/Ca2+ exchangers 0.9996849 chemical cleaner0 2023-07-12T15:31:21Z CHEBI: Na+ 0.9996843 chemical cleaner0 2023-07-12T15:31:36Z CHEBI: Ca2+ 0.9996164 chemical cleaner0 2023-07-12T15:31:38Z CHEBI: Ca2+ 0.9994154 experimental_method cleaner0 2023-07-12T16:59:23Z MESH: structural analysis 0.9996061 protein_type cleaner0 2023-07-12T16:55:56Z MESH: exchanger 0.99922407 species cleaner0 2023-07-12T15:32:42Z MESH: Methanococcus jannaschii protein PR: cleaner0 2023-07-12T15:33:37Z NCX_Mj 0.9994753 protein_state cleaner0 2023-07-12T19:41:51Z DUMMY: bound to 0.9996884 chemical cleaner0 2023-07-12T15:31:28Z CHEBI: Na+ 0.99972093 chemical cleaner0 2023-07-12T15:31:30Z CHEBI: Ca2+ 0.99971426 chemical cleaner0 2023-07-12T15:31:33Z CHEBI: Sr2+ 0.99966574 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo chemical CHEBI: cleaner0 2023-07-12T15:30:34Z Na+ chemical CHEBI: cleaner0 2023-07-12T15:30:58Z Ca2+ 0.9962363 evidence cleaner0 2023-07-12T19:18:10Z DUMMY: conformational free-energy landscape protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj protein_state DUMMY: cleaner0 2023-07-12T19:42:15Z ion-occupancy 0.99939805 experimental_method cleaner0 2023-07-12T16:59:30Z MESH: enhanced-sampling molecular-dynamics simulations 0.99956983 evidence cleaner0 2023-07-12T19:18:15Z DUMMY: crystal structures 0.9994036 experimental_method cleaner0 2023-07-12T16:59:40Z MESH: calculations 0.99183875 protein_state cleaner0 2023-07-12T19:42:18Z DUMMY: outward protein_state DUMMY: cleaner0 2023-07-12T19:42:39Z inward 0.99943435 chemical cleaner0 2023-07-12T15:31:40Z CHEBI: Na+ 0.9923915 chemical cleaner0 2023-07-12T15:31:42Z CHEBI: Ca2+ INTRO title_1 1257 Introduction INTRO paragraph 1270 Na+/Ca2+ exchangers (NCX) play physiologically essential roles in Ca2+ signaling and homeostasis. 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. This reaction is, however, inherently reversible, its direction being dictated only by the transmembrane electrochemical ion gradients . The mechanism of NCX proteins is therefore highly likely to be consistent with the alternating-access model of secondary-active transport. The basic functional unit for ion transport in NCX consists of ten membrane-spanning segments, comprising two homologous halves. 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). 0.99797577 protein_type cleaner0 2023-07-12T15:31:57Z MESH: Na+/Ca2+ exchangers 0.9995409 protein_type cleaner0 2023-07-12T15:33:12Z MESH: NCX 0.9995762 chemical cleaner0 2023-07-12T16:53:44Z CHEBI: Ca2+ 0.99959725 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9996245 chemical cleaner0 2023-07-12T16:53:47Z CHEBI: Ca2+ 0.9996516 chemical cleaner0 2023-07-12T16:53:50Z CHEBI: Na+ 0.9993729 chemical cleaner0 2023-07-12T16:53:53Z CHEBI: Na+ 0.9993702 chemical cleaner0 2023-07-12T16:53:56Z CHEBI: Ca2+ protein_type MESH: cleaner0 2023-07-12T15:33:13Z NCX 0.99957067 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9994636 structure_element cleaner0 2023-07-12T19:36:18Z SO: membrane-spanning segments 0.99272573 structure_element cleaner0 2023-07-12T19:36:35Z SO: halves 0.9981975 structure_element cleaner0 2023-07-12T19:36:37Z SO: halves 0.9995371 protein_state cleaner0 2023-07-12T19:42:43Z DUMMY: highly conserved 0.99968433 structure_element cleaner0 2023-07-12T19:36:51Z SO: α-repeat 0.99947935 taxonomy_domain cleaner0 2023-07-12T15:32:26Z DUMMY: eukaryotic 0.99958783 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.99172944 structure_element cleaner0 2023-07-12T19:36:48Z SO: halves structure_element SO: cleaner0 2023-07-12T19:37:14Z intracellular regulatory domain 0.9465155 protein_state cleaner0 2023-07-12T19:42:48Z DUMMY: absent 0.9995036 taxonomy_domain cleaner0 2023-07-12T15:32:31Z DUMMY: microbial 0.9995828 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX INTRO paragraph 2260 Despite a long history of physiological and functional studies, the molecular mechanism of NCX has been elusive, owing to the lack of structural information. Our recent atomic-resolution structure of NCX_Mj from Methanococcus jannaschii provided the first view of the basic functional unit of an NCX protein. 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). 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. 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. 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. An independent analysis based on molecular-dynamics simulations demonstrates that the structures capture mechanistically relevant states. 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. 0.99948573 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9964077 evidence cleaner0 2023-07-12T19:18:28Z DUMMY: structure protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99919784 species cleaner0 2023-07-12T15:32:44Z MESH: Methanococcus jannaschii 0.99958605 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.99796706 evidence cleaner0 2023-07-12T19:18:31Z DUMMY: structure 0.9991055 protein_type cleaner0 2023-07-12T16:56:01Z MESH: exchanger 0.9994218 protein_state cleaner0 2023-07-12T15:36:59Z DUMMY: outward-facing 0.9995973 site cleaner0 2023-07-12T19:32:32Z SO: ion-binding sites site SO: cleaner0 2023-07-12T15:35:34Z internal 0.9518084 site cleaner0 2023-07-12T15:35:41Z SO: Sint site SO: cleaner0 2023-07-12T15:35:54Z external 0.9600976 site cleaner0 2023-07-12T15:36:02Z SO: Sext 0.99758303 site cleaner0 2023-07-12T15:36:15Z SO: Ca2+-binding 0.9913696 site cleaner0 2023-07-12T15:36:28Z SO: SCa site SO: cleaner0 2023-07-12T15:36:41Z middle 0.99600405 site cleaner0 2023-07-12T15:36:47Z SO: Smid 0.8781682 protein_state cleaner0 2023-07-12T19:43:07Z DUMMY: occluded from 0.99951184 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99919754 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9993592 evidence cleaner0 2023-07-12T19:18:33Z DUMMY: structures 0.9994232 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing 0.9995788 protein_state cleaner0 2023-07-12T15:37:05Z DUMMY: wild-type protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9991128 protein_state cleaner0 2023-07-12T15:37:10Z DUMMY: in complex with 0.99974465 chemical cleaner0 2023-07-12T15:37:15Z CHEBI: Na+ 0.9997424 chemical cleaner0 2023-07-12T15:37:18Z CHEBI: Ca2+ 0.99974084 chemical cleaner0 2023-07-12T15:37:20Z CHEBI: Sr2+ 0.9977774 evidence cleaner0 2023-07-12T19:18:36Z DUMMY: structures 0.9994773 experimental_method cleaner0 2023-07-12T16:59:50Z MESH: molecular-dynamics simulations 0.9992453 evidence cleaner0 2023-07-12T19:18:38Z DUMMY: structures 0.997436 experimental_method cleaner0 2023-07-12T16:59:53Z MESH: calculations 0.9993314 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing 0.9994099 protein_type cleaner0 2023-07-12T16:56:05Z MESH: exchanger 0.9994784 protein_state cleaner0 2023-07-12T15:37:25Z DUMMY: inward-facing RESULTS title_1 3934 Results RESULTS title_2 3942 Extracellular Na+ binding 0.9997164 chemical cleaner0 2023-07-12T15:37:33Z CHEBI: Na+ RESULTS paragraph 3968 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. To conclusively clarify this assignment, we first set out to examine the Na+ occupancy of these sites without Ca2+. Crystals were grown in 150 mM NaCl using the lipidic cubic phase (LCP) technique. The crystallization solutions around the LCP droplets were then slowly replaced by solutions containing different concentrations of NaCl and EGTA (Methods). 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). 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). 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). The Na+ occupation at SCa, compounded with the expected 3Na+:1Ca2+ stoichiometry, implies our previous assignment of the Smid site must be re-evaluated. Indeed, two observations indicate that a water molecule rather than a Na+ ion occupies Smid, as was predicted in a recent simulation study. First, the electron density at Smid does not depend significantly on the Na+ concentration. Second, the protein coordination geometry at Smid is clearly suboptimal for Na+ (Supplementary Fig. 1d). 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. 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. 0.99960774 site cleaner0 2023-07-12T19:32:39Z SO: central binding sites protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.999382 evidence cleaner0 2023-07-12T19:18:44Z DUMMY: structure 0.9995949 chemical cleaner0 2023-07-12T15:37:44Z CHEBI: Na+ 0.999593 chemical cleaner0 2023-07-12T15:37:47Z CHEBI: Ca2+ 0.9987638 evidence cleaner0 2023-07-12T19:18:48Z DUMMY: crystals 0.9990246 chemical cleaner0 2023-07-12T15:37:52Z CHEBI: Na+ 0.9995868 chemical cleaner0 2023-07-12T15:37:54Z CHEBI: Ca2+ 0.73599255 evidence cleaner0 2023-07-12T17:00:39Z DUMMY: Crystals 0.9994336 chemical cleaner0 2023-07-12T15:37:49Z CHEBI: NaCl 0.99660176 experimental_method cleaner0 2023-07-12T17:00:43Z MESH: lipidic cubic phase 0.9660596 experimental_method cleaner0 2023-07-12T15:55:37Z MESH: LCP experimental_method MESH: cleaner0 2023-07-12T15:55:37Z LCP 0.9997323 chemical cleaner0 2023-07-12T15:37:57Z CHEBI: NaCl 0.9997514 chemical cleaner0 2023-07-12T15:37:59Z CHEBI: EGTA 0.99931455 experimental_method cleaner0 2023-07-12T17:00:46Z MESH: X-ray diffraction 0.83355284 evidence cleaner0 2023-07-12T17:01:02Z DUMMY: crystals 0.9719577 chemical cleaner0 2023-07-12T15:38:01Z CHEBI: Na+ 0.99949425 evidence cleaner0 2023-07-12T19:18:58Z DUMMY: electron-density distribution 0.8793344 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.96133804 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.96933526 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9910625 chemical cleaner0 2023-07-12T15:38:14Z CHEBI: Na+ 0.9995426 experimental_method cleaner0 2023-07-12T17:01:06Z MESH: Occupancy refinement 0.9994962 chemical cleaner0 2023-07-12T15:38:04Z CHEBI: Na+ 0.9791804 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.98667175 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.9993845 chemical cleaner0 2023-07-12T15:40:15Z CHEBI: Na+ 0.9625437 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9994303 chemical cleaner0 2023-07-12T15:38:06Z CHEBI: Na+ 0.8532711 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9990901 evidence cleaner0 2023-07-12T19:19:01Z DUMMY: density 0.99952185 chemical cleaner0 2023-07-12T15:38:57Z CHEBI: Na+ 0.5988609 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.99950314 chemical cleaner0 2023-07-12T15:38:09Z CHEBI: Na+ 0.97880304 chemical cleaner0 2023-07-12T15:38:11Z CHEBI: Na+ 0.97307587 site cleaner0 2023-07-12T15:36:29Z SO: SCa chemical CHEBI: cleaner0 2023-07-12T16:52:45Z Na+ chemical CHEBI: cleaner0 2023-07-12T16:52:57Z Ca2+ site SO: cleaner0 2023-07-12T15:36:47Z Smid 0.9997707 chemical cleaner0 2023-07-12T15:38:52Z CHEBI: water 0.99955773 chemical cleaner0 2023-07-12T15:38:47Z CHEBI: Na+ 0.70465773 site cleaner0 2023-07-12T15:36:47Z SO: Smid 0.94124186 experimental_method cleaner0 2023-07-12T17:01:10Z MESH: simulation 0.9995507 evidence cleaner0 2023-07-12T19:19:03Z DUMMY: electron density 0.5928501 site cleaner0 2023-07-12T15:36:47Z SO: Smid 0.99941397 chemical cleaner0 2023-07-12T15:38:49Z CHEBI: Na+ 0.5664469 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.9995431 chemical cleaner0 2023-07-12T15:38:37Z CHEBI: Na+ 0.99977714 chemical cleaner0 2023-07-12T15:38:54Z CHEBI: water 0.49353516 site cleaner0 2023-07-12T15:36:48Z SO: Smid bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bonds 0.9993975 protein_state cleaner0 2023-07-12T19:43:19Z DUMMY: highly conserved 0.99989235 residue_name_number cleaner0 2023-07-12T15:40:29Z DUMMY: Glu54 0.9998932 residue_name_number cleaner0 2023-07-12T15:40:34Z DUMMY: Glu213 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinate 0.99929106 chemical cleaner0 2023-07-12T16:54:02Z CHEBI: Na+ 0.92876875 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.98581064 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.9932243 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.999899 residue_name_number cleaner0 2023-07-12T15:40:30Z DUMMY: Glu54 0.99989676 residue_name_number cleaner0 2023-07-12T15:40:34Z DUMMY: Glu213 0.9998975 residue_name_number cleaner0 2023-07-12T15:40:40Z DUMMY: Asp240 0.8588451 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.99919647 residue_name cleaner0 2023-07-12T15:40:22Z SO: Asn 0.99947625 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.5852442 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.97600526 protein_state cleaner0 2023-07-12T19:43:29Z DUMMY: protonated 0.9843647 experimental_method cleaner0 2023-07-12T17:01:14Z MESH: simulation RESULTS title_2 5981 Na+-dependent conformational change 0.99453074 chemical cleaner0 2023-07-12T16:54:06Z CHEBI: Na+ RESULTS paragraph 6017 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). 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). TM7b occludes the four central binding sites from the external solution, with the backbone carbonyl of Ala206 coordinating the Na+ ion (Fig. 2b-d). 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). TM7a also forms hydrophobic contacts with the C-terminal half of TM6. These contacts are absent in the structure with Na+ at Sext, in which there is an open gap between the two helices (Fig. 2b). 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. 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). Thus, the structures at high and low Na+ concentrations represent the outward-facing occluded and partially open states, respectively. This conformational change is dependent on the Na+ occupancy of Sext and occurs when Na+ already occupies Sint and SCa. 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. This structurally-derived Na+ affinity agrees well with the external Na+ concentration required for NCX activation in eukaryotes. 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. protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99961394 evidence cleaner0 2023-07-12T19:19:09Z DUMMY: structures 0.9994112 chemical cleaner0 2023-07-12T15:41:06Z CHEBI: Na+ 0.9996445 chemical cleaner0 2023-07-12T15:41:09Z CHEBI: Na+ 0.8975322 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9996025 chemical cleaner0 2023-07-12T15:41:11Z CHEBI: Na+ 0.97438496 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.63517 protein_state cleaner0 2023-07-12T19:43:51Z DUMMY: high 0.99961746 structure_element cleaner0 2023-07-12T19:37:23Z SO: N-terminal half 0.9997969 structure_element cleaner0 2023-07-12T19:37:26Z SO: TM7 0.9983444 structure_element cleaner0 2023-07-12T19:37:29Z SO: short helices 0.9997923 structure_element cleaner0 2023-07-12T15:41:22Z SO: TM7a 0.999793 structure_element cleaner0 2023-07-12T15:41:27Z SO: TM7b 0.9997937 structure_element cleaner0 2023-07-12T15:41:27Z SO: TM7b 0.99953514 site cleaner0 2023-07-12T19:32:45Z SO: central binding sites 0.9999001 residue_name_number cleaner0 2023-07-12T15:41:02Z DUMMY: Ala206 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinating 0.99943876 chemical cleaner0 2023-07-12T15:41:14Z CHEBI: Na+ 0.5882469 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9966037 protein_state cleaner0 2023-07-12T19:44:00Z DUMMY: empty 0.9698598 protein_state cleaner0 2023-07-12T19:44:04Z DUMMY: low 0.99899304 chemical cleaner0 2023-07-12T15:41:17Z CHEBI: Na+ 0.9997869 structure_element cleaner0 2023-07-12T15:41:21Z SO: TM7a 0.9997874 structure_element cleaner0 2023-07-12T15:41:26Z SO: TM7b 0.7902682 structure_element cleaner0 2023-07-12T19:37:33Z SO: helix 0.9999031 residue_name_number cleaner0 2023-07-12T15:41:03Z DUMMY: Ala206 0.99979156 structure_element cleaner0 2023-07-12T15:41:22Z SO: TM7a bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrophobic contacts 0.9996227 structure_element cleaner0 2023-07-12T19:37:42Z SO: C-terminal half 0.99979216 structure_element cleaner0 2023-07-12T15:41:32Z SO: TM6 0.99959534 evidence cleaner0 2023-07-12T19:19:13Z DUMMY: structure 0.99957615 chemical cleaner0 2023-07-12T15:41:53Z CHEBI: Na+ 0.988373 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9690582 structure_element cleaner0 2023-07-12T19:38:00Z SO: helices 0.99979764 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.99980515 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9991763 protein_type cleaner0 2023-07-12T16:56:11Z MESH: transporter 0.99814576 protein_state cleaner0 2023-07-12T15:37:26Z DUMMY: inward-facing 0.9997955 structure_element cleaner0 2023-07-12T15:41:46Z SO: TM7ab 0.95291543 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.98256505 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.5734766 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.9609776 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9988845 protein_state cleaner0 2023-07-12T19:44:24Z DUMMY: occluded 0.9995701 evidence cleaner0 2023-07-12T19:19:15Z DUMMY: structures 0.9383757 protein_state cleaner0 2023-07-12T19:44:28Z DUMMY: high 0.95977247 protein_state cleaner0 2023-07-12T19:44:31Z DUMMY: low 0.9989848 chemical cleaner0 2023-07-12T16:54:13Z CHEBI: Na+ 0.9461078 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing 0.9996234 protein_state cleaner0 2023-07-12T19:44:37Z DUMMY: occluded 0.9722863 protein_state cleaner0 2023-07-12T19:44:41Z DUMMY: partially open 0.99948144 chemical cleaner0 2023-07-12T15:41:59Z CHEBI: Na+ 0.61460423 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9996245 chemical cleaner0 2023-07-12T15:42:01Z CHEBI: Na+ 0.9835104 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9705 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.9994615 experimental_method cleaner0 2023-07-12T17:01:20Z MESH: crystallographic titration experiment 0.9962109 evidence cleaner0 2023-07-12T19:19:46Z DUMMY: K1/2 0.9995229 chemical cleaner0 2023-07-12T15:42:03Z CHEBI: Na+ 0.5956819 site cleaner0 2023-07-12T15:36:03Z SO: Sext protein_state DUMMY: cleaner0 2023-07-12T19:45:09Z partially occupied protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9996499 evidence cleaner0 2023-07-12T19:19:49Z DUMMY: crystal 0.9996519 protein_state cleaner0 2023-07-12T19:45:13Z DUMMY: occluded 0.75721264 protein_state cleaner0 2023-07-12T19:45:16Z DUMMY: partially open 0.972502 evidence cleaner0 2023-07-12T19:19:52Z DUMMY: Na+ affinity 0.99967176 chemical cleaner0 2023-07-12T15:42:10Z CHEBI: Na+ 0.999126 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9995185 taxonomy_domain cleaner0 2023-07-12T16:53:35Z DUMMY: eukaryotes 0.99950695 chemical cleaner0 2023-07-12T15:42:06Z CHEBI: Na+ 0.99931836 protein_type cleaner0 2023-07-12T16:56:14Z MESH: transporter 0.9992928 evidence cleaner0 2023-07-12T19:19:54Z DUMMY: Hill coefficient 0.9994451 chemical cleaner0 2023-07-12T15:42:08Z CHEBI: Na+ 0.99952483 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.9994234 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX RESULTS title_2 8043 Extracellular Ca2+ and Sr2+ binding and their competition with Na+ 0.9997628 chemical cleaner0 2023-07-12T15:42:21Z CHEBI: Ca2+ 0.9997602 chemical cleaner0 2023-07-12T15:42:24Z CHEBI: Sr2+ 0.9997529 chemical cleaner0 2023-07-12T15:42:26Z CHEBI: Na+ RESULTS paragraph 8110 To determine how Ca2+ binds to NCX_Mj and competes with Na+, we first titrated the crystals with Sr2+ (Methods). Sr2+ is transported by NCX similarly to Ca2+ , and is distinguishable from Na+ by its greater electron-density intensity. 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). The Sr2+-loaded NCX_Mj structure adopts the partially open conformation observed at low Na+ concentrations. Binding of Sr2+, however, excludes Na+ entirely. 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). 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. 0.9996861 chemical cleaner0 2023-07-12T15:42:29Z CHEBI: Ca2+ protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9995922 chemical cleaner0 2023-07-12T15:42:41Z CHEBI: Na+ experimental_method MESH: cleaner0 2023-07-12T17:01:45Z titrated the crystals 0.9996915 chemical cleaner0 2023-07-12T15:42:43Z CHEBI: Sr2+ 0.999686 chemical cleaner0 2023-07-12T15:42:46Z CHEBI: Sr2+ 0.9997907 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9996725 chemical cleaner0 2023-07-12T15:42:54Z CHEBI: Ca2+ 0.9996459 chemical cleaner0 2023-07-12T15:42:52Z CHEBI: Na+ 0.9994124 evidence cleaner0 2023-07-12T19:20:00Z DUMMY: electron-density intensity experimental_method MESH: cleaner0 2023-07-12T17:02:07Z Protein crystals soaked 0.9996846 chemical cleaner0 2023-07-12T15:42:50Z CHEBI: Sr2+ 0.9996888 chemical cleaner0 2023-07-12T15:42:48Z CHEBI: Na+ 0.99926555 evidence cleaner0 2023-07-12T19:20:03Z DUMMY: electron-density peak 0.5260014 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.999609 chemical cleaner0 2023-07-12T15:42:57Z CHEBI: Sr2+ 0.99643576 protein_state cleaner0 2023-07-12T15:43:47Z DUMMY: Sr2+-loaded protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99959177 evidence cleaner0 2023-07-12T19:20:05Z DUMMY: structure 0.9993584 protein_state cleaner0 2023-07-12T19:45:23Z DUMMY: partially open 0.9996485 chemical cleaner0 2023-07-12T15:43:09Z CHEBI: Na+ 0.99964035 chemical cleaner0 2023-07-12T15:43:11Z CHEBI: Sr2+ 0.9994898 chemical cleaner0 2023-07-12T15:43:13Z CHEBI: Na+ 0.99958897 experimental_method cleaner0 2023-07-12T19:13:20Z MESH: Crystal titrations 0.84637284 experimental_method cleaner0 2023-07-12T19:13:24Z MESH: decreasing 0.99964565 chemical cleaner0 2023-07-12T15:43:15Z CHEBI: Sr2+ 0.95324624 experimental_method cleaner0 2023-07-12T19:13:26Z MESH: increasing 0.9996711 chemical cleaner0 2023-07-12T15:43:17Z CHEBI: Na+ 0.99968195 chemical cleaner0 2023-07-12T15:43:33Z CHEBI: Sr2+ protein_state DUMMY: cleaner0 2023-07-12T15:37:00Z outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99965817 chemical cleaner0 2023-07-12T15:43:36Z CHEBI: Na+ 0.999688 chemical cleaner0 2023-07-12T15:44:05Z CHEBI: Na+ 0.9996972 chemical cleaner0 2023-07-12T16:54:20Z CHEBI: Sr2+ 0.9996567 chemical cleaner0 2023-07-12T15:44:03Z CHEBI: Na+ 0.99961853 chemical cleaner0 2023-07-12T15:44:01Z CHEBI: Sr2+ protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99837416 protein_state cleaner0 2023-07-12T15:43:52Z DUMMY: Na+-loaded 0.99951965 protein_state cleaner0 2023-07-12T15:43:57Z DUMMY: fully occluded RESULTS paragraph 9050 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). 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). The partial Ca2+ occupancy at Smid is likely caused by Asp240, which flanks this site and can in principle coordinate Ca2+. Previous functional and computational studies, however, indicate Asp240 becomes protonated during transport. Indeed, in most NCX proteins Asp240 is substituted by Asn, which would likely weaken or abrogate Ca2+ binding to Smid. SCa is therefore the functional Ca2+ site. 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). 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. 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). 0.83672285 experimental_method cleaner0 2023-07-12T19:13:30Z MESH: titration experiments 0.99967515 chemical cleaner0 2023-07-12T15:44:35Z CHEBI: Ca2+ 0.9996866 chemical cleaner0 2023-07-12T15:44:38Z CHEBI: Sr2+ protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9995885 evidence cleaner0 2023-07-12T19:20:08Z DUMMY: electron density distribution 0.99763757 experimental_method cleaner0 2023-07-12T19:13:41Z MESH: crystals soaked in 0.79457146 protein_state cleaner0 2023-07-12T19:45:41Z DUMMY: high 0.99966097 chemical cleaner0 2023-07-12T15:44:41Z CHEBI: Ca2+ 0.90524435 protein_state cleaner0 2023-07-12T19:45:44Z DUMMY: low 0.9996723 chemical cleaner0 2023-07-12T15:44:44Z CHEBI: Na+ 0.9996927 chemical cleaner0 2023-07-12T15:44:46Z CHEBI: Ca2+ 0.7841132 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.8154444 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.8182291 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.99968505 chemical cleaner0 2023-07-12T15:44:49Z CHEBI: Ca2+ 0.99975723 chemical cleaner0 2023-07-12T15:45:03Z CHEBI: water bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinating protein_state DUMMY: cleaner0 2023-07-12T19:45:57Z partial 0.99937546 chemical cleaner0 2023-07-12T15:44:59Z CHEBI: Ca2+ protein_state DUMMY: cleaner0 2023-07-12T19:46:08Z occupancy 0.611224 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.9999026 residue_name_number cleaner0 2023-07-12T15:40:41Z DUMMY: Asp240 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinate 0.99966395 chemical cleaner0 2023-07-12T15:45:05Z CHEBI: Ca2+ 0.9973443 experimental_method cleaner0 2023-07-12T19:13:46Z MESH: functional and computational studies 0.9998989 residue_name_number cleaner0 2023-07-12T15:40:41Z DUMMY: Asp240 protein_state DUMMY: cleaner0 2023-07-12T19:46:31Z protonated protein_type MESH: cleaner0 2023-07-12T15:33:13Z NCX 0.9998833 residue_name_number cleaner0 2023-07-12T15:40:41Z DUMMY: Asp240 experimental_method MESH: cleaner0 2023-07-12T19:46:42Z substituted 0.9993026 residue_name cleaner0 2023-07-12T16:52:19Z SO: Asn 0.99957514 chemical cleaner0 2023-07-12T15:45:14Z CHEBI: Ca2+ 0.81406647 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.580456 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.9995466 site cleaner0 2023-07-12T19:32:50Z SO: Ca2+ site 0.9997176 chemical cleaner0 2023-07-12T15:45:36Z CHEBI: Sr2+ 0.9997066 chemical cleaner0 2023-07-12T15:45:34Z CHEBI: Ca2+ evidence DUMMY: cleaner0 2023-07-12T19:20:37Z affinity protein_state DUMMY: cleaner0 2023-07-12T15:37:00Z outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99970555 chemical cleaner0 2023-07-12T15:45:17Z CHEBI: Na+ evidence DUMMY: cleaner0 2023-07-12T19:47:02Z physiological and biochemical data 0.99922514 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.99955577 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj evidence DUMMY: cleaner0 2023-07-12T15:46:08Z Ca2+ affinity 0.99957705 experimental_method cleaner0 2023-07-12T19:13:51Z MESH: crystallographic titration assay 0.99969375 chemical cleaner0 2023-07-12T16:54:26Z CHEBI: Ca2+ evidence DUMMY: cleaner0 2023-07-12T19:21:05Z affinity evidence DUMMY: cleaner0 2023-07-12T15:46:28Z Ca2+ affinities 0.99935716 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9995334 evidence cleaner0 2023-07-12T19:20:17Z DUMMY: Km protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99948776 evidence cleaner0 2023-07-12T19:20:20Z DUMMY: Km RESULTS paragraph 10498 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. 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. 0.9993302 experimental_method cleaner0 2023-07-12T19:14:09Z MESH: crystal titration experiments 0.8698213 site cleaner0 2023-07-12T19:32:57Z SO: binding sites 0.9966097 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99553925 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9974414 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.99956393 chemical cleaner0 2023-07-12T15:47:05Z CHEBI: Na+ 0.99726665 site cleaner0 2023-07-12T15:36:29Z SO: SCa 0.99862236 chemical cleaner0 2023-07-12T15:47:08Z CHEBI: Ca2+ 0.9997227 chemical cleaner0 2023-07-12T15:47:10Z CHEBI: Na+ 0.9992256 experimental_method cleaner0 2023-07-12T19:14:14Z MESH: simulation 0.99973094 chemical cleaner0 2023-07-12T15:47:12Z CHEBI: Sr2+ 0.9943116 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.9997344 chemical cleaner0 2023-07-12T15:47:17Z CHEBI: Ca2+ 0.99609 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.99974436 chemical cleaner0 2023-07-12T15:47:15Z CHEBI: water 0.9996326 site cleaner0 2023-07-12T19:33:00Z SO: ion-binding sites protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99972224 chemical cleaner0 2023-07-12T15:47:19Z CHEBI: Na+ 0.99973035 chemical cleaner0 2023-07-12T15:47:21Z CHEBI: Na+ 0.9997362 chemical cleaner0 2023-07-12T15:47:24Z CHEBI: Ca2+ 0.9997355 chemical cleaner0 2023-07-12T15:47:26Z CHEBI: Sr2+ 0.99949324 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.99963903 protein_type cleaner0 2023-07-12T16:56:19Z MESH: exchangers RESULTS title_2 11126 A structure of NCX_Mj without Na+ or Ca2+ bound 0.99962306 evidence cleaner0 2023-07-12T19:23:58Z DUMMY: structure protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99943596 protein_state cleaner0 2023-07-12T19:47:20Z DUMMY: without 0.99964356 chemical cleaner0 2023-07-12T15:47:49Z CHEBI: Na+ 0.99968123 chemical cleaner0 2023-07-12T15:47:51Z CHEBI: Ca2+ 0.5086934 protein_state cleaner0 2023-07-12T19:47:22Z DUMMY: bound RESULTS paragraph 11174 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). 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). This structure is similar to the partially open structure with two Na+ or either one Ca2+ or one Sr2+ ion, with two noticeable differences. 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. 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). 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. This apo structure might therefore represent the unloaded, open state of outward-facing NCX_Mj. Alternatively, this structure might capture a fully protonated state of the transporter, to which Na+ and Ca2+ cannot bind. Such interpretation would be consistent with the computer simulations reported below. Indeed, transport assays of NCX_Mj have shown that even in the presence of Na+ or Ca2+, low pH inactivates the transport cycle. 0.99966514 protein_state cleaner0 2023-07-12T15:48:05Z DUMMY: apo 0.9870203 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9996991 chemical cleaner0 2023-07-12T15:48:28Z CHEBI: Na+ 0.9996948 chemical cleaner0 2023-07-12T15:48:30Z CHEBI: Ca2+ 0.99965405 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.9995609 evidence cleaner0 2023-07-12T19:24:30Z DUMMY: structure protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99966407 experimental_method cleaner0 2023-07-12T19:14:19Z MESH: crystallizing 0.6531592 protein_state cleaner0 2023-07-12T19:47:30Z DUMMY: lower pH 0.9994112 protein_state cleaner0 2023-07-12T19:47:33Z DUMMY: absence of 0.99970734 chemical cleaner0 2023-07-12T15:48:33Z CHEBI: Na+ 0.9994924 evidence cleaner0 2023-07-12T19:24:26Z DUMMY: structure 0.9994571 protein_state cleaner0 2023-07-12T19:47:37Z DUMMY: partially open 0.99934405 evidence cleaner0 2023-07-12T19:24:28Z DUMMY: structure 0.99962556 chemical cleaner0 2023-07-12T15:48:36Z CHEBI: Na+ 0.9996591 chemical cleaner0 2023-07-12T15:48:38Z CHEBI: Ca2+ 0.9996749 chemical cleaner0 2023-07-12T15:48:41Z CHEBI: Sr2+ 0.9998122 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.9994032 structure_element cleaner0 2023-07-12T19:38:06Z SO: extracellular half 0.99981755 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.999819 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9996153 site cleaner0 2023-07-12T19:33:04Z SO: binding sites 0.9999018 residue_name_number cleaner0 2023-07-12T15:40:30Z DUMMY: Glu54 0.9998983 residue_name_number cleaner0 2023-07-12T15:40:34Z DUMMY: Glu213 0.9996121 site cleaner0 2023-07-12T19:33:07Z SO: binding sites bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z ion coordination protein_state DUMMY: cleaner0 2023-07-12T19:48:06Z fully Na+-loaded 0.99958795 evidence cleaner0 2023-07-12T19:24:23Z DUMMY: structure 0.9996027 site cleaner0 2023-07-12T19:33:09Z SO: binding sites protein_state DUMMY: cleaner0 2023-07-12T19:48:24Z fully accessible 0.999437 evidence cleaner0 2023-07-12T19:24:15Z DUMMY: electron density 0.99965954 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.9995679 evidence cleaner0 2023-07-12T19:24:18Z DUMMY: structure 0.99966276 protein_state cleaner0 2023-07-12T19:48:46Z DUMMY: unloaded 0.9996495 protein_state cleaner0 2023-07-12T19:48:49Z DUMMY: open 0.99581814 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99950194 evidence cleaner0 2023-07-12T19:24:20Z DUMMY: structure 0.99955976 protein_state cleaner0 2023-07-12T19:48:53Z DUMMY: fully protonated 0.9995828 protein_type cleaner0 2023-07-12T16:56:22Z MESH: transporter 0.999714 chemical cleaner0 2023-07-12T15:49:00Z CHEBI: Na+ 0.99970317 chemical cleaner0 2023-07-12T15:49:02Z CHEBI: Ca2+ 0.99953717 experimental_method cleaner0 2023-07-12T19:14:23Z MESH: computer simulations 0.99950814 experimental_method cleaner0 2023-07-12T19:14:26Z MESH: transport assays protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.98821247 protein_state cleaner0 2023-07-12T19:48:56Z DUMMY: presence of 0.9997245 chemical cleaner0 2023-07-12T15:49:04Z CHEBI: Na+ 0.99972236 chemical cleaner0 2023-07-12T15:49:07Z CHEBI: Ca2+ 0.94008267 protein_state cleaner0 2023-07-12T19:48:59Z DUMMY: low pH protein_state DUMMY: cleaner0 2023-07-12T19:49:11Z inactivates RESULTS title_2 12609 Ion occupancy determines the free-energy landscape of NCX_Mj protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj RESULTS paragraph 12670 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. 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. The reason why only specific occupancy states permit this transition in a given system, thereby determining its biological function, remains unclear. 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. This computational analysis was based solely on the published structure of NCX_Mj, independently of the crystallographic studies described above. 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. 0.9995427 protein_type cleaner0 2023-07-12T16:56:30Z MESH: secondary-active transporters 0.99890316 protein_state cleaner0 2023-07-12T19:49:25Z DUMMY: outward- 0.9992698 protein_state cleaner0 2023-07-12T19:49:28Z DUMMY: inward-open 0.99877745 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9996822 chemical cleaner0 2023-07-12T16:54:39Z CHEBI: Na+ 0.99968994 chemical cleaner0 2023-07-12T16:54:43Z CHEBI: Ca2+ 0.9989229 protein_type cleaner0 2023-07-12T16:56:33Z MESH: antiporter 0.9994962 protein_type cleaner0 2023-07-12T16:56:36Z MESH: symporters 0.5239756 protein_state cleaner0 2023-07-12T19:49:39Z DUMMY: bound 0.9996517 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.7642773 evidence cleaner0 2023-07-12T19:18:11Z DUMMY: conformational free-energy landscape protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99953496 experimental_method cleaner0 2023-07-12T19:14:31Z MESH: molecular dynamics 0.9996784 experimental_method cleaner0 2023-07-12T19:14:33Z MESH: MD 0.99583435 experimental_method cleaner0 2023-07-12T19:14:36Z MESH: simulations 0.99943846 evidence cleaner0 2023-07-12T19:25:12Z DUMMY: structure protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9952549 experimental_method cleaner0 2023-07-12T19:14:38Z MESH: crystallographic studies 0.9989248 evidence cleaner0 2023-07-12T19:25:14Z DUMMY: structures protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj RESULTS paragraph 13965 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. 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). That is, Na+ ions occupy Sext, SCa, and Sint, while D240 is protonated and a water molecule occupies Smid. 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. The Na+ ions at SCa and Sint were displaced subsequently, and an analogous simulation was then carried out. These initial simulations revealed noticeable changes in the transporter, consistent with those observed in the new crystal structures. The most notable change upon displacement of Na+ from Sext was the straightening of TM7ab (Fig. 4a). 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). 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). 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. Displacement of Na+ from SCa and Sint induces further changes (Fig. 4c). 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). TM1 and TM6 also slide further towards the membrane center, relative to the outward-occluded state (Fig. 4c). Together, these changes open a second aqueous channel leading directly into SCa and Sint (Fig. 4f, Fig. 4h-i). The transporter thus becomes fully outward-open. experimental_method MESH: cleaner0 2023-07-12T15:50:21Z MD simulations protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.9995326 evidence cleaner0 2023-07-12T19:25:16Z DUMMY: structure 0.9996343 site cleaner0 2023-07-12T19:33:14Z SO: ion-binding sites 0.9993299 experimental_method cleaner0 2023-07-12T19:14:45Z MESH: simulated 0.99959403 protein_state cleaner0 2023-07-12T15:51:40Z DUMMY: outward-occluded protein_state DUMMY: cleaner0 2023-07-12T19:25:43Z high-Na+ 0.99953187 evidence cleaner0 2023-07-12T19:25:19Z DUMMY: crystal structure 0.99958265 chemical cleaner0 2023-07-12T15:50:41Z CHEBI: Na+ 0.9526997 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9144155 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9161612 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99989283 residue_name_number cleaner0 2023-07-12T15:50:33Z DUMMY: D240 0.99949884 protein_state cleaner0 2023-07-12T19:49:45Z DUMMY: protonated 0.9997693 chemical cleaner0 2023-07-12T15:50:46Z CHEBI: water 0.9149831 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.9996277 chemical cleaner0 2023-07-12T15:50:48Z CHEBI: Na+ 0.92753726 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9996302 chemical cleaner0 2023-07-12T15:50:50Z CHEBI: Na+ 0.940987 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.93242574 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99955755 experimental_method cleaner0 2023-07-12T19:14:51Z MESH: simulation 0.9996125 experimental_method cleaner0 2023-07-12T19:14:54Z MESH: simulations 0.99883944 protein_type cleaner0 2023-07-12T16:56:40Z MESH: transporter 0.9995551 evidence cleaner0 2023-07-12T19:25:47Z DUMMY: crystal structures 0.999622 chemical cleaner0 2023-07-12T15:50:53Z CHEBI: Na+ 0.8926937 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.99979156 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.99964416 chemical cleaner0 2023-07-12T15:50:56Z CHEBI: Na+ 0.9992055 protein_state cleaner0 2023-07-12T19:49:53Z DUMMY: bound 0.9997819 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.99874955 structure_element cleaner0 2023-07-12T19:38:12Z SO: α-helical fragments bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bonds 0.9999049 residue_name_number cleaner0 2023-07-12T15:51:02Z DUMMY: F202 0.9999025 residue_name_number cleaner0 2023-07-12T15:51:06Z DUMMY: A206 0.9999051 residue_name_number cleaner0 2023-07-12T15:51:11Z DUMMY: T203 0.999905 residue_name_number cleaner0 2023-07-12T15:51:16Z DUMMY: F207 0.7172006 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9996556 chemical cleaner0 2023-07-12T15:51:24Z CHEBI: Na+ 0.99990416 residue_name_number cleaner0 2023-07-12T15:51:07Z DUMMY: A206 0.9238429 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.99892056 protein_state cleaner0 2023-07-12T19:50:09Z DUMMY: empty 0.999783 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.9995744 structure_element cleaner0 2023-07-12T19:38:15Z SO: α-helix 0.9997913 structure_element cleaner0 2023-07-12T15:54:55Z SO: TM3 0.99978286 structure_element cleaner0 2023-07-12T19:38:20Z SO: TM7 0.9997751 chemical cleaner0 2023-07-12T15:51:22Z CHEBI: water 0.9077475 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.99258596 protein_type cleaner0 2023-07-12T16:56:45Z MESH: transporter protein_state DUMMY: cleaner0 2023-07-12T19:50:41Z no longer occluded 0.9996489 chemical cleaner0 2023-07-12T15:51:27Z CHEBI: Na+ 0.92651975 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9055643 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9997913 structure_element cleaner0 2023-07-12T19:38:22Z SO: TM7 0.999793 structure_element cleaner0 2023-07-12T19:38:26Z SO: TM2 0.99963284 chemical cleaner0 2023-07-12T15:51:31Z CHEBI: Na+ 0.84332246 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9997956 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9997918 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.9996016 protein_state cleaner0 2023-07-12T15:51:39Z DUMMY: outward-occluded 0.993948 site cleaner0 2023-07-12T19:33:18Z SO: aqueous channel 0.94592416 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.93095547 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.9969367 protein_type cleaner0 2023-07-12T16:56:48Z MESH: transporter protein_state DUMMY: cleaner0 2023-07-12T15:52:01Z fully outward-open RESULTS paragraph 16116 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). 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+. 0.9995615 protein_type cleaner0 2023-07-12T16:56:51Z MESH: exchanger 0.7485032 experimental_method cleaner0 2023-07-12T19:15:10Z MESH: MD calculations 0.99957466 protein_state cleaner0 2023-07-12T15:51:40Z DUMMY: outward-occluded 0.9760635 protein_state cleaner0 2023-07-12T15:52:00Z DUMMY: fully outward-open 0.83825004 evidence cleaner0 2023-07-12T19:25:54Z DUMMY: free-energy landscape 0.9996829 chemical cleaner0 2023-07-12T15:52:17Z CHEBI: Na+ site SO: cleaner0 2023-07-12T15:36:03Z Sext 0.97782946 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.96249205 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99969685 chemical cleaner0 2023-07-12T15:52:20Z CHEBI: Na+ 0.9897099 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.98150855 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.91960526 protein_state cleaner0 2023-07-12T19:50:56Z DUMMY: without 0.9996451 chemical cleaner0 2023-07-12T15:52:15Z CHEBI: Na+ RESULTS paragraph 16630 These calculations demonstrate that the Na+ occupancy state of the transporter has a profound effect on its conformational free-energy landscape. 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). 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). 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). 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. 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). Crucially, though, the free-energy landscape for this partially occupied state demonstrates that the occluded conformation is no longer energetically feasible (Fig. 5a). 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). The transition to the occluded state in this apo state is again energetically unfeasible. 0.99476874 experimental_method cleaner0 2023-07-12T19:15:21Z MESH: calculations 0.9994221 chemical cleaner0 2023-07-12T15:52:29Z CHEBI: Na+ 0.99880683 protein_type cleaner0 2023-07-12T16:56:55Z MESH: transporter evidence DUMMY: cleaner0 2023-07-12T19:18:11Z conformational free-energy landscape 0.9963434 site cleaner0 2023-07-12T19:33:22Z SO: Na+ sites 0.9136924 evidence cleaner0 2023-07-12T19:26:02Z DUMMY: free-energy minimum 0.99976987 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.99978584 structure_element cleaner0 2023-07-12T19:38:32Z SO: TM2 0.999782 structure_element cleaner0 2023-07-12T15:54:55Z SO: TM3 0.99960315 site cleaner0 2023-07-12T19:33:26Z SO: binding sites 0.9988123 protein_type cleaner0 2023-07-12T16:56:59Z MESH: transporter 0.9926402 protein_state cleaner0 2023-07-12T19:51:05Z DUMMY: metastable 0.99951047 protein_state cleaner0 2023-07-12T19:51:08Z DUMMY: half-open 0.9997415 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.99897313 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9991191 protein_state cleaner0 2023-07-12T19:51:15Z DUMMY: open 0.9996369 chemical cleaner0 2023-07-12T15:52:39Z CHEBI: Na+ 0.9821666 site cleaner0 2023-07-12T15:36:03Z SO: Sext protein_state DUMMY: cleaner0 2023-07-12T19:51:28Z fully coordinated 0.99978906 chemical cleaner0 2023-07-12T15:52:36Z CHEBI: water 0.9998319 residue_name_number cleaner0 2023-07-12T15:51:07Z DUMMY: A206 0.9997695 residue_name_number cleaner0 2023-07-12T15:51:03Z DUMMY: F202 0.99910873 residue_name_number cleaner0 2023-07-12T15:51:07Z DUMMY: A206 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bond 0.9994727 protein_state cleaner0 2023-07-12T19:51:40Z DUMMY: semi-open 0.9996475 chemical cleaner0 2023-07-12T15:52:44Z CHEBI: Na+ 0.9954594 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.99499726 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99963343 chemical cleaner0 2023-07-12T15:52:42Z CHEBI: Na+ 0.9996406 chemical cleaner0 2023-07-12T15:52:47Z CHEBI: Na+ 0.9855064 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.98678976 protein_state cleaner0 2023-07-12T19:51:48Z DUMMY: metastable 0.954656 site cleaner0 2023-07-12T19:33:31Z SO: aqueous channel 0.9846718 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.99977773 structure_element cleaner0 2023-07-12T19:38:37Z SO: TM7 0.999772 structure_element cleaner0 2023-07-12T19:38:39Z SO: TM2 0.99951386 structure_element cleaner0 2023-07-12T19:38:43Z SO: gating helices 0.99977654 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9997745 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.999376 protein_state cleaner0 2023-07-12T19:52:05Z DUMMY: open 0.9921392 evidence cleaner0 2023-07-12T19:26:05Z DUMMY: free-energy landscape 0.9701071 protein_state cleaner0 2023-07-12T19:52:10Z DUMMY: partially occupied 0.99963486 protein_state cleaner0 2023-07-12T19:52:13Z DUMMY: occluded 0.99961114 chemical cleaner0 2023-07-12T15:52:50Z CHEBI: Na+ 0.9882245 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9884404 site cleaner0 2023-07-12T15:35:42Z SO: Sint evidence DUMMY: cleaner0 2023-07-12T19:52:30Z free-energy landscape 0.9990018 protein_type cleaner0 2023-07-12T16:57:02Z MESH: transporter 0.9993917 protein_state cleaner0 2023-07-12T19:52:37Z DUMMY: fully open 0.99727106 site cleaner0 2023-07-12T19:33:33Z SO: aqueous channels 0.99966455 protein_state cleaner0 2023-07-12T19:52:43Z DUMMY: occluded 0.99969745 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo RESULTS paragraph 18408 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. 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. 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. 0.9996661 protein_state cleaner0 2023-07-12T19:52:48Z DUMMY: open 0.99954325 protein_state cleaner0 2023-07-12T19:52:51Z DUMMY: semi-open 0.99965084 chemical cleaner0 2023-07-12T15:53:08Z CHEBI: Na+ 0.99968684 chemical cleaner0 2023-07-12T15:52:56Z CHEBI: Na+ evidence DUMMY: cleaner0 2023-07-12T19:53:04Z free-energy landscape 0.99940515 protein_type cleaner0 2023-07-12T16:57:04Z MESH: transporter 0.99961746 protein_state cleaner0 2023-07-12T19:54:05Z DUMMY: occluded protein_state DUMMY: cleaner0 2023-07-12T19:53:49Z Na+ occupancy is complete 0.99970305 chemical cleaner0 2023-07-12T15:53:06Z CHEBI: Na+ 0.99950075 protein_state cleaner0 2023-07-12T15:53:45Z DUMMY: Ca2+-bound 0.99932635 protein_type cleaner0 2023-07-12T16:57:06Z MESH: transporter 0.9995554 protein_state cleaner0 2023-07-12T15:51:48Z DUMMY: outward-open 0.9997084 chemical cleaner0 2023-07-12T15:53:00Z CHEBI: Ca2+ 0.99964285 protein_state cleaner0 2023-07-12T19:54:10Z DUMMY: occluded 0.9995422 protein_state cleaner0 2023-07-12T19:54:24Z DUMMY: semi-open 0.9993727 protein_state cleaner0 2023-07-12T19:54:29Z DUMMY: inward-open 0.9997597 chemical cleaner0 2023-07-12T15:53:03Z CHEBI: H+ 0.99954563 protein_state cleaner0 2023-07-12T19:54:33Z DUMMY: semi-open 0.9995364 protein_state cleaner0 2023-07-12T19:54:37Z DUMMY: fully open RESULTS paragraph 19431 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). 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). 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). 0.9993738 experimental_method cleaner0 2023-07-12T19:15:28Z MESH: enhanced-sampling simulations 0.99746 protein_state cleaner0 2023-07-12T19:54:46Z DUMMY: Ca2+ 0.9994981 protein_state cleaner0 2023-07-12T15:53:39Z DUMMY: H+-bound protein_state DUMMY: cleaner0 2023-07-12T15:37:00Z outward-facing protein PR: cleaner0 2023-07-12T15:33:38Z NCX_Mj 0.99955744 chemical cleaner0 2023-07-12T16:54:48Z CHEBI: Na+ 0.9979315 evidence cleaner0 2023-07-12T19:26:11Z DUMMY: structures 0.99948627 protein_state cleaner0 2023-07-12T15:53:45Z DUMMY: Ca2+-bound 0.7329998 experimental_method cleaner0 2023-07-12T19:15:35Z MESH: calculated 0.99619526 evidence cleaner0 2023-07-12T19:26:14Z DUMMY: free-energy landscape 0.9994836 protein_state cleaner0 2023-07-12T15:53:45Z DUMMY: Ca2+-bound protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99962956 chemical cleaner0 2023-07-12T16:54:51Z CHEBI: Ca2+ protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99964595 chemical cleaner0 2023-07-12T16:54:54Z CHEBI: Ca2+ 0.99966574 protein_state cleaner0 2023-07-12T19:54:56Z DUMMY: occluded 0.99966586 protein_state cleaner0 2023-07-12T19:54:59Z DUMMY: dehydrated 0.99929 protein_type cleaner0 2023-07-12T16:57:11Z MESH: exchanger 0.9995785 protein_state cleaner0 2023-07-12T19:55:09Z DUMMY: semi-open 0.9996244 protein_state cleaner0 2023-07-12T19:55:13Z DUMMY: open 0.9996137 chemical cleaner0 2023-07-12T16:54:58Z CHEBI: Ca2+ 0.99942195 chemical cleaner0 2023-07-12T16:55:02Z CHEBI: Na+ 0.9995045 site cleaner0 2023-07-12T19:33:38Z SO: aqueous access channels site SO: cleaner0 2023-07-12T15:36:03Z Sext site SO: cleaner0 2023-07-12T15:36:30Z SCa protein_state DUMMY: cleaner0 2023-07-12T19:55:27Z protonation 0.9998983 residue_name_number cleaner0 2023-07-12T15:40:30Z DUMMY: Glu54 0.9998963 residue_name_number cleaner0 2023-07-12T15:40:34Z DUMMY: Glu213 0.999652 protein_state cleaner0 2023-07-12T19:55:49Z DUMMY: occluded protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj chemical CHEBI: cleaner0 2023-07-12T19:55:44Z protons 0.99950355 protein_state cleaner0 2023-07-12T15:53:40Z DUMMY: H+-bound 0.99924624 protein_type cleaner0 2023-07-12T16:57:14Z MESH: exchanger 0.99956053 protein_state cleaner0 2023-07-12T19:55:53Z DUMMY: semi-open 0.9990244 protein_state cleaner0 2023-07-12T19:55:59Z DUMMY: low pH 0.9996755 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.99960977 evidence cleaner0 2023-07-12T19:26:16Z DUMMY: crystal structure 0.9995214 chemical cleaner0 2023-07-12T16:55:18Z CHEBI: H+ RESULTS paragraph 20516 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. 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. 0.9936283 experimental_method cleaner0 2023-07-12T19:15:38Z MESH: systematic computational analysis 0.99354166 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99871165 protein_type cleaner0 2023-07-12T15:54:34Z MESH: Na+/Ca2+ exchanger evidence DUMMY: cleaner0 2023-07-12T19:56:12Z free-energy landscape 0.9996339 protein_state cleaner0 2023-07-12T19:56:03Z DUMMY: occluded 0.99963164 protein_state cleaner0 2023-07-12T19:56:51Z DUMMY: occluded 0.9996307 protein_state cleaner0 2023-07-12T19:56:54Z DUMMY: outward 0.99335724 protein_state cleaner0 2023-07-12T19:56:56Z DUMMY: inward-open protein_state DUMMY: cleaner0 2023-07-12T19:56:26Z dehydration protein_state DUMMY: cleaner0 2023-07-12T19:56:38Z complete occupancy 0.99958426 site cleaner0 2023-07-12T19:33:42Z SO: binding sites DISCUSS title_1 21130 Discussion DISCUSS paragraph 21141 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. This control mechanism is functionally crucial, as it precludes the backflow of the species that is transported uphill, and also prevents the dissipation of the driving electrochemical gradients. 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. Similarly puzzling is that a given antiporter will undergo this transition upon recognition of substrates of different charge, size and number. 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. 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. 0.9995727 protein_state cleaner0 2023-07-12T19:57:01Z DUMMY: outward 0.98898214 protein_state cleaner0 2023-07-12T19:57:04Z DUMMY: inward-open protein_state DUMMY: cleaner0 2023-07-12T19:57:20Z secondary-active 0.9989353 protein_type cleaner0 2023-07-12T16:57:17Z MESH: transporter 0.9996537 protein_type cleaner0 2023-07-12T16:57:21Z MESH: antiporters protein_type MESH: cleaner0 2023-07-12T16:57:35Z membrane symporters 0.9996426 protein_type cleaner0 2023-07-12T16:58:16Z MESH: antiporter 0.99962723 protein_type cleaner0 2023-07-12T16:58:19Z MESH: antiporter 0.99869835 protein_type cleaner0 2023-07-12T16:58:21Z MESH: symporter 0.9563345 site cleaner0 2023-07-12T19:33:47Z SO: alternating-access switch 0.9995649 experimental_method cleaner0 2023-07-12T19:15:44Z MESH: structural studies 0.99950796 experimental_method cleaner0 2023-07-12T19:15:46Z MESH: quantitative molecular simulations 0.99925137 protein_type cleaner0 2023-07-12T15:54:33Z MESH: Na+/Ca2+ exchanger DISCUSS paragraph 22278 Specifically, our studies of NCX_Mj reveal the mechanism of forward ion exchange (Fig. 7). 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. 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. 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. 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. We anticipate that the intracellular ion-exchange process involves analogous conformational changes. protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9728699 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99804515 protein_state cleaner0 2023-07-12T15:37:26Z DUMMY: inward-facing 0.9996087 evidence cleaner0 2023-07-12T19:26:22Z DUMMY: crystal structures 0.999475 protein_type cleaner0 2023-07-12T16:58:26Z MESH: Ca2+/H+ exchangers protein_type MESH: cleaner0 2023-07-12T15:33:13Z NCX 0.9997992 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.99979264 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.9995315 protein_type cleaner0 2023-07-12T16:58:30Z MESH: transporter 0.6976584 structure_element cleaner0 2023-07-12T19:38:48Z SO: extracellular region 0.9995459 structure_element cleaner0 2023-07-12T16:58:38Z SO: TM2-TM3 0.99947095 structure_element cleaner0 2023-07-12T19:38:52Z SO: TM7-TM8 bundle 0.9995149 structure_element cleaner0 2023-07-12T19:38:55Z SO: N-terminal half 0.999793 structure_element cleaner0 2023-07-12T19:38:59Z SO: TM7 0.9997991 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.99979347 structure_element cleaner0 2023-07-12T19:39:02Z SO: TM2 0.9997917 structure_element cleaner0 2023-07-12T15:54:54Z SO: TM3 0.9997948 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.9987703 site cleaner0 2023-07-12T19:33:51Z SO: aqueous channels 0.99962413 site cleaner0 2023-07-12T19:33:55Z SO: ion-binding sites 0.9997807 structure_element cleaner0 2023-07-12T19:39:04Z SO: TM7 0.9996387 site cleaner0 2023-07-12T19:33:59Z SO: ion-binding sites 0.99978393 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.9997807 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.99978846 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9989407 protein_state cleaner0 2023-07-12T15:37:26Z DUMMY: inward-facing DISCUSS paragraph 23451 The crystal structures of NCX_Mj reported here, with either Na+, Ca2+, Sr2+ or H+ bound, capture the exchanger in different conformational states. 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. 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). 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. The simulations also demonstrate how this landscape is drastically re-shaped upon each ion-binding event. 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. 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. 0.99962485 evidence cleaner0 2023-07-12T19:26:25Z DUMMY: crystal structures protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9406617 chemical cleaner0 2023-07-12T15:55:01Z CHEBI: Na+, 0.9671022 chemical cleaner0 2023-07-12T15:55:03Z CHEBI: Ca2+, 0.9996768 chemical cleaner0 2023-07-12T16:55:22Z CHEBI: Sr2+ 0.9996743 chemical cleaner0 2023-07-12T15:55:06Z CHEBI: H+ 0.9984199 protein_state cleaner0 2023-07-12T19:57:59Z DUMMY: bound 0.9995522 protein_type cleaner0 2023-07-12T16:58:43Z MESH: exchanger 0.99961483 protein_type cleaner0 2023-07-12T16:58:46Z MESH: transporter 0.99953866 evidence cleaner0 2023-07-12T19:26:28Z DUMMY: crystal protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.59371054 experimental_method cleaner0 2023-07-12T15:55:36Z MESH: LCP 0.9996481 structure_element cleaner0 2023-07-12T19:39:08Z SO: extracellular half 0.9996382 structure_element cleaner0 2023-07-12T19:39:10Z SO: gating helices 0.99981445 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.99981624 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.999647 site cleaner0 2023-07-12T19:34:03Z SO: ion-binding sites 0.9996816 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.9993656 protein_state cleaner0 2023-07-12T19:58:06Z DUMMY: low pH 0.9993604 evidence cleaner0 2023-07-12T19:26:31Z DUMMY: structure evidence DUMMY: cleaner0 2023-07-12T19:28:04Z calculated free-energy landscapes 0.7967147 evidence cleaner0 2023-07-12T19:26:59Z DUMMY: crystallized structures 0.9994253 experimental_method cleaner0 2023-07-12T19:15:55Z MESH: simulations 0.9996013 protein_state cleaner0 2023-07-12T19:58:15Z DUMMY: occluded protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj chemical CHEBI: cleaner0 2023-07-12T16:53:16Z Na+ chemical CHEBI: cleaner0 2023-07-12T16:53:28Z Ca2+ 0.999537 protein_type cleaner0 2023-07-12T16:58:51Z MESH: transporters 0.9981036 protein_type cleaner0 2023-07-12T16:58:55Z MESH: symporters 0.99957913 protein_type cleaner0 2023-07-12T16:58:58Z MESH: antiporters 0.99960095 protein_state cleaner0 2023-07-12T19:58:17Z DUMMY: occluded evidence DUMMY: cleaner0 2023-07-12T19:27:18Z protein conformational free-energy landscape DISCUSS paragraph 24901 In multiple ways, our findings provide an explanation for, existing functional, biochemical and biophysical data for both NCX_Mj and its eukaryotic homologues. 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. 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). 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. 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. 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. Interestingly, binding of Ca2+ to Smid appears to be also possible, but available evidence indicates that this event transiently blocks the exchange cycle. 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. protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99939287 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.99909866 evidence cleaner0 2023-07-12T19:27:22Z DUMMY: ion-binding affinities 0.9746627 experimental_method cleaner0 2023-07-12T19:15:58Z MESH: crystallographic titrations 0.99950063 evidence cleaner0 2023-07-12T19:27:24Z DUMMY: Km 0.9809807 evidence cleaner0 2023-07-12T19:27:27Z DUMMY: K1/2 values experimental_method MESH: cleaner0 2023-07-12T19:16:21Z functional assays protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99977225 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99919945 evidence cleaner0 2023-07-12T19:27:30Z DUMMY: structures 0.9939589 evidence cleaner0 2023-07-12T19:27:32Z DUMMY: crystallographic data 0.9993855 taxonomy_domain cleaner0 2023-07-12T15:32:27Z DUMMY: eukaryotic 0.997542 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9985592 protein_type cleaner0 2023-07-12T15:54:34Z MESH: Na+/Ca2+ exchanger 0.8415257 experimental_method cleaner0 2023-07-12T19:16:25Z MESH: crystal titrations 0.82438564 chemical cleaner0 2023-07-12T15:56:26Z CHEBI: Na+ 0.9428204 chemical cleaner0 2023-07-12T15:56:28Z CHEBI: Ca2+ 0.79499984 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.7342383 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9997239 chemical cleaner0 2023-07-12T15:56:31Z CHEBI: Ca2+ 0.99970806 chemical cleaner0 2023-07-12T15:56:33Z CHEBI: Na+ 0.9997103 chemical cleaner0 2023-07-12T15:56:35Z CHEBI: Na+ 0.9997045 chemical cleaner0 2023-07-12T15:56:37Z CHEBI: Na+ 0.99971414 chemical cleaner0 2023-07-12T15:56:40Z CHEBI: Na+ 0.9997874 chemical cleaner0 2023-07-12T15:56:42Z CHEBI: water 0.9237341 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.99967635 chemical cleaner0 2023-07-12T15:56:45Z CHEBI: Ca2+ 0.9980756 evidence cleaner0 2023-07-12T19:27:37Z DUMMY: Hill coefficient 0.99949723 chemical cleaner0 2023-07-12T15:56:47Z CHEBI: Na+ 0.99964225 chemical cleaner0 2023-07-12T15:56:49Z CHEBI: Ca2+ site SO: cleaner0 2023-07-12T15:36:03Z Sext 0.9995297 site cleaner0 2023-07-12T19:35:00Z SO: activation site 0.999643 chemical cleaner0 2023-07-12T15:56:57Z CHEBI: Na+ 0.9996819 chemical cleaner0 2023-07-12T15:56:54Z CHEBI: Na+ 0.9996492 chemical cleaner0 2023-07-12T15:56:52Z CHEBI: Na+ 0.99957913 protein_state cleaner0 2023-07-12T19:58:34Z DUMMY: occluded 0.9996916 chemical cleaner0 2023-07-12T15:57:00Z CHEBI: Ca2+ 0.970009 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.99948275 evidence cleaner0 2023-07-12T19:27:39Z DUMMY: structures protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9992466 protein_state cleaner0 2023-07-12T19:58:37Z DUMMY: bound to 0.999743 chemical cleaner0 2023-07-12T15:57:02Z CHEBI: Cd2+ 0.99974525 chemical cleaner0 2023-07-12T15:57:04Z CHEBI: Mn2+ 0.9707498 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.99974155 chemical cleaner0 2023-07-12T15:57:07Z CHEBI: Sr2+ 0.9855223 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9986142 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.99973387 chemical cleaner0 2023-07-12T15:57:09Z CHEBI: calcium DISCUSS paragraph 26753 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. Specifically, saturating amounts of Ca2+ or Na+ resulted in a noticeable slowdown in the HDX rate for extracellular portions of the α-repeat helices. 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. 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. 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. 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. protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9997053 chemical cleaner0 2023-07-12T15:57:45Z CHEBI: Ca2+ 0.9996995 chemical cleaner0 2023-07-12T15:57:47Z CHEBI: Na+ 0.98209786 experimental_method cleaner0 2023-07-12T19:16:30Z MESH: hydrogen-deuterium exchange 0.97860384 experimental_method cleaner0 2023-07-12T19:16:34Z MESH: HDX protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.83848304 protein_state cleaner0 2023-07-12T19:58:42Z DUMMY: presence 0.9987177 protein_state cleaner0 2023-07-12T19:58:44Z DUMMY: absence of 0.9976303 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing 0.999722 chemical cleaner0 2023-07-12T15:57:50Z CHEBI: Ca2+ 0.9997134 chemical cleaner0 2023-07-12T15:57:53Z CHEBI: Na+ 0.7904098 evidence cleaner0 2023-07-12T15:58:26Z DUMMY: HDX rate 0.99959975 structure_element cleaner0 2023-07-12T19:39:14Z SO: α-repeat helices 0.9996377 site cleaner0 2023-07-12T19:35:08Z SO: ion-binding sites 0.9996598 protein_state cleaner0 2023-07-12T19:58:54Z DUMMY: occluded evidence DUMMY: cleaner0 2023-07-12T15:58:25Z HDX rate 0.9785595 evidence cleaner0 2023-07-12T15:58:26Z DUMMY: HDX rate 0.999727 chemical cleaner0 2023-07-12T15:57:55Z CHEBI: Na+ 0.99972725 chemical cleaner0 2023-07-12T15:57:57Z CHEBI: Ca2+ evidence DUMMY: cleaner0 2023-07-12T19:28:03Z calculated free-energy landscapes 0.99972034 chemical cleaner0 2023-07-12T15:57:59Z CHEBI: Na+ 0.9997157 chemical cleaner0 2023-07-12T15:58:02Z CHEBI: Ca2+ 0.99941885 protein_type cleaner0 2023-07-12T16:59:05Z MESH: transporter 0.999541 protein_state cleaner0 2023-07-12T19:59:00Z DUMMY: ion-bound 0.977703 protein_state cleaner0 2023-07-12T19:59:10Z DUMMY: fully open METHODS title_1 28248 Methods METHODS title_2 28256 Protein expression, purification and crystallization METHODS paragraph 28309 NCX_Mj was expressed, purified and crystallized as previously described. Briefly, the NCX_Mj gene with a C-terminal hexa-histidine tag was subcloned into the pQE60 vector and expressed in Escherichia coli BL21(DE3)plysS. Harvested cells were homogenized and incubated in buffer containing 50 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, 40 mM DDM. After incubation at room temperature (RT) for 3.5 hours, the supernatant was collected by centrifugation and loaded onto a Talon Co2+ affinity column (Clontech). The non-specifically bound contaminates on the column were washed with buffer containing 50 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, 15 mM imidazole, and 1 mM DDM. The bound NCX_Mj was eluted by increasing the imidazole concentration to 300 mM. The eluate was treated with thrombin to remove the hexa-histidine tag and dialyzed against 20 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2, and 1 mM DDM at RT overnight. After overnight digestion the sample was loaded onto a second Co2+ affinity column to remove any free hexa-histidine tag and contaminant proteins. NCX_Mj in the flow-through was collected and further purified by gel filtration using a Superdex-200 (10/300) column (GE Healthcare) in 20 mM HEPES pH 7.2, 50 mM NaCl, 12 mM KCl, 10 mM CaCl2 and 0.5 mM DDM. The purified protein was then concentrated to 40 mg/ml for crystallization. METHODS paragraph 29691 Native NCX_Mj was crystallized using the lipidic cubic phase (LCP) technique, as previously described. Concentrated NCX_Mj was first reconstituted into 1-oleoyl-rac-glycerol (Sigma) in a protein:lipid weight ratio of 1:1.5, using the two-syringe method. Protein-laden LCP droplets of 35 nL were dispensed onto Corning 96-well protein-crystallization plates and overlaid with 5 μL of precipitant solution containing 40-42% PEG 400, 100 mM MES pH 6.5, 100 mM NaAc. Crystals were observed after 48 hours and grew to full size after 2 weeks. The native crystals belong to space group P212121 with a cell dimension of a=49.5Å, b=72.9Å and c=96.2Å, and contain one subunit per asymmetric unit. As the LCP droplet accounts for less than 1% of the total crystallization volume, the salt composition in the crystallization condition was determined mainly by the overlaying solution, and estimated to have 150 mM Na+ (from MES buffer and NaAc) and 30 μM Ca2+ (from LCP droplet). In these concentration conditions (high Na+ and low Ca2+) Ca2+ does not bind to NCX_Mj (as shown in our crystallographic titration experiments) and thus this native crystal structure represents NCX_Mj in 150 mM Na+. The native crystals were used in all subsequent titration experiments to define low-Na+, Ca2+ and Sr2+-loaded structures. METHODS paragraph 31003 To obtain the apo crystal form, the protein was first purified in a solution containing 20 mM Hepes-Tris pH 7.2, 100 mM NMDG, 10 mM CaCl2 and 0.5 mM DDM. The crystals were obtained in LCP with crystallization solution containing 200 mM KAc, pH 4.0, 35% PEG400. The apo NCX_Mj crystals belong to space group C2 with a cell dimension of a=164.2Å, b=46.8Å, c=97.0 Å and β=106.2°, and contain two protein subunits per asymmetric unit. METHODS title_2 31439 Crystal titrations METHODS paragraph 31458 Once the native crystals reached their full size, the crystallization solutions overlaying lipid/protein droplets were gradually replaced by titration solutions through multiple steps of solution exchange. In general, 2-3 μL of existing crystallization solutions (normally in 5 μL) were replaced by the same volume of titration solutions, followed by overnight equilibration. The same procedures were repeated 6-10 times until the ion components in the crystal drops reached the targeted concentrations. For titration experiments to define concentration-dependent Na+ binding, the titration solutions contained 100 mM MES-Tris pH 6.5, 44% PEG400, 10 mM EGTA and a 100 mM mixture of NaAc and CsAc, in the following proportions: 100 mM CsAc; 90 mM CsAc and 10 mM NaAc; 80 mM CsAc and 20 mM NaAc; and 100 mM NaAc. Note that Cs+ does not bind NCX proteins and is commonly used as a Na+ substituent to maintain the ionic strength of the solutions. As complete removal of Na+ would deteriorate the crystals, we had to maintain a minimum Na+ concentration of about 2.5 mM in the crystal drops. The final Na+ concentrations in this set of titration experiments were about 2.5, 10, 20 and 100 mM, respectively. It is worth noting that the observed Na+-dependent conformational change occurs while the proteins are in crystal form and embedded in lipid. METHODS paragraph 32804 In the titration experiments carried out to define the mode of divalent cation binding and competition with Na+, the soaking solutions contained 100 mM MES-Tris pH 6.5, 44% PEG400, 100 mM mixture of CsAc and NaAc and various concentrations of XCl2, where X=Ca2+ or Sr2+, in the following proportions: 100 mM CsAc and 10 mM XCl2; 100 mM CsAc and 1 mM XCl2; 100 mM CsAc and 0.1 mM XCl2; 90 mM CsAc, 10mM NaAc and 10mM XCl2; and 100 mM NaAc and 10 mM XCl2. After multiple steps of solution exchanges, the final soaking conditions contained 0.1, 1, or 10 mM of X2+ together with 2.5 mM Na+; or 10 mM X2+ together with 2.5, 10 or 100 mM Na+. METHODS title_2 33441 Data collection and structure determination METHODS paragraph 33485 After soaking crystals were mounted on 100-μm Mitegen Microloops and frozen in liquid nitrogen. All diffraction data were collected at the Advanced Photon Source (APS) GM/CA-CAT beamlines 23ID-B or 23ID-D using a beam size of 35 μm × 50 μm. Data were processed and scaled using HKL2000 and the structures were determined by molecular replacement in PHASER using our previously published NCX_Mj structure (PDB code 3V5U) as a search model. Model building was completed using COOT and structure refinement was performed with PHENIX. The data sets from crystals soaked in solutions containing 2.5 to 100 mM Na+ were collected using an X-ray wavelength of 1.033Å; the crystal grown with 150 mM Na+, and those soaked with Ca2+ and Sr2+ solutions, were obtained with a wavelength of 0.9793 Å. Lastly, the data from the crystal grown at low pH with no Na+ or Ca2+ were collected with a 2.0-Å wavelength beam. The resulting statistics for data collection and refinement are shown in Tables 2-4. All structure figures were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.). The ion passageways in low- and high-Na+ structures as well as the apo state were analyzed using the program CAVER. Due to the variation in diffraction resolution and intensity among crystals, ion-occupancy comparisons were made on the basis of the diffraction data obtained in the titration experiments scaled against a common reference data before map calculation. The NCX_Mj crystal obtained with 2.5 mm Na+ only was used as the reference. METHODS title_2 35042 Conventional molecular dynamics (MD) simulations METHODS paragraph 35091 Conventional (i.e. not enhanced) MD simulations were carried out with NAMD 2.7-2.9 at constant temperature (298 K), pressure (1 atm), and membrane surface area (~69 Å2 per lipid),and with periodic boundaries in all directions. All calculations used the standard CHARMM27/CMAP force field, except for NBFIX corrections for the interaction between carboxylate-oxygens and Na+ interaction or Ca2+ (Supplementary Note 3, Supplementary Fig. 5). Electrostatic interactions were calculated using PME with a real-space cut-off of 12 Å; the same cut-off distance was used for all van der Waals interactions. METHODS paragraph 35693 Five ion-occupancy states of the transporter were considered, namely with 3 Na+, 2 Na+, 2 H+ or 1 Ca2+, and with no ions bound; in all cases Asp240 is protonated. For the 3×Na+ state, we reanalyzed a 200-ns trajectory of NCX_Mj reported previously. NCX_Mj had been embedded in a POPC lipid membrane using GRIFFIN. The initial configuration of the 2×Na+ state was generated from an equilibrated configuration of 3×Na+ state, from which the Na+ ion at Sext was displaced by means of a slow alchemical transformation that annihilates the bound ion and recreates it in the bulk solution (in the same simulation box). The resulting 2×Na+ state was simulated for 250 ns. Similarly, the state with no Na+ bound was generated from an equilibrated configuration of the 2×Na+ state, from which the remaining Na+ ions were displaced; this state was again simulated for 250 ns. For the 2×H+ state, an initial configuration was generated from an equilibrated configuration of the 3×Na+ state, by gradually annihilating the Na+ ions from the binding sites and creating protonated E54 and E213 side chains; concurrently, acetic acid molecules in the bulk solution (in the same simulation box) were deprotonated and Na+ ions introduced. A second initial configuration of the 2×H+ state was obtained from an equilibrated configuration of the simulation with no ions bound, by slowly transforming deprotonated E54 and E213 into their protonated form, while doing the opposite to acetic acid molecules in the bulk solution. These two initial configurations of the 2×H+ state were then equilibrated for 800 ns. All annihilation/creation simulations were carried out using the FEP module of NAMD; and comprised 32-50 intermediate simulations of 400 ps each, for each transformation. A soft-core van der Waals potential with a radius-shifting coefficient of 2 Å2 was used. The annihilated Na+ ions were confined within their corresponding binding sites using flat-bottom distance restraints. Specifically, the Na+ in Sext was concurrently maintained within 4 Å of E54:Cδ, A206:C, S77:Cβ, T209:Cβ and S210:Cβ. The Na+ ions in SCa and Sint were concurrently kept within 4 Å of the E213:Cδ and A47:C, respectively. The Na+ ions and acetic acid molecules in the bulk solution were kept at a distance greater than 37 Å from the membrane center. Finally, the initial configuration of the Ca2+ state was generated on the basis of the published NCX_Mj X-ray structure by placing Ca2+ in the SCa site and two water molecules coordinating Ca2+ at and near the Smid site, so as to satisfy the expected coordination geometry (see Supplementary Note 2, Supplementary Fig. 3-4). This configuration was initially equilibrated through a series of simulations in which RMSD-based restraints of gradually diminishing strength were applied to the protein Cα atoms as well the side-chains involved in Ca2+ coordination. A 250 ns equilibration was then carried without any restraints. METHODS title_2 38656 Enhanced-sampling MD simulations METHODS paragraph 38689 Free-energy landscapes were calculated using Bias-Exchange Well-Tempered Metadynamics (BE-WT-MetaD), using GROMACS4.5.5/PLUMED. The force-field and simulation conditions were equivalent to those employed in the unbiased MD simulations. The accumulated simulation time for each of the ion-occupancy states studied was 1.6 μs. Each of these calculations consisted of 16 concurrent, interdependent simulations (or replicas); in 15 of these replicas, a WT-MetaD biasing potential was applied on different subsets of collective variables, as specified below, while the remaining replica was unbiased. Attempts to exchange coordinate configurations among replicas were made every 2-5 ps, using the Metropolis criterion. The inputs for each calculation were equilibrated configurations extracted from the unbiased MD simulations. METHODS paragraph 39513 The choice of collective variables to be biased in the BE-WT-MetaD simulations was also based on analysis of the unbiased MD trajectories (Fig. 4g-i). Specifically, to enhance the reversible opening and closing of the water channels reaching from the extracellular bulk solution into either the Sext or the SCa binding sites, we employed the following time-dependent collective variable (Fig. 4h-i): where ri denotes the distance between the oxygen atom of each water molecule in the system i and the center of the binding site considered (V1 for Sext,V2 for SCa), and β is 10-100 nm. When the binding site was occupied, the ion was used to define its center. If the site was empty, its center was defined as the center-of-mass of the oxygen atoms coordinating the ion if bound. Bound water molecules at or near Smid (coordinating the Na+ or Ca2+ ions) were not considered. METHODS paragraph 40391 To enhance the reversible formation and disruption of selected backbone hydrogen bonds in TM7ab (Fig. 4g), we used an analogous collective variable: In this case, the index i denotes atoms P202:O and T203:O, while the index j denotes atoms A206:N and P207:N. To preclude the artificial unraveling of TM7ab driven by this bias, an upper-bound V3max equal to 0.7 nm was imposed with a boundary potential of the form k (V3(t) – V3max) if V3(t) > V3max, where k = 105 nm−4 kJ/mol. In addition, to control the bending and straightening of TM7ab more globally, we used the following path-collective variables: where d1 and d2 denote the mean-square-differences between the conformation of TM7ab and either the straight or bent conformations, respectively, and λ = 100 nm−2. Note that V 4 is by definition confined between a lower (V4 ~ 1) and upper bound (V4 ~ 2); to confine the exploration of V5, an upper value V5max of 0.020-0.025 nm2 was imposed with boundary potential of the form k (V5(t) – V5max) if V5(t) > V5max, where k = 1011 nm−8 kJ/mol. The mean-square-differences d1 and d2 comprise the backbone atoms of residues 198-211 as well as the side chain carbon atoms of residues P207 and L211. METHODS paragraph 41601 A boundary potential was also applied to confine the ions and water molecule bound to Sext, SCa, Sint and Smid to their corresponding binding sites. Specifically, the variable confined was: where ri denotes the distance between the ion and each of its coordinating oxygen atoms; r0 was set to 0.24 nm for the Na+ ions, and to 0.30 nm for Ca2+. Note that the upper-bound value of Vc is, by definition, approximately the coordination number in the bound state, whereas Vc becomes 0 as the ion becomes unbound. For the Na+ ion bound to Sext and Sint, therefore, a lower bound value Vcmin = 4.3 was imposed with potential of the form k (Vc(t) – Vcmin) when Vc(t) < Vcmin, where k = 2500 kJ/mol. An analogous restraint was used for the Na+ ion at SCa, with Vcmin = 4.75. Similarly, for the Ca2+ ion at SCa a lower bound value Vcmin = 7.4 was imposed with a potential of the form k (Vc(t) – Vcmin) when Vc(t) < Vcmin, with k = 400 kJ/mol. Note that these restraints do not perturb the chemical structure of the ion-coordination sphere when the ion is bound, i.e. Vc(t) > Vcmin. The displacement of the bound water molecules in the ion-coordination sphere by equivalent water molecules in the solvent was prevented similarly. METHODS paragraph 42825 The specific sets of collective variables biased in each of the replica WT-MetaD simulations, as well as further details on the biasing potentials introduced, are specified in Supplementary Table 1. METHODS title_2 43024 Derivation of conformational free-energy landscapes METHODS paragraph 43076 To translate the data gathered in the BE-WT-MetaD simulations into conformational free-energy landscapes, we sought to identify a low-dimensional representation of the data that is nevertheless also intuitive and representative. We ultimately settled on two structure-based descriptors of the degree of opening of the each of the aqueous channels leading to the ion-binding sites (Fig. 4), defined as: where rij denotes a set of pairwise distances for specific Cα atoms in the protein, for a given simulation snapshot. For S1, index i refers to the Cα of residues 198 to 209 in TM7, while index j refers to those in residues 66 to 80 and 290 to 297, in TM3 and TM10, respectively. For S2, index i refers to the Cα of residues 51 to 64 in TM2, while index j refers to those in residues 177 to 193 and 198 to 209, in TM6 and TM7, respectively. Therefore, S1 describes the effective separation between TM7 and TM3/TM10, on the extracellular half of the protein, and thus reports on the accessibility to the Sext site. Analogously, S2 measures the separation between TM2 and TM6-TM7, also on the extracellular side, and thus reports on the accessibility to SCa. METHODS paragraph 44247 The conformational free energy of NCX_Mj as a function of S1 and S2 was then computed for each ion-occupancy state separately (Figs. 5a, 6a). These landscapes were obtained through reweighting of the biased probability distribution from the BE-WT-MetaD sampling, using the WHAM method; through this approach we combine the statistics gathered in all replicas, and can consider alternate free-energy projections. METHODS paragraph 44659 To correct the landscape calculated for the Ca2+-bound state (Fig. 6a) on account of the excess amount of charge transferred from the ion to the protein (Supplementary Note 4, Supplementary Fig. 6), we reprocessed all the sampling obtained during the original BE-WT-MetaD simulations, introducing in the WHAM equations a re-weighting factor w for each configuration X: where U denotes the ‘uncorrected’ CHARMM27/NBFIX potential-energy function, and Uc denotes the corrected function. To calculate Uc (X), the charge of Ca2+ was reduced to +1.8e from its standard value of +2e, and the difference was distributed among the surrounding protein residues (as specified in Supplementary Note 4). To minimally alter the charge-distribution used in the original CHARMM27 force-field, the charge added to each protein atom was proportional to the absolute value of its uncorrected charge. METHODS paragraph 45545 The statistical errors for all free-energy landscapes are provided in Supplementary Fig. 7. METHODS title_2 45637 Derivation of representative structures and water-density maps METHODS paragraph 45700 Representative structures and water-density iso-surfaces (Fig. 5b-c, Fig. 6b-c) were derived for each BE-WT-MetaD simulation by clustering all sampling in the multi-dimensional space of V1, V2, and V4 (Eq. 1-3), plus a descriptor S3 of the proximity between TM6-TM7 and TM2-TM3/TM10, on the extracellular side of the protein. More precisely: where r0 = 7.5 Å, and rij denotes a specific set of pairwise Cα distances, for a given simulation snapshot. Similarly to S1 and S2 (Eq. 6), index i refers to Cα atoms in the extracellular halves of TM6 and TM7, while index j refers to Cα atoms in the extracellular halves of TM2, TM3, and TM10. We thus obtained ~2,000 clusters for each of the simulation systems (using RMSD cut-off values of 1.3 Å for V1 and V2, 0.1 Å for V4; and 6.25 Å for S3). Using the WHAM equations, we calculated the relative free energy of each of these clusters, and then identified the major basins in this space with the MCL method (with p = 1.4.) Water occupancy maps were calculated for each of these major free-energy basins, only using the sampling gathered by the unbiased replicas. SUPPL title_1 46822 Supplementary Material SUPPL footnote 46845 Author Contributions SUPPL footnote 46866 J.L. and Y.J. designed the experimental studies and analyzed the resulting data. J.L., C.L. and Y.H. performed the experimental research. F.M and J.D.F.G. designed the computational research and analyzed the corresponding data. F.M. performed the computational work. J.L., Y.J. F.M. and J.D.F.G. wrote the paper. The authors declare no competing financial interests. SUPPL footnote 47233 Accession codes SUPPL footnote 47249 Atomic coordinates and structural factors have been deposited in the Protein Data Bank, with accession numbers 5HWX, 5HWY, 5HXC, 5HXE, 5HYA, 5HXH, 5HXS and 5HXR, as specified in Tables 2-4. 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Edit ref 38 1999 51922 Peptide folding: When simulation meets experiment. in press surname:Branduardi;given-names:D surname:Marinelli;given-names:F REF J. Comput. Chem ref 2015 51973 Faraldo-Gómez, J.D. Atomic-resolution dissection of the energetics and mechanism of isomerization of hydrated ATP-Mg through the SOMA string method. 1575 1584 surname:Enright;given-names:AJ surname:Van Dongen;given-names:S surname:Ouzounis;given-names:CA 11917018 REF Nucleic Acids Res ref 30 2002 52123 An efficient algorithm for large-scale detection of protein families. nihms-779827-f0001.jpg F1 FIG fig_caption 52193 Na+ binding to outward-facing NCX_Mj. (a) Overall structure of native outward-facing NCX_Mj from crystals grown in 150 mM Na+. N- and C-terminal halves are colored yellow and cyan, respectively. Colored spheres represent the bound Na+ (green) and water (red). (b) Structural details and definition of the four central binding sites. Only residues flanking these sites are shown for clarity (same for all other figures). 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). Further details are shown in Supplementary Fig. 1. (c) Concentration-dependent change in Na+ occupancy (see also Table 1). All Fo – Fc ion-omit maps are calculated to 2.4 Å and contoured at 3σ for comparison. 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). At 20 mM Na+, both conformations co-exist. No significant changes were observed in the side-chains involved in ion or water coordination at the SCa, Sint and Smid sites. 0.99970114 chemical cleaner0 2023-07-12T16:07:44Z CHEBI: Na+ protein_state DUMMY: cleaner0 2023-07-12T15:37:00Z outward-facing protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9735729 evidence cleaner0 2023-07-12T19:28:28Z DUMMY: structure 0.99929404 protein_state cleaner0 2023-07-12T19:59:19Z DUMMY: native protein_state DUMMY: cleaner0 2023-07-12T15:37:00Z outward-facing protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.99713457 experimental_method cleaner0 2023-07-12T19:16:50Z MESH: crystals grown 0.9996817 chemical cleaner0 2023-07-12T16:07:46Z CHEBI: Na+ 0.9996953 chemical cleaner0 2023-07-12T16:07:48Z CHEBI: Na+ 0.9995254 chemical cleaner0 2023-07-12T16:07:50Z CHEBI: water 0.99958056 site cleaner0 2023-07-12T19:35:13Z SO: central binding sites 0.99955356 evidence cleaner0 2023-07-12T19:28:30Z DUMMY: electron density 0.9991879 evidence cleaner0 2023-07-12T19:28:33Z DUMMY: Fo-Fc ion omit map 0.7673661 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.9964263 chemical cleaner0 2023-07-12T16:07:52Z CHEBI: water 0.9494199 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9231161 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.8831704 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99964094 chemical cleaner0 2023-07-12T16:07:54Z CHEBI: Na+ 0.9990984 chemical cleaner0 2023-07-12T16:07:57Z CHEBI: Na+ 0.999241 evidence cleaner0 2023-07-12T19:28:36Z DUMMY: Fo – Fc ion-omit maps 0.9998784 residue_name_number cleaner0 2023-07-12T15:51:07Z DUMMY: A206 0.999323 chemical cleaner0 2023-07-12T16:07:59Z CHEBI: Na+ 0.9931774 protein_state cleaner0 2023-07-12T19:59:23Z DUMMY: partially open 0.99960274 protein_state cleaner0 2023-07-12T19:59:25Z DUMMY: occluded 0.9996083 chemical cleaner0 2023-07-12T16:08:03Z CHEBI: Na+ 0.9996876 chemical cleaner0 2023-07-12T16:08:01Z CHEBI: Na+ 0.99939406 chemical cleaner0 2023-07-12T16:08:05Z CHEBI: water 0.7865566 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.6614779 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.6314445 site cleaner0 2023-07-12T15:36:48Z SO: Smid nihms-779827-f0002.jpg F2 FIG fig_caption 53361 Na+-occupancy dependent conformational change in NCX_Mj. (a) Superimposition of the NCX_Mj crystal structures obtained in high (100 mM, cyan cylinders) and low (10 mM, brown cylinders) Na+ concentrations. (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). Residues forming van-der-Waals contacts in the structure at low Na+ concentration are shown in detail. (c) Close-up view of the Na+-binding sites. The vacant Sext site in the structure at low Na+ concentration is indicated with a white sphere. Residues surrounding this site are also indicated; note A206 (labeled in red) coordinates Na+ at Sext via its backbone carbonyl oxygen. (d) Extracellular solvent accessibility of the ion binding sites in the structures at high and low [Na+]. Putative solvent channels are represented as light-purple surfaces. 0.9972726 chemical cleaner0 2023-07-12T16:09:12Z CHEBI: Na+ protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9996729 experimental_method cleaner0 2023-07-12T19:16:58Z MESH: Superimposition protein PR: cleaner0 2023-07-12T15:33:39Z NCX_Mj 0.9995926 evidence cleaner0 2023-07-12T19:28:38Z DUMMY: crystal structures 0.99920523 chemical cleaner0 2023-07-12T16:09:15Z CHEBI: Na+ 0.9993622 site cleaner0 2023-07-12T19:35:19Z SO: interface 0.99981266 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.99981123 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.99962425 evidence cleaner0 2023-07-12T19:28:40Z DUMMY: structures 0.9994711 chemical cleaner0 2023-07-12T16:09:20Z CHEBI: Na+ 0.99940884 evidence cleaner0 2023-07-12T19:28:43Z DUMMY: structure 0.8585934 protein_state cleaner0 2023-07-12T19:59:42Z DUMMY: low 0.999156 chemical cleaner0 2023-07-12T16:09:17Z CHEBI: Na+ 0.9996513 site cleaner0 2023-07-12T19:35:22Z SO: Na+-binding sites site SO: cleaner0 2023-07-12T15:36:03Z Sext 0.9996259 evidence cleaner0 2023-07-12T19:28:45Z DUMMY: structure 0.9406699 protein_state cleaner0 2023-07-12T19:59:50Z DUMMY: low 0.9995061 chemical cleaner0 2023-07-12T16:09:22Z CHEBI: Na+ 0.99989617 residue_name_number cleaner0 2023-07-12T15:51:07Z DUMMY: A206 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinates 0.99944687 chemical cleaner0 2023-07-12T16:09:24Z CHEBI: Na+ site SO: cleaner0 2023-07-12T15:36:03Z Sext 0.9996469 site cleaner0 2023-07-12T19:35:26Z SO: ion binding sites 0.9995679 evidence cleaner0 2023-07-12T19:28:48Z DUMMY: structures 0.7218978 protein_state cleaner0 2023-07-12T19:59:46Z DUMMY: high 0.56791896 protein_state cleaner0 2023-07-12T19:59:48Z DUMMY: low 0.99950194 chemical cleaner0 2023-07-12T16:09:26Z CHEBI: Na+ 0.99904966 site cleaner0 2023-07-12T19:35:28Z SO: solvent channels nihms-779827-f0003.jpg F3 FIG fig_caption 54284 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). Residues involved in Sr2+ coordination are labeled. There are no significant changes in the side-chains involved in ion coordination, relative to the Na+-bound state. T50 and T209 (labeled in red) coordinate Sr2+ through their backbone carbonyl-oxygen atoms. High Na+ concentration (100 mM) completely displaces Sr2+ and reverts NCX_Mj to the occluded state (right panel). 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. (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). 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. The side chains of E54 and E213 from the low Na+ structure are also shown (light brown) for comparison. White spheres indicate the location Sint, Smid SCa. (e) Extracellular solvent accessibility of the ion-binding sites in apo NCX_Mj. 0.9996723 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.9992555 evidence cleaner0 2023-07-12T19:28:51Z DUMMY: structure protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.999681 chemical cleaner0 2023-07-12T16:42:37Z CHEBI: Sr2+ 0.38059822 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.94249403 experimental_method cleaner0 2023-07-12T19:17:06Z MESH: crystals titrated 0.9996698 chemical cleaner0 2023-07-12T16:42:40Z CHEBI: Sr2+ 0.9996922 chemical cleaner0 2023-07-12T16:42:42Z CHEBI: Na+ 0.9994567 chemical cleaner0 2023-07-12T16:42:44Z CHEBI: Sr2+ 0.9994848 protein_state cleaner0 2023-07-12T20:00:04Z DUMMY: Na+-bound 0.99990046 residue_name_number cleaner0 2023-07-12T16:44:39Z DUMMY: T50 0.9999018 residue_name_number cleaner0 2023-07-12T16:44:44Z DUMMY: T209 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z coordinate 0.9995192 chemical cleaner0 2023-07-12T16:42:49Z CHEBI: Sr2+ 0.9996927 chemical cleaner0 2023-07-12T16:55:27Z CHEBI: Na+ 0.9996083 chemical cleaner0 2023-07-12T16:42:52Z CHEBI: Sr2+ protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.9996699 protein_state cleaner0 2023-07-12T20:00:08Z DUMMY: occluded 0.9992844 evidence cleaner0 2023-07-12T19:28:57Z DUMMY: Fo – Fc omit map 0.9996213 evidence cleaner0 2023-07-12T19:28:59Z DUMMY: structure 0.9996326 chemical cleaner0 2023-07-12T16:43:01Z CHEBI: Na+ 0.9992766 evidence cleaner0 2023-07-12T19:29:03Z DUMMY: density 0.99966663 chemical cleaner0 2023-07-12T16:42:54Z CHEBI: Na+ 0.9991315 chemical cleaner0 2023-07-12T16:42:56Z CHEBI: H2O 0.9997431 chemical cleaner0 2023-07-12T16:42:59Z CHEBI: Ca2+ 0.82117546 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.96461064 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.8262321 experimental_method cleaner0 2023-07-12T19:17:09Z MESH: crystals titrated 0.99973714 chemical cleaner0 2023-07-12T16:43:03Z CHEBI: Ca2+ 0.99970067 chemical cleaner0 2023-07-12T16:43:06Z CHEBI: Na+ 0.9996834 experimental_method cleaner0 2023-07-12T19:17:12Z MESH: Superimposition protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.9996061 evidence cleaner0 2023-07-12T19:29:09Z DUMMY: structures 0.9995919 chemical cleaner0 2023-07-12T16:43:09Z CHEBI: Na+ 0.999273 protein_state cleaner0 2023-07-12T20:00:13Z DUMMY: absence of 0.9996879 chemical cleaner0 2023-07-12T16:43:13Z CHEBI: Na+ 0.85081136 protein_state cleaner0 2023-07-12T20:00:19Z DUMMY: pH 4 0.99968123 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo 0.99961984 site cleaner0 2023-07-12T19:35:34Z SO: ion-binding sites 0.9996793 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo protein_state DUMMY: cleaner0 2023-07-12T16:43:44Z high H+ 0.99989545 residue_name_number cleaner0 2023-07-12T16:44:30Z DUMMY: E54 0.99989486 residue_name_number cleaner0 2023-07-12T16:44:35Z DUMMY: E213 0.8679526 protein_state cleaner0 2023-07-12T16:43:47Z DUMMY: low Na+ 0.9995432 evidence cleaner0 2023-07-12T19:29:06Z DUMMY: structure 0.69025 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.52867126 site cleaner0 2023-07-12T15:36:48Z SO: Smid 0.6491887 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9996259 site cleaner0 2023-07-12T19:35:37Z SO: ion-binding sites 0.99967813 protein_state cleaner0 2023-07-12T15:48:06Z DUMMY: apo protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj nihms-779827-f0004.jpg F4 FIG fig_caption 55691 Spontaneous changes in the structure of outward-occluded, fully Na+-occupied NCX_Mj (PDB code 3V5U) upon sequential displacement of Na+. (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). Black lines indicate (i, i + 4) carbonyl-amide pairs along the helix; specific O-N distances are indicated, in Å (magenta). (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). (d) Close-up of the ion-binding region in the fully Na+-occupied 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. (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). These descriptors were employed as collective variables in the Bias-Exchange Metadynamics simulations (Methods). (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). 0.99937314 evidence cleaner0 2023-07-12T19:29:16Z DUMMY: structure 0.9995842 protein_state cleaner0 2023-07-12T15:51:40Z DUMMY: outward-occluded protein_state DUMMY: cleaner0 2023-07-12T16:46:18Z fully Na+-occupied protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.9996201 chemical cleaner0 2023-07-12T16:45:25Z CHEBI: Na+ 0.9994692 experimental_method cleaner0 2023-07-12T19:17:22Z MESH: simulation protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.9996111 chemical cleaner0 2023-07-12T16:45:30Z CHEBI: Na+ 0.9904822 protein_state cleaner0 2023-07-12T20:00:30Z DUMMY: bound at 0.98990685 site cleaner0 2023-07-12T15:36:03Z SO: Sext 0.9873886 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.98048145 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99961627 chemical cleaner0 2023-07-12T16:45:27Z CHEBI: Na+ 0.9982833 protein_state cleaner0 2023-07-12T20:00:34Z DUMMY: bound only at 0.98023087 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9603746 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.99787515 structure_element cleaner0 2023-07-12T19:39:19Z SO: N-terminal half 0.99968886 structure_element cleaner0 2023-07-12T19:39:22Z SO: TM7 0.9995776 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab chemical CHEBI: cleaner0 2023-07-12T16:45:46Z Na+ evidence DUMMY: cleaner0 2023-07-12T19:29:41Z simulation snapshots 0.9996201 chemical cleaner0 2023-07-12T16:45:14Z CHEBI: Na+ 0.9945813 protein_state cleaner0 2023-07-12T20:00:38Z DUMMY: bound at 0.98243904 site cleaner0 2023-07-12T15:36:30Z SO: SCa 0.9753448 site cleaner0 2023-07-12T15:35:42Z SO: Sint 0.8014012 protein_state cleaner0 2023-07-12T20:00:41Z DUMMY: without 0.9995755 chemical cleaner0 2023-07-12T16:55:34Z CHEBI: Na+ 0.99646664 protein_state cleaner0 2023-07-12T20:00:45Z DUMMY: bound 0.999531 site cleaner0 2023-07-12T19:35:45Z SO: ion-binding region protein_state DUMMY: cleaner0 2023-07-12T16:46:17Z fully Na+-occupied 0.99973565 structure_element cleaner0 2023-07-12T19:39:25Z SO: TM2 0.99972385 structure_element cleaner0 2023-07-12T15:54:55Z SO: TM3 0.9997049 structure_element cleaner0 2023-07-12T19:39:27Z SO: TM7 0.9995299 site cleaner0 2023-07-12T19:35:49Z SO: ion-binding region protein_state DUMMY: cleaner0 2023-07-12T16:46:46Z partially Na+-occupied 0.9995466 site cleaner0 2023-07-12T19:35:52Z SO: ion-binding region 0.9994396 protein_state cleaner0 2023-07-12T16:45:52Z DUMMY: Na+-free 0.9995689 experimental_method cleaner0 2023-07-12T19:17:30Z MESH: simulations 0.7984423 protein_state cleaner0 2023-07-12T20:00:49Z DUMMY: Na+-occupancy experimental_method MESH: cleaner0 2023-07-12T19:17:49Z Bias-Exchange Metadynamics simulations 0.9994446 evidence cleaner0 2023-07-12T19:29:48Z DUMMY: Probability distributions bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bonding 0.9996165 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab site SO: cleaner0 2023-07-12T15:36:03Z Sext site SO: cleaner0 2023-07-12T15:36:30Z SCa nihms-779827-f0005.jpg F5 FIG fig_caption 57342 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. 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). Contours correspond to 1 kcal/mol intervals. 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. (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. Na+ ions are shown as green spheres. The two inverted-topology repeats in the transporter structure (transparent cartoons) are colored differently (TM1-5, orange; TM6-10, marine). (c) Close-up views of the ion-binding region in the same conformational free-energy minima. Key residues involved in Na+ and water coordination (W) are highlighted (sticks, black lines). The water-density maps in (b) is shown here as a grey mesh. Note D240 is protonated, while E54 and E213 are ionized. 0.9983607 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX evidence DUMMY: cleaner0 2023-07-12T19:30:24Z Calculated conformational free-energy landscapes 0.9370653 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.94464195 chemical cleaner0 2023-07-12T16:47:30Z CHEBI: Na+ protein_state DUMMY: cleaner0 2023-07-12T20:01:13Z no ions bound 0.99926555 evidence cleaner0 2023-07-12T19:30:31Z DUMMY: free energy 0.9853114 site cleaner0 2023-07-12T19:35:57Z SO: aqueous channels 0.99879116 site cleaner0 2023-07-12T15:36:03Z SO: Sext site SO: cleaner0 2023-07-12T15:36:30Z SCa 0.9995549 evidence cleaner0 2023-07-12T19:30:34Z DUMMY: X-ray structures protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj protein_state DUMMY: cleaner0 2023-07-12T20:01:38Z high protein_state DUMMY: cleaner0 2023-07-12T20:01:47Z low 0.9995905 chemical cleaner0 2023-07-12T16:47:28Z CHEBI: Na+ 0.9859681 protein_state cleaner0 2023-07-12T20:01:51Z DUMMY: low pH 0.99949825 evidence cleaner0 2023-07-12T19:30:38Z DUMMY: Density isosurfaces 0.9997478 chemical cleaner0 2023-07-12T16:47:44Z CHEBI: water 0.99945337 site cleaner0 2023-07-12T19:35:59Z SO: ion-binding region 0.93207103 evidence cleaner0 2023-07-12T19:30:42Z DUMMY: conformational free-energy minima 0.9996631 chemical cleaner0 2023-07-12T16:47:46Z CHEBI: Na+ 0.9994018 structure_element cleaner0 2023-07-12T19:39:33Z SO: inverted-topology repeats 0.9994667 protein_type cleaner0 2023-07-12T16:59:10Z MESH: transporter 0.9994167 evidence cleaner0 2023-07-12T19:30:59Z DUMMY: structure 0.999639 structure_element cleaner0 2023-07-12T16:48:04Z SO: TM1-5 0.99954796 structure_element cleaner0 2023-07-12T16:48:07Z SO: TM6-10 0.9993889 site cleaner0 2023-07-12T19:36:03Z SO: ion-binding region 0.8357873 evidence cleaner0 2023-07-12T19:30:52Z DUMMY: conformational free-energy minima 0.99955535 chemical cleaner0 2023-07-12T16:47:48Z CHEBI: Na+ 0.9997657 chemical cleaner0 2023-07-12T16:47:51Z CHEBI: water 0.99945056 evidence cleaner0 2023-07-12T19:30:55Z DUMMY: water-density maps 0.99990237 residue_name_number cleaner0 2023-07-12T15:50:34Z DUMMY: D240 0.9999039 residue_name_number cleaner0 2023-07-12T16:44:31Z DUMMY: E54 0.9999013 residue_name_number cleaner0 2023-07-12T16:44:36Z DUMMY: E213 nihms-779827-f0006.jpg F6 FIG fig_caption 58654 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. The free energy is plotted as in Fig. 5. 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). 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+). Black circles map the crystal structures obtained at high Ca2+ concentration and at low pH (or high H+) reported in this study. (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. 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. Key residues involved in Ca2+ and water coordination (W) are highlighted (sticks, black lines). The water-density maps in (b) are shown here as a grey mesh. 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. 0.797733 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX evidence DUMMY: cleaner0 2023-07-12T19:28:04Z Calculated free-energy landscapes 0.94660574 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing protein PR: cleaner0 2023-07-12T15:33:40Z NCX_Mj 0.9996346 chemical cleaner0 2023-07-12T16:48:25Z CHEBI: Ca2+ 0.99955344 protein_state cleaner0 2023-07-12T20:02:03Z DUMMY: fully protonated 0.9993578 evidence cleaner0 2023-07-12T19:31:13Z DUMMY: free energy 0.9996518 chemical cleaner0 2023-07-12T16:48:30Z CHEBI: Ca2+ 0.9995228 evidence cleaner0 2023-07-12T19:31:18Z DUMMY: map 0.9993074 evidence cleaner0 2023-07-12T19:31:20Z DUMMY: map 0.9995147 evidence cleaner0 2023-07-12T19:31:23Z DUMMY: map 0.99963975 protein_state cleaner0 2023-07-12T20:02:09Z DUMMY: open 0.9994335 protein_state cleaner0 2023-07-12T20:02:12Z DUMMY: semi-open 0.9996573 protein_state cleaner0 2023-07-12T20:02:14Z DUMMY: occluded 0.99516165 protein_state cleaner0 2023-07-12T20:02:26Z DUMMY: bound to 0.9997256 chemical cleaner0 2023-07-12T16:48:28Z CHEBI: Na+ 0.99962234 evidence cleaner0 2023-07-12T19:31:26Z DUMMY: crystal structures 0.9996813 chemical cleaner0 2023-07-12T16:48:35Z CHEBI: Ca2+ 0.9968871 protein_state cleaner0 2023-07-12T20:02:29Z DUMMY: low pH protein_state DUMMY: cleaner0 2023-07-12T16:49:28Z high H+ 0.9993633 evidence cleaner0 2023-07-12T19:31:29Z DUMMY: Water-density isosurfaces 0.6663834 evidence cleaner0 2023-07-12T19:31:32Z DUMMY: free-energy minima 0.9995218 evidence cleaner0 2023-07-12T19:31:34Z DUMMY: free-energy maps 0.9996765 chemical cleaner0 2023-07-12T16:49:32Z CHEBI: Ca2+ 0.99904287 site cleaner0 2023-07-12T19:36:07Z SO: ion-binding region evidence DUMMY: cleaner0 2023-07-12T19:31:50Z conformational free-energy minima 0.9996734 chemical cleaner0 2023-07-12T16:49:34Z CHEBI: Ca2+ 0.9997689 chemical cleaner0 2023-07-12T16:49:36Z CHEBI: water 0.9995133 evidence cleaner0 2023-07-12T19:31:53Z DUMMY: water-density maps 0.99966085 protein_state cleaner0 2023-07-12T20:02:44Z DUMMY: occluded 0.99963033 chemical cleaner0 2023-07-12T16:49:41Z CHEBI: Ca2+ 0.98349607 protein_state cleaner0 2023-07-12T20:02:48Z DUMMY: bound 0.99950075 structure_element cleaner0 2023-07-12T19:40:58Z SO: helix 0.9997874 structure_element cleaner0 2023-07-12T15:41:47Z SO: TM7ab 0.9995513 protein_state cleaner0 2023-07-12T20:02:52Z DUMMY: fully occupied 0.99937093 chemical cleaner0 2023-07-12T16:49:43Z CHEBI: Na+ 0.99990904 residue_name_number cleaner0 2023-07-12T15:41:03Z DUMMY: Ala206 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:17:39Z hydrogen-bonding interaction 0.99990845 residue_name_number cleaner0 2023-07-12T16:52:06Z DUMMY: Ser210 nihms-779827-f0007.jpg F7 FIG fig_caption 60134 Structural mechanism of extracellular forward ion exchange in NCX. 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. The green open cylinders represent the gating helices TM1 and TM6. Asterisks mark the states whose crystal structures have been determined in this study. 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. 0.61637443 protein_type cleaner0 2023-07-12T15:33:13Z MESH: NCX 0.9999052 residue_name_number cleaner0 2023-07-12T16:52:12Z DUMMY: Ala47 0.9998196 structure_element cleaner0 2023-07-12T19:41:05Z SO: TM2b 0.9999049 residue_name_number cleaner0 2023-07-12T15:41:03Z DUMMY: Ala206 0.9998172 structure_element cleaner0 2023-07-12T15:41:27Z SO: TM7b 0.9999027 residue_name_number cleaner0 2023-07-12T15:40:30Z DUMMY: Glu54 0.9998171 structure_element cleaner0 2023-07-12T19:41:08Z SO: TM2c 0.99990034 residue_name_number cleaner0 2023-07-12T15:40:34Z DUMMY: Glu213 0.99981946 structure_element cleaner0 2023-07-12T19:41:11Z SO: TM7c 0.9995922 structure_element cleaner0 2023-07-12T19:41:13Z SO: gating helices 0.999819 structure_element cleaner0 2023-07-12T15:41:41Z SO: TM1 0.9998191 structure_element cleaner0 2023-07-12T15:41:33Z SO: TM6 0.99955064 evidence cleaner0 2023-07-12T19:32:00Z DUMMY: crystal structures 0.9958951 evidence cleaner0 2023-07-12T19:28:04Z DUMMY: calculated free-energy landscapes protein_state DUMMY: cleaner0 2023-07-12T16:50:06Z Ca2+-loaded 0.94450617 protein_state cleaner0 2023-07-12T15:37:00Z DUMMY: outward-facing 0.99857485 protein_state cleaner0 2023-07-12T20:03:00Z DUMMY: occluded 0.99964225 protein_state cleaner0 2023-07-12T20:03:03Z DUMMY: unloaded 0.9994731 protein_state cleaner0 2023-07-12T20:03:06Z DUMMY: fully open T1.xml T1 TABLE table_caption 60772 Concentration-dependent Na+ occupancy of outward-facing NCX_Mj T1.xml T1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Sites \ [Na⁺]</th><th align="center" valign="top" rowspan="1" colspan="1">2.5 mM</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM</th><th align="center" valign="top" rowspan="1" colspan="1">20 mM</th><th align="center" valign="top" rowspan="1" colspan="1">100 mM</th><th align="center" valign="top" rowspan="1" colspan="1">150 mM</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">S_int</td><td align="center" valign="top" rowspan="1" colspan="1">0.84</td><td align="center" valign="top" rowspan="1" colspan="1">0.92</td><td align="center" valign="top" rowspan="1" colspan="1">0.99</td><td align="center" valign="top" rowspan="1" colspan="1">0.96</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">S_Ca</td><td align="center" valign="top" rowspan="1" colspan="1">0.75</td><td align="center" valign="top" rowspan="1" colspan="1">0.87</td><td align="center" valign="top" rowspan="1" colspan="1">0.97</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td><td align="center" valign="top" rowspan="1" colspan="1">1.00</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">S_ext</td><td align="center" valign="top" rowspan="1" colspan="1">-</td><td align="center" valign="top" rowspan="1" colspan="1">-</td><td align="center" valign="top" rowspan="1" colspan="1">0.59</td><td align="center" valign="top" rowspan="1" colspan="1">0.79</td><td align="center" valign="top" rowspan="1" colspan="1">0.93</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><hr/></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Total</td><td align="center" valign="top" rowspan="1" colspan="1">1.58</td><td align="center" valign="top" rowspan="1" colspan="1">1.79</td><td align="center" valign="top" rowspan="1" colspan="1">2.55</td><td align="center" valign="top" rowspan="1" colspan="1">2.75</td><td align="center" valign="top" rowspan="1" colspan="1">2.93</td></tr><tr><td colspan="6" align="left" valign="top" rowspan="1"><hr/></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Conformation</td><td align="center" valign="top" rowspan="1" colspan="1">Partially open</td><td align="center" valign="top" rowspan="1" colspan="1">Partially open</td><td align="center" valign="top" rowspan="1" colspan="1">Mixed</td><td align="center" valign="top" rowspan="1" colspan="1">Occluded</td><td align="center" valign="top" rowspan="1" colspan="1">Occluded</td></tr></tbody></table> 60835 Sites \ [Na+] 2.5 mM 10 mM 20 mM 100 mM 150 mM Sint 0.84 0.92 0.99 0.96 1.00 SCa 0.75 0.87 0.97 1.00 1.00 Sext - - 0.59 0.79 0.93 Total 1.58 1.79 2.55 2.75 2.93 Conformation Partially open Partially open Mixed Occluded Occluded T2.xml T2 TABLE table_caption 61082 Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+, and no Ca2+, and at low pH and no Na+ or Ca2+. T2.xml T2 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Na⁺]</th><th align="center" valign="top" rowspan="1" colspan="1">2.5 mM PDB 5HWX</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM PDB 5HWY</th><th align="center" valign="top" rowspan="1" colspan="1">20 mM PDB 5HXC</th><th align="center" valign="top" rowspan="1" colspan="1">100 mM PDB 5HXE</th><th align="center" valign="top" rowspan="1" colspan="1">150 mM PDB 5HYA</th><th align="center" valign="top" rowspan="1" colspan="1">0 mM PDB 5HXH</th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Data collection</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="5" align="center" valign="top" rowspan="1">P212121</td><td align="center" valign="top" rowspan="1" colspan="1">C2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.70, 72.28, 95.78</td><td align="center" valign="top" rowspan="1" colspan="1">46.21, 71.97, 95.63</td><td align="center" valign="top" rowspan="1" colspan="1">49.75, 72.56, 95.78</td><td align="center" valign="top" rowspan="1" colspan="1">49.77, 72.85, 96.36</td><td align="center" valign="top" rowspan="1" colspan="1">49.49, 72.88, 96.21</td><td align="center" valign="top" rowspan="1" colspan="1">164.18, 46.83, 96.96</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 106.20, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.40 (2.44-2.40)</td><td align="center" valign="top" rowspan="1" colspan="1">2.10 (2.14-2.10)</td><td align="center" valign="top" rowspan="1" colspan="1">2.10 (2.14-2.10)</td><td align="center" valign="top" rowspan="1" colspan="1">2.28 (2.32-2.28)</td><td align="center" valign="top" rowspan="1" colspan="1">1.90 (1.93-1.90)</td><td align="center" valign="top" rowspan="1" colspan="1">2.80 (2.85-2.80)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_sym (%)</td><td align="center" valign="top" rowspan="1" colspan="1">9.4 (99.4)</td><td align="center" valign="top" rowspan="1" colspan="1">9.3 (69.8)</td><td align="center" valign="top" rowspan="1" colspan="1">10.5 (99.6)</td><td align="center" valign="top" rowspan="1" colspan="1">9.9 (56.3)</td><td align="center" valign="top" rowspan="1" colspan="1">8.6 (88.0)</td><td align="center" valign="top" rowspan="1" colspan="1">10.5 (94.3)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">24.2 (1.8)</td><td align="center" valign="top" rowspan="1" colspan="1">24.3 (2.7)</td><td align="center" valign="top" rowspan="1" colspan="1">20.6 (1.9)</td><td align="center" valign="top" rowspan="1" colspan="1">15.9 (2.8)</td><td align="center" valign="top" rowspan="1" colspan="1">32.9 (2.4)</td><td align="center" valign="top" rowspan="1" colspan="1">17.7 (1.2)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC_1/2</td><td align="center" valign="top" rowspan="1" colspan="1">(0.625)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.819)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.602)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.722)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.786)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.561)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">99.8 (99.1)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (99.8)</td><td align="center" valign="top" rowspan="1" colspan="1">96.4 (98.6)</td><td align="center" valign="top" rowspan="1" colspan="1">98.9 (97.8)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (99.9)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">6.9 (6.7)</td><td align="center" valign="top" rowspan="1" colspan="1">6.1 (6.0)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (7.0)</td><td align="center" valign="top" rowspan="1" colspan="1">3.4 (3.3)</td><td align="center" valign="top" rowspan="1" colspan="1">9.3 (8.9)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (5.7)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Refinement</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.4</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.1</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.1</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.3</td><td align="center" valign="top" rowspan="1" colspan="1">50-1.9</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.80</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">13977</td><td align="center" valign="top" rowspan="1" colspan="1">19254</td><td align="center" valign="top" rowspan="1" colspan="1">20739</td><td align="center" valign="top" rowspan="1" colspan="1">15767</td><td align="center" valign="top" rowspan="1" colspan="1">27923</td><td align="center" valign="top" rowspan="1" colspan="1">21489</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_work/R_free</td><td align="center" valign="top" rowspan="1" colspan="1">0.21/0.25</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.22</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.23</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.24</td><td align="center" valign="top" rowspan="1" colspan="1">0.179/0.207</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.26</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>No. atoms</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2206</td><td align="center" valign="top" rowspan="1" colspan="1">2274</td><td align="center" valign="top" rowspan="1" colspan="1">2366</td><td align="center" valign="top" rowspan="1" colspan="1">2229</td><td align="center" valign="top" rowspan="1" colspan="1">2229</td><td align="center" valign="top" rowspan="1" colspan="1">4410</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">56/3</td><td align="center" valign="top" rowspan="1" colspan="1">154/2</td><td align="center" valign="top" rowspan="1" colspan="1">161/5</td><td align="center" valign="top" rowspan="1" colspan="1">162/6</td><td align="center" valign="top" rowspan="1" colspan="1">257/4</td><td align="center" valign="top" rowspan="1" colspan="1">121/2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">18</td><td align="center" valign="top" rowspan="1" colspan="1">36</td><td align="center" valign="top" rowspan="1" colspan="1">67</td><td align="center" valign="top" rowspan="1" colspan="1">67</td><td align="center" valign="top" rowspan="1" colspan="1">100</td><td align="center" valign="top" rowspan="1" colspan="1">39</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>B-factors</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">53.25</td><td align="center" valign="top" rowspan="1" colspan="1">34.28</td><td align="center" valign="top" rowspan="1" colspan="1">34.91</td><td align="center" valign="top" rowspan="1" colspan="1">39.75</td><td align="center" valign="top" rowspan="1" colspan="1">26.05</td><td align="center" valign="top" rowspan="1" colspan="1">42.98</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">62.94/47.70</td><td align="center" valign="top" rowspan="1" colspan="1">55.86/31.75</td><td align="center" valign="top" rowspan="1" colspan="1">55.25/36.47</td><td align="center" valign="top" rowspan="1" colspan="1">58.24/38.93</td><td align="center" valign="top" rowspan="1" colspan="1">46.11/22.13</td><td align="center" valign="top" rowspan="1" colspan="1">54.29/63.47</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">58.12</td><td align="center" valign="top" rowspan="1" colspan="1">41.16</td><td align="center" valign="top" rowspan="1" colspan="1">46.97</td><td align="center" valign="top" rowspan="1" colspan="1">49.74</td><td align="center" valign="top" rowspan="1" colspan="1">37.80</td><td align="center" valign="top" rowspan="1" colspan="1">33.17</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>R.m.s deviations</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.006</td><td align="center" valign="top" rowspan="1" colspan="1">0.008</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td><td align="center" valign="top" rowspan="1" colspan="1">0.006</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.819</td><td align="center" valign="top" rowspan="1" colspan="1">0.915</td><td align="center" valign="top" rowspan="1" colspan="1">1.269</td><td align="center" valign="top" rowspan="1" colspan="1">1.024</td><td align="center" valign="top" rowspan="1" colspan="1">0.966</td><td align="center" valign="top" rowspan="1" colspan="1">0.705</td></tr></tbody></table> 61256 [Na+] 2.5 mM PDB 5HWX 10 mM PDB 5HWY 20 mM PDB 5HXC 100 mM PDB 5HXE 150 mM PDB 5HYA 0 mM PDB 5HXH Data collection Space group P212121 C2 Cell dimensions a, b, c (Å) 49.70, 72.28, 95.78 46.21, 71.97, 95.63 49.75, 72.56, 95.78 49.77, 72.85, 96.36 49.49, 72.88, 96.21 164.18, 46.83, 96.96 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 106.20, 90 Resolution (Å) 2.40 (2.44-2.40) 2.10 (2.14-2.10) 2.10 (2.14-2.10) 2.28 (2.32-2.28) 1.90 (1.93-1.90) 2.80 (2.85-2.80) Rsym (%) 9.4 (99.4) 9.3 (69.8) 10.5 (99.6) 9.9 (56.3) 8.6 (88.0) 10.5 (94.3) I/σI 24.2 (1.8) 24.3 (2.7) 20.6 (1.9) 15.9 (2.8) 32.9 (2.4) 17.7 (1.2) CC1/2 (0.625) (0.819) (0.602) (0.722) (0.786) (0.561) Completeness (%) 99.8 (99.1) 99.9 (100) 99.9 (99.8) 96.4 (98.6) 98.9 (97.8) 99.9 (99.9) Redundancy 6.9 (6.7) 6.1 (6.0) 7.1 (7.0) 3.4 (3.3) 9.3 (8.9) 7.1 (5.7) Refinement Resolution (Å) 50-2.4 50-2.1 50-2.1 50-2.3 50-1.9 50-2.80 No. reflections 13977 19254 20739 15767 27923 21489 Rwork/Rfree 0.21/0.25 0.19/0.22 0.19/0.23 0.19/0.24 0.179/0.207 0.20/0.26 No. atoms Protein 2206 2274 2366 2229 2229 4410 Ligand/Ion 56/3 154/2 161/5 162/6 257/4 121/2 Water 18 36 67 67 100 39 B-factors Protein 53.25 34.28 34.91 39.75 26.05 42.98 Ligand/Ion 62.94/47.70 55.86/31.75 55.25/36.47 58.24/38.93 46.11/22.13 54.29/63.47 Water 58.12 41.16 46.97 49.74 37.80 33.17 R.m.s deviations Bond lengths (Å) 0.004 0.006 0.008 0.003 0.006 0.003 Bond angles (°) 0.819 0.915 1.269 1.024 0.966 0.705 T2.xml T2 TABLE table_foot 62823 Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. T3.xml T3 TABLE table_caption 62986 Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+ and Sr2+. T3.xml T3 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Sr²⁺] / [Na⁺]</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 2.5 mM PDB 5HXS</th><th align="center" valign="top" rowspan="1" colspan="1">1 mM / 2.5 mM N/A^{<xref ref-type="table-fn" rid="TFN1">*</xref>}</th><th align="center" valign="top" rowspan="1" colspan="1">0.1 mM / 2.5 mM N/A^{<xref ref-type="table-fn" rid="TFN1">*</xref>}</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 10 mM N/A^{<xref ref-type="table-fn" rid="TFN1">*</xref>}</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 100 mM N/A^{<xref ref-type="table-fn" rid="TFN2">#</xref>}</th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Data collection</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="5" align="center" valign="top" rowspan="1">P212121</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.52, 72.35, 96.00</td><td align="center" valign="top" rowspan="1" colspan="1">49.76, 72.62, 95.62</td><td align="center" valign="top" rowspan="1" colspan="1">49.80, 72.27, 94.84</td><td align="center" valign="top" rowspan="1" colspan="1">49.67, 72.46, 96.43</td><td align="center" valign="top" rowspan="1" colspan="1">49.88, 72.43, 95.91</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.80 (2.85-2.80)</td><td align="center" valign="top" rowspan="1" colspan="1">2.90 (2.95-2.90)</td><td align="center" valign="top" rowspan="1" colspan="1">2.54 (2.58-2.54)</td><td align="center" valign="top" rowspan="1" colspan="1">2.30 (2.34-2.30)</td><td align="center" valign="top" rowspan="1" colspan="1">2.50 (2.54-2.50)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_sym (%)</td><td align="center" valign="top" rowspan="1" colspan="1">12.3 (88.0)</td><td align="center" valign="top" rowspan="1" colspan="1">12.3 (91.0)</td><td align="center" valign="top" rowspan="1" colspan="1">11.9 (88.5)</td><td align="center" valign="top" rowspan="1" colspan="1">13.7 (95.8)</td><td align="center" valign="top" rowspan="1" colspan="1">10.9 (96.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">19.0 (1.8)</td><td align="center" valign="top" rowspan="1" colspan="1">19.2 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">21.5 (1.9)</td><td align="center" valign="top" rowspan="1" colspan="1">18.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">19.7 (1.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC_1/2</td><td align="center" valign="top" rowspan="1" colspan="1">(0.674)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.686)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.768)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.420)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.767)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">98.8 (90.4)</td><td align="center" valign="top" rowspan="1" colspan="1">94.9 (96.1)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.8 (99.9)</td><td align="center" valign="top" rowspan="1" colspan="1">99.6 (99.7)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">6.5 (5.4)</td><td align="center" valign="top" rowspan="1" colspan="1">6.7 (6.9)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (6.8)</td><td align="center" valign="top" rowspan="1" colspan="1">6.2 (4.5)</td><td align="center" valign="top" rowspan="1" colspan="1">5.9 (6.0)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Refinement</bold> </td><td colspan="5" align="center" valign="top" rowspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.80</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.90</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.54</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.3</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.5</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">8927</td><td align="center" valign="top" rowspan="1" colspan="1">7611</td><td align="center" valign="top" rowspan="1" colspan="1">11483</td><td align="center" valign="top" rowspan="1" colspan="1">16488</td><td align="center" valign="top" rowspan="1" colspan="1">12665</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_work/R_free</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.27</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.27</td><td align="center" valign="top" rowspan="1" colspan="1">0.23/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.25</td><td align="center" valign="top" rowspan="1" colspan="1">0.21/0.24</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>No. atoms</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2227</td><td align="center" valign="top" rowspan="1" colspan="1">2249</td><td align="center" valign="top" rowspan="1" colspan="1">2217</td><td align="center" valign="top" rowspan="1" colspan="1">2271</td><td align="center" valign="top" rowspan="1" colspan="1">2223</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">110/2</td><td align="center" valign="top" rowspan="1" colspan="1">133/3</td><td align="center" valign="top" rowspan="1" colspan="1">155/3</td><td align="center" valign="top" rowspan="1" colspan="1">101/3</td><td align="center" valign="top" rowspan="1" colspan="1">104/6</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">10</td><td align="center" valign="top" rowspan="1" colspan="1">2</td><td align="center" valign="top" rowspan="1" colspan="1">5</td><td align="center" valign="top" rowspan="1" colspan="1">34</td><td align="center" valign="top" rowspan="1" colspan="1">17</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>B-factors</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">61.55</td><td align="center" valign="top" rowspan="1" colspan="1">67.82</td><td align="center" valign="top" rowspan="1" colspan="1">60.83</td><td align="center" valign="top" rowspan="1" colspan="1">46.42</td><td align="center" valign="top" rowspan="1" colspan="1">56.75</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">74.56/63.32</td><td align="center" valign="top" rowspan="1" colspan="1">85.56/70.47</td><td align="center" valign="top" rowspan="1" colspan="1">80.33/90.41</td><td align="center" valign="top" rowspan="1" colspan="1">64.79/38.25</td><td align="center" valign="top" rowspan="1" colspan="1">71.27/53.50</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">59.22</td><td align="center" valign="top" rowspan="1" colspan="1">61.31</td><td align="center" valign="top" rowspan="1" colspan="1">68.58</td><td align="center" valign="top" rowspan="1" colspan="1">51.39</td><td align="center" valign="top" rowspan="1" colspan="1">55.07</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>R.m.s deviations</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.005</td><td align="center" valign="top" rowspan="1" colspan="1">0.005</td><td align="center" valign="top" rowspan="1" colspan="1">0.007</td><td align="center" valign="top" rowspan="1" colspan="1">0.003</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.887</td><td align="center" valign="top" rowspan="1" colspan="1">1.076</td><td align="center" valign="top" rowspan="1" colspan="1">0.963</td><td align="center" valign="top" rowspan="1" colspan="1">1.057</td><td align="center" valign="top" rowspan="1" colspan="1">0.733</td></tr></tbody></table> 63122 [Sr2+] / [Na+] 10 mM / 2.5 mM PDB 5HXS 1 mM / 2.5 mM N/A* 0.1 mM / 2.5 mM N/A* 10 mM / 10 mM N/A* 10 mM / 100 mM N/A# Data collection Space group P212121 Cell dimensions a, b, c (Å) 49.52, 72.35, 96.00 49.76, 72.62, 95.62 49.80, 72.27, 94.84 49.67, 72.46, 96.43 49.88, 72.43, 95.91 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 2.80 (2.85-2.80) 2.90 (2.95-2.90) 2.54 (2.58-2.54) 2.30 (2.34-2.30) 2.50 (2.54-2.50) Rsym (%) 12.3 (88.0) 12.3 (91.0) 11.9 (88.5) 13.7 (95.8) 10.9 (96.8) I/σI 19.0 (1.8) 19.2 (1.6) 21.5 (1.9) 18.4 (1.6) 19.7 (1.8) CC1/2 (0.674) (0.686) (0.768) (0.420) (0.767) Completeness (%) 98.8 (90.4) 94.9 (96.1) 99.9 (100.0) 99.8 (99.9) 99.6 (99.7) Redundancy 6.5 (5.4) 6.7 (6.9) 7.1 (6.8) 6.2 (4.5) 5.9 (6.0) Refinement Resolution (Å) 50-2.80 50-2.90 50-2.54 50-2.3 50-2.5 No. reflections 8927 7611 11483 16488 12665 Rwork/Rfree 0.22/0.27 0.22/0.27 0.23/0.26 0.20/0.25 0.21/0.24 No. atoms Protein 2227 2249 2217 2271 2223 Ligand/Ion 110/2 133/3 155/3 101/3 104/6 Water 10 2 5 34 17 B-factors Protein 61.55 67.82 60.83 46.42 56.75 Ligand/Ion 74.56/63.32 85.56/70.47 80.33/90.41 64.79/38.25 71.27/53.50 Water 59.22 61.31 68.58 51.39 55.07 R.m.s deviations Bond lengths (Å) 0.004 0.005 0.005 0.007 0.003 Bond angles (°) 0.887 1.076 0.963 1.057 0.733 T3.xml T3 TABLE table_foot 64519 Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. T3.xml T3 TABLE table_footnote 64682 These structures are virtually identical to that resolved with 10 mM Sr2+ (PDB 5HXS), except for the weakened electron-density signal for the divalent ion, and were therefore not deposited in the PDB. T3.xml T3 TABLE table_footnote 64883 This structure is virtually identical to that resolved with 100 mM Na+ without Ca2+ or Sr2+ (Table 1, PDB 5HXE) and was therefore not deposited in the PDB. T4.xml T4 TABLE table_caption 65039 Data collection and refinement statistics for the NCX_Mj structures obtained from crystals soaked with varying amounts of Na+ and Ca2+. T4.xml T4 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="box" rules="all"><thead><tr><th align="center" valign="top" rowspan="1" colspan="1">[Ca²⁺] / [Na⁺]</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 2.5 mM PDB 5HXR</th><th align="center" valign="top" rowspan="1" colspan="1">1 mM / 2.5 mM N/A^{<xref ref-type="table-fn" rid="TFN3">*</xref>}</th><th align="center" valign="top" rowspan="1" colspan="1">0.1 mM / 2.5 mM N/A^{<xref ref-type="table-fn" rid="TFN3">*</xref>}</th><th align="center" valign="top" rowspan="1" colspan="1">10 mM / 10 mM N/A^{<xref ref-type="table-fn" rid="TFN3">*</xref>}</th></tr></thead><tbody><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Data collection</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Space group</td><td colspan="4" align="center" valign="top" rowspan="1">P212121</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Cell dimensions</td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">a, b, c (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">49.70, 72.52, 96.94</td><td align="center" valign="top" rowspan="1" colspan="1">49.80, 72.26, 95.80</td><td align="center" valign="top" rowspan="1" colspan="1">49.48, 72.47, 96.30</td><td align="center" valign="top" rowspan="1" colspan="1">49.88, 72.22, 96.10</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">α, β, γ (°)</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td><td align="center" valign="top" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">2.45 (2.49-2.45)</td><td align="center" valign="top" rowspan="1" colspan="1">2.65 (2.70-2.65)</td><td align="center" valign="top" rowspan="1" colspan="1">2.40 (2.44-2.40)</td><td align="center" valign="top" rowspan="1" colspan="1">2.20 (2.24-2.20)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_sym (%)</td><td align="center" valign="top" rowspan="1" colspan="1">11.8 (94.2)</td><td align="center" valign="top" rowspan="1" colspan="1">11.1 (92.1)</td><td align="center" valign="top" rowspan="1" colspan="1">11.2 (91.3)</td><td align="center" valign="top" rowspan="1" colspan="1">10.3 (99.1)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">I/σI</td><td align="center" valign="top" rowspan="1" colspan="1">24.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">20.9 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">20.4 (1.6)</td><td align="center" valign="top" rowspan="1" colspan="1">22.4 (1.8)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">CC_1/2</td><td align="center" valign="top" rowspan="1" colspan="1">(0.611)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.696)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.632)</td><td align="center" valign="top" rowspan="1" colspan="1">(0.549)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Completeness (%)</td><td align="center" valign="top" rowspan="1" colspan="1">99.7 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.9 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.2 (100.0)</td><td align="center" valign="top" rowspan="1" colspan="1">99.7 (100.0)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Redundancy</td><td align="center" valign="top" rowspan="1" colspan="1">7.8 (7.8)</td><td align="center" valign="top" rowspan="1" colspan="1">7.0 (6.5)</td><td align="center" valign="top" rowspan="1" colspan="1">6.9 (7.0)</td><td align="center" valign="top" rowspan="1" colspan="1">7.1 (7.1)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>Refinement</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Resolution (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.45</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.65</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.40</td><td align="center" valign="top" rowspan="1" colspan="1">50-2.2</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">No. reflections</td><td align="center" valign="top" rowspan="1" colspan="1">12996</td><td align="center" valign="top" rowspan="1" colspan="1">10548</td><td align="center" valign="top" rowspan="1" colspan="1">13736</td><td align="center" valign="top" rowspan="1" colspan="1">18080</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">R_work/R_free</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.22/0.28</td><td align="center" valign="top" rowspan="1" colspan="1">0.20/0.26</td><td align="center" valign="top" rowspan="1" colspan="1">0.19/0.24</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>No. atoms</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">2211</td><td align="center" valign="top" rowspan="1" colspan="1">2225</td><td align="center" valign="top" rowspan="1" colspan="1">2228</td><td align="center" valign="top" rowspan="1" colspan="1">2284</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">114/3</td><td align="center" valign="top" rowspan="1" colspan="1">130/3</td><td align="center" valign="top" rowspan="1" colspan="1">162/3</td><td align="center" valign="top" rowspan="1" colspan="1">164/4</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">16</td><td align="center" valign="top" rowspan="1" colspan="1">20</td><td align="center" valign="top" rowspan="1" colspan="1">42</td><td align="center" valign="top" rowspan="1" colspan="1">57</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>B-factors</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Protein</td><td align="center" valign="top" rowspan="1" colspan="1">64.97</td><td align="center" valign="top" rowspan="1" colspan="1">71.22</td><td align="center" valign="top" rowspan="1" colspan="1">53.50</td><td align="center" valign="top" rowspan="1" colspan="1">45.16</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Ligand/Ion</td><td align="center" valign="top" rowspan="1" colspan="1">78.98/60.53</td><td align="center" valign="top" rowspan="1" colspan="1">90.03/72.90</td><td align="center" valign="top" rowspan="1" colspan="1">70.67/45.53</td><td align="center" valign="top" rowspan="1" colspan="1">69.09/42.92</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Water</td><td align="center" valign="top" rowspan="1" colspan="1">68.57</td><td align="center" valign="top" rowspan="1" colspan="1">81.02</td><td align="center" valign="top" rowspan="1" colspan="1">64.12</td><td align="center" valign="top" rowspan="1" colspan="1">55.45</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1"> <bold>R.m.s deviations</bold> </td><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1"/></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="center" valign="top" rowspan="1" colspan="1">0.002</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td><td align="center" valign="top" rowspan="1" colspan="1">0.002</td><td align="center" valign="top" rowspan="1" colspan="1">0.004</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Bond angles (°)</td><td align="center" valign="top" rowspan="1" colspan="1">0.630</td><td align="center" valign="top" rowspan="1" colspan="1">1.143</td><td align="center" valign="top" rowspan="1" colspan="1">0.686</td><td align="center" valign="top" rowspan="1" colspan="1">0.650</td></tr></tbody></table> 65175 [Ca2+] / [Na+] 10 mM / 2.5 mM PDB 5HXR 1 mM / 2.5 mM N/A* 0.1 mM / 2.5 mM N/A* 10 mM / 10 mM N/A* Data collection Space group P212121 Cell dimensions a, b, c (Å) 49.70, 72.52, 96.94 49.80, 72.26, 95.80 49.48, 72.47, 96.30 49.88, 72.22, 96.10 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 2.45 (2.49-2.45) 2.65 (2.70-2.65) 2.40 (2.44-2.40) 2.20 (2.24-2.20) Rsym (%) 11.8 (94.2) 11.1 (92.1) 11.2 (91.3) 10.3 (99.1) I/σI 24.4 (1.6) 20.9 (1.6) 20.4 (1.6) 22.4 (1.8) CC1/2 (0.611) (0.696) (0.632) (0.549) Completeness (%) 99.7 (100.0) 99.9 (100.0) 99.2 (100.0) 99.7 (100.0) Redundancy 7.8 (7.8) 7.0 (6.5) 6.9 (7.0) 7.1 (7.1) Refinement Resolution (Å) 50-2.45 50-2.65 50-2.40 50-2.2 No. reflections 12996 10548 13736 18080 Rwork/Rfree 0.22/0.26 0.22/0.28 0.20/0.26 0.19/0.24 No. atoms Protein 2211 2225 2228 2284 Ligand/Ion 114/3 130/3 162/3 164/4 Water 16 20 42 57 B-factors Protein 64.97 71.22 53.50 45.16 Ligand/Ion 78.98/60.53 90.03/72.90 70.67/45.53 69.09/42.92 Water 68.57 81.02 64.12 55.45 R.m.s deviations Bond lengths (Å) 0.002 0.004 0.002 0.004 Bond angles (°) 0.630 1.143 0.686 0.650 T4.xml T4 TABLE table_foot 66383 Values in parenthesis are for highest resolution shell. 5% of the data was used in the Rfree calculation. ‘Ligand’ atoms are from lipids, PEG400 and acetates. T4.xml T4 TABLE table_footnote 66546 These structures are virtually identical to that resolved with 10 mM Ca2+ and 2.5 mM Na+ (PDB 5HXR), except for the weakened electron-density signal for the divalent ion, and were therefore not deposited in the PDB.