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- EMDB-23343: Cryo-EM Structure of KdpFABC in E2Pi state with AlF4 -

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Basic information

Entry
Database: EMDB / ID: EMD-23343
TitleCryo-EM Structure of KdpFABC in E2Pi state with AlF4
Map dataSharpened map from Non-Uniform Refinement in cryoSPARC
Sample
  • Complex: KdpFABC in E2Pi state with AlF4 in sample buffer
    • Protein or peptide: Potassium-transporting ATPase potassium-binding subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit
    • Protein or peptide: Potassium-transporting ATPase KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit
Function / homology
Function and homology information


P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity ...P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Potassium-transporting ATPase potassium-binding subunit / Potassium-transporting ATPase ATP-binding subunit / Potassium-transporting ATPase KdpC subunit / Potassium-transporting ATPase KdpF subunit
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsSweet ME / Larsen C / Pedersen BP / Stokes DL
Funding support United States, Denmark, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM108043 United States
Danish Council for Independent ResearchDFF-8021-00161 Denmark
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural basis for potassium transport in prokaryotes by KdpFABC.
Authors: Marie E Sweet / Casper Larsen / Xihui Zhang / Michael Schlame / Bjørn P Pedersen / David L Stokes /
Abstract: KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K ...KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K or water and which define a pathway for K transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K to the cytoplasm.
History
DepositionJan 25, 2021-
Header (metadata) releaseFeb 10, 2021-
Map releaseFeb 10, 2021-
UpdateAug 11, 2021-
Current statusAug 11, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23343.png

Downloads & links

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Map

FileDownload / File: emd_23343.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map from Non-Uniform Refinement in cryoSPARC
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.6419113 - 1.0509913
Average (Standard dev.)0.00023759042 (±0.01917086)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 338.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z338.880338.880338.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.6421.0510.000

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Supplemental data

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Half map: Un-sharpened half-map A from Non-Uniform Refinement in cryoSPARC

Fileemd_23343_half_map_1.map
AnnotationUn-sharpened half-map A from Non-Uniform Refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Un-sharpened half-map B from Non-Uniform Refinement in cryoSPARC

Fileemd_23343_half_map_2.map
AnnotationUn-sharpened half-map B from Non-Uniform Refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : KdpFABC in E2Pi state with AlF4 in sample buffer

EntireName: KdpFABC in E2Pi state with AlF4 in sample buffer
Components
  • Complex: KdpFABC in E2Pi state with AlF4 in sample buffer
    • Protein or peptide: Potassium-transporting ATPase potassium-binding subunit
    • Protein or peptide: Potassium-transporting ATPase ATP-binding subunit
    • Protein or peptide: Potassium-transporting ATPase KdpC subunit
    • Protein or peptide: Potassium-transporting ATPase KdpF subunit

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Supramolecule #1: KdpFABC in E2Pi state with AlF4 in sample buffer

SupramoleculeName: KdpFABC in E2Pi state with AlF4 in sample buffer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: KdpFABC complex solublized in decyl maltopyranoside (DM) detergent.
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: TK2498 / Recombinant plasmid: pSD107
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: Potassium-transporting ATPase potassium-binding subunit

MacromoleculeName: Potassium-transporting ATPase potassium-binding subunit
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV ...String:
MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV DLLRITLWVL VPVALLIALF FIQQGALQNF LPYQAVNTVE GAQQLLPMGP VASQEAIKML GTNGGGFFNA NS SHPFENP TALTNFVQML AIFLIPTALC FAFGEVMGDR RQGRMLLWAM SVIFVICVGV VMWAEVQGNP HLLALGTDSS INM EGKESR FGVLVSSLFA VVTTAASCGA VIAMHDSFTA LGGMVPMWLM QIGEVVFGGV GSGLYGMMLF VLLAVFIAGL MIGR TPEYL GKKIDVREMK LTALAILVTP TLVLMGAALA MMTDAGRSAM LNPGPHGFSE VLYAVSSAAN NNGSAFAGLS ANSPF WNCL LAFCMFVGRF GVIIPVMAIA GSLVSKKSQA ASSGTLPTHG PLFVGLLIGT VLLVGALTFI PALALGPVAE YLS

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Macromolecule #2: Potassium-transporting ATPase ATP-binding subunit

MacromoleculeName: Potassium-transporting ATPase ATP-binding subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: P-type K+ transporter
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE ...String:
MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE SGGDFASVTG GTRILSDWLV IECSVNPGET FLDRMIAMVE GAQRRKTPNE IALTILLIAL TIVFLLATAT LW PFSAWGG NAVSVTVLVA LLVCLIPTTI GGLLSAIGVA GMSRMLGANV IATSGRAVEA AGDVDVLLLD KTGTITLGNR QAS EFIPAQ GVDEKTLADA AQLASLADET PEGRSIVILA KQRFNLRERD VQSLHATFVP FTAQSRMSGI NIDNRMIRKG SVDA IRRHV EANGGHFPTD VDQKVDQVAR QGATPLVVVE GSRVLGVIAL KDIVKGGIKE RFAQLRKMGI KTVMITGDNR LTAAA IAAE AGVDDFLAEA TPEAKLALIR QYQAEGRLVA MTGDGTNDAP ALAQADVAVA MNSGTQAAKE AGNMVDLDSN PTKLIE VVH IGKQMLMTRG SLTTFSIAND VAKYFAIIPA AFAATYPQLN ALNIMCLHSP DSAILSAVIF NALIIVFLIP LALKGVS YK PLTASAMLRR NLWIYGLGGL LVPFIGIKVI DLLLTVCGLV

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Macromolecule #3: Potassium-transporting ATPase KdpC subunit

MacromoleculeName: Potassium-transporting ATPase KdpC subunit / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV ...String:
MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV KYIGQPVVNI VELNLALDKL DEGTGLVPRG SSHHHHHHHH

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Macromolecule #4: Potassium-transporting ATPase KdpF subunit

MacromoleculeName: Potassium-transporting ATPase KdpF subunit / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSAGVITGVL LVFLLLGYLV YALINAEAF

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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

Concentration4.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2tris(hydroxymethyl)aminomethane
200.0 mMNaClSodium chloridesodium chloride
5.0 mMMgCl2magnesium chloride
1.0 mMC9H15O6Ptris(2-carboxyethyl)phosphine
1.5 mg/mLC22H42O11decyl maltopyranoside
1.0 mMAlF4aluminum fluorideAluminium fluoride

Details: AlF4 was added to the protein after size-exclusion chromatography and incubated 1 hr prior to freezing
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: PELCO easiGlow Glow Discharge Cleaning System used for glow discharge with default settings.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0, no wait before plunging..
DetailsDM-solublized KdpFABC complex purified through SEC the same day as grid freezing. Peak fraction diluted to 4.5 mg/mL and complexed with AlF4 for 1 hr at room temperature prior to application to grid.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2636 / Average exposure time: 2.7 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1202004 / Details: template picking
CTF correctionSoftware - Name: cryoSPARC (ver. 2.9.0)
Startup modelType of model: NONE / Details: ab initio
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.9.0)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 2.9.0) / Details: Heterogenous Refinement.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.9.0) / Number images used: 300627
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Cross-correlation

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