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- EMDB-49233: Open conformation of ArsA from L. ferriphilum in presence of MgATP -

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Basic information

Entry
Database: EMDB / ID: EMD-49233
TitleOpen conformation of ArsA from L. ferriphilum in presence of MgATP
Map data
Sample
  • Complex: ArsA D46N/D364N in presence of MgATP
    • Protein or peptide: ArsA D46N/D36N (non-hydrolyzing mutant) from L. ferriphilum
KeywordsATPase / open conformation / ATP / Intradimeric Walker A / HYDROLASE
Biological speciesLeptospirillum ferriphilum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsMahajan S / Rees DC / Clemons WM
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Nucleotide- and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA.
Authors: Shivansh Mahajan / Ashley E Pall / Yancheng E Li / Timothy L Stemmler / Douglas C Rees / William M Clemons /
Abstract: Arsenite (As) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As resistance mechanism in prokaryotes involves proteins encoded by the operon. A ...Arsenite (As) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As resistance mechanism in prokaryotes involves proteins encoded by the operon. A central component of the operon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of As by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-bound states, as well as a distinct MgATP-bound state liganded to As. X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of As to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by As. This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.
History
DepositionFeb 14, 2025-
Header (metadata) releaseAug 20, 2025-
Map releaseAug 20, 2025-
UpdateSep 10, 2025-
Current statusSep 10, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49233.png

Downloads & links

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Map

FileDownload / File: emd_49233.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 320 pix.
= 208. Å		0.65 Å/pix.
x 320 pix.
= 208. Å		0.65 Å/pix.
x 320 pix.
= 208. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.26327786 - 0.41858694
Average (Standard dev.)0.00023111263 (±0.0109988535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 208.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_49233_half_map_1.map
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Half map: #2

Fileemd_49233_half_map_2.map
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Sample components

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Entire : ArsA D46N/D364N in presence of MgATP

EntireName: ArsA D46N/D364N in presence of MgATP
Components
  • Complex: ArsA D46N/D364N in presence of MgATP
    • Protein or peptide: ArsA D46N/D36N (non-hydrolyzing mutant) from L. ferriphilum

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Supramolecule #1: ArsA D46N/D364N in presence of MgATP

SupramoleculeName: ArsA D46N/D364N in presence of MgATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: ArsA D46N/D364N incubated with 2 mM each of MgCl2 and ATP for 3 hours
Source (natural)Organism: Leptospirillum ferriphilum (bacteria) / Strain: ML-04

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Macromolecule #1: ArsA D46N/D36N (non-hydrolyzing mutant) from L. ferriphilum

MacromoleculeName: ArsA D46N/D36N (non-hydrolyzing mutant) from L. ferriphilum
type: protein_or_peptide / ID: 1 / Details: ArsA D46N/D364N (non-hydrolyzing mutant) / Enantiomer: LEVO
Source (natural)Organism: Leptospirillum ferriphilum (bacteria) / Strain: ML-04
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGMKFLQLPP RFMFFTGKGG VGKTSIACAT SIQLANAGKR VLLVSTNPAS NVGQVFGVDI GNRVTPIPAV PHLSALEIDP EAAASAYRER LVGPVRGVLP DDVVKGIEES LSGACTTEIA AFDEFTALLT NAVLTADYQH IIFDTAPTGH TIRLLQLPGA WSGFLEAGKG ...String:
MGMKFLQLPP RFMFFTGKGG VGKTSIACAT SIQLANAGKR VLLVSTNPAS NVGQVFGVDI GNRVTPIPAV PHLSALEIDP EAAASAYRER LVGPVRGVLP DDVVKGIEES LSGACTTEIA AFDEFTALLT NAVLTADYQH IIFDTAPTGH TIRLLQLPGA WSGFLEAGKG DASCLGPLAG LEKQRTQYKA AVEALADPLQ TRLVLVARAQ QATLREVART HEELATIGIK QQHLVINGIL PSAEAANDPL AAAIHEREQT ALKNIPATLT SLPRDLVQLK PFNLVGLDAL RQLLTDLPLQ AHVAADAPIE LDEPGMGDLV DGIEADGHGL VMLMGKGGVG KTTLAAAIAV ELAHRGLPVH LTTSNPAAHL TDTLEASLDN LTVSRIDPHA ETERYRQHVL ETKGAQLDAE GRALLEEDLH SPCTEEIAVF QAFSRIIREA GKKFVVMDTA PTGHTLLLLD ATGAYHREVR RQMGNKGTHF TTPMMQLRDP NQTKVLVVTL AETTPVLEAA KLQADLRRAG IEPWAWIINT SVAAASAKSP LLRQRAANEL REINAVANHH ADRYAVVPLL KEEPIGAERL RALIHPQTHH HHHH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsArsA incubated with 2 mM each of MgCl2 and ADP and 5 mM of sodium arsenite for 3 hours

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48382
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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