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- PDB-9rh5: Cryo-EM structure of the inward-facing apo NhaA in the unplugged ... -

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Basic information

Entry
Database: PDB / ID: 9rh5
TitleCryo-EM structure of the inward-facing apo NhaA in the unplugged state at pH 6.3
Components
  • Fv6F9 heavy chain
  • Fv6F9 light chain
  • Na(+)/H(+) antiporter NhaA
KeywordsTRANSPORT PROTEIN / NhaA / sodium proton exchanger
Function / homology
Function and homology information


response to alkaline pH / sodium:proton antiporter activity / cardiolipin binding / response to salt stress / regulation of intracellular pH / plasma membrane
Similarity search - Function
Na+/H+ antiporter NhaA / Na+/H+ antiporter domain superfamily / Na+/H+ antiporter 1
Similarity search - Domain/homology
Na(+)/H(+) antiporter NhaA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsWeng, T.-H. / Safarian, S. / Michel, H.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2026
Title: pH-dependent activation of the Na/H antiporter NhaA and conformational dynamics of its N-terminus.
Authors: Tsai-Hsuan Weng / Balázs Fábián / Elena Olkhova / Sonja Welsch / Sarah Luise Schmidt / Tsafi Danieli / Yael Keren / Abraham Rimon / Schara Safarian / Gerhard Hummer / Etana Padan / Hartmut Michel /
Abstract: Na⁺/H⁺ antiporters are vital for regulating intracellular pH and sodium ion levels across all domains of life. In Escherichia coli, NhaA is the principal Na⁺/H⁺ antiporter, exhibiting strong ...Na⁺/H⁺ antiporters are vital for regulating intracellular pH and sodium ion levels across all domains of life. In Escherichia coli, NhaA is the principal Na⁺/H⁺ antiporter, exhibiting strong pH sensitivity and rapid turnover, yet the structural transitions underlying its activation and substrate recognition have remained obscure. Here, we use single-particle cryo-electron microscopy to determine the conformational ensemble of NhaA across a physiological pH range and in the presence of Na⁺, complemented by constant-pH molecular dynamics simulations. High-resolution structures of apo and Na⁺-bound NhaA reconstituted in lipid nanodiscs reveal progressive opening of the cytoplasmic funnel with increasing pH. We also visualize the previously unresolved N-terminal tail, which forms a dynamic plug at the cytoplasmic entrance under low-pH conditions and disengages at alkaline pH, coinciding with activation. The Na⁺-bound structure captures Na⁺ coordination at the ion-binding site, and simulations suggest potential roles for the conserved charged residues. Together, these findings illuminate how pH sensing, N-terminal gating, and substrate binding are structurally coordinated in NhaA, providing a framework for understanding Na⁺/H⁺ antiporter activation and regulation, and the basis for targeting clinical important antiporters.
History
DepositionJun 8, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 17, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Jun 17, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Jul 1, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jul 1, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Na(+)/H(+) antiporter NhaA
B: Fv6F9 heavy chain
C: Fv6F9 light chain


Theoretical massNumber of molelcules
Total (without water)70,7143
Polymers70,7143
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Na(+)/H(+) antiporter NhaA / Na(+)/H(+) exchanger / Na(+)/Li(+)/H(+) antiporter / Sodium/proton antiporter NhaA


Mass: 43585.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: nhaA, ant, b0019, JW0018 / Production host: Escherichia coli (E. coli) / References: UniProt: P13738
#2: Antibody Fv6F9 heavy chain


Mass: 13934.411 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#3: Antibody Fv6F9 light chain


Mass: 13193.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1NhaA in complex with Fv6F9COMPLEXall0MULTIPLE SOURCES
2NhaACOMPLEX#11RECOMBINANT
3Fv6F9COMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.07 MDaNO
210.043 MDaNO
310.027 MDaNO
43
52
63
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
33Mus musculus (house mouse)10090
42Escherichia coli (E. coli)562
53Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 6.3
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5aparticle selection
2PHENIX1.20.1_4487:model refinement
3EPU3.6image acquisition
4RELION4CTF correction
5ISOLDEmodel fitting
6Coot0.9.8model fitting
11RELION4initial Euler assignment
12RELION4final Euler assignment
13RELION4classification
14RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87709 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingPDB-ID: 9RH1

9rh1


Accession code: 9RH1 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034895
ELECTRON MICROSCOPYf_angle_d0.6976622
ELECTRON MICROSCOPYf_dihedral_angle_d10.271765
ELECTRON MICROSCOPYf_chiral_restr0.04765
ELECTRON MICROSCOPYf_plane_restr0.009803

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