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- PDB-8uyc: Magnesium transporter MgtA dimer from E. coli in 5 mM MgCl2 and 5... -

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Basic information

Entry
Database: PDB / ID: 8uyc
TitleMagnesium transporter MgtA dimer from E. coli in 5 mM MgCl2 and 5 mM ADP
ComponentsMagnesium-transporting ATPase, P-type 1
KeywordsMEMBRANE PROTEIN / magnesium / transport / dimer / oligomer / cryo-EM / P-type ATPase / ion translocation
Function / homology
Function and homology information


P-type Mg2+ transporter / P-type magnesium transporter activity / magnesium ion transmembrane transport / P-type ion transporter activity / cellular response to magnesium ion / ATPase-coupled monoatomic cation transmembrane transporter activity / intracellular membrane-bounded organelle / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane
Similarity search - Function
P-type ATPase, subfamily IIIB / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N ...P-type ATPase, subfamily IIIB / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / 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
Magnesium-transporting ATPase, P-type 1
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsZeinert, R. / Zhou, F. / Cavalcanti Franco, P.H. / Zoeller, J. / Lessen, H. / Iyer, A. / Langer, J.D. / Sodt, A.J. / Storz, G. / Matthies, D.
Funding support United States, Germany, 6items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)ZIA HD008998 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)ZIA HD008855 United States
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)ZIA HD008955 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)LM594244 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1FI2GM146628-01 United States
German Research Foundation (DFG)3542/1-1 Germany
CitationJournal: To Be Published
Title: Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase
Authors: Zeinert, R. / Zhou, F. / Cavalcanti Franco, P.H. / Zoeller, J. / Lessen, H. / Iyer, A. / Langer, J.D. / Sodt, A.J. / Storz, G. / Matthies, D.
History
DepositionNov 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Magnesium-transporting ATPase, P-type 1
B: Magnesium-transporting ATPase, P-type 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,9328
Polymers200,7862
Non-polymers1466
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Magnesium-transporting ATPase, P-type 1


Mass: 100393.062 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: Escherichia coli K-12 / Gene: mgtA / Plasmid: pETDuet-1
Details (production host): pETDuet-1 is designed for the coexpression of two target genes. pETDuet-1 carries the pBR322-derived ColE1 replicon, the lacI gene and the ampicillin resistance gene.
Production host: Escherichia coli (E. coli) / Strain (production host): Escherichia coli C43 (DE3) / References: UniProt: P0ABB8
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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

ComponentName: Magnesium transporter MgtA from Escherichia coli in the dimeric form in the presence of 5 mM MgCl2 and 5 mM ADP
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.201 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Escherichia coli K-12
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Escherichia coli C43 (DE3) / Plasmid: pETDuet-1
Buffer solutionpH: 7.5
Details: 50 mM Tris/HCl, 50 mM K2SO4, 5 mM MgCl2, 0.007% glycol-diosgenin, 2 mM DTT, 5 mM ADP
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: E. coli MgtA purified with a C-terminal His-tag
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 88 % / Chamber temperature: 278 K

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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 magnification: 105000 X / Calibrated magnification: 60241 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9626
Details: A first dataset was collected with a dose rate of 9 A/px/s (~7.5 on the camera through the sample), exposure time 0.076 s/frame (~1 e-/A2) and total exposure time of 3.795 s per movie (~50 e- ...Details: A first dataset was collected with a dose rate of 9 A/px/s (~7.5 on the camera through the sample), exposure time 0.076 s/frame (~1 e-/A2) and total exposure time of 3.795 s per movie (~50 e-/A2), resulting in a total of 1897 movies with 50 frames each. A second dataset was collected with a dose rate of 9 A/px/s (~7.5 on the camera through the sample), exposure time 0.076 s/frame (~1 e-/A2) and total exposure time of 3.795 s per movie (~50 e-/A2), resulting in a total of 7082 movies with 50 frames each.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryFitting-ID
1cryoSPARCparticle selection
3SerialEM3.8-11image acquisition
5cryoSPARCCTF correction
8UCSF Chimera8.6.10model fitting1
13Coot0.9.8.7model fitting2
14Coot0.9.8.7model refinement2
15PHENIX1.20.1-4487model fitting3
16PHENIX1.20.1-4487model refinement3
17cryoSPARCinitial Euler assignment
19cryoSPARCfinal Euler assignment
21cryoSPARCclassification
23cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1456447
Details: 302,981 particles were initially selected from dataset 1 (1,897 selected movies from 2,519 total). 1,153,466 particles were initially selected from dataset 2 (7,082 selected movies from 7,107 total).
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28666 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1RIGID BODY FITREAL
2FLEXIBLE FITREAL
3FLEXIBLE FITREAL
Atomic model building
ID 3D fitting-IDSource nameType
11AlphaFoldin silico model
22AlphaFoldin silico model
33AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214110
ELECTRON MICROSCOPYf_angle_d0.45519220
ELECTRON MICROSCOPYf_dihedral_angle_d3.671908
ELECTRON MICROSCOPYf_chiral_restr0.0392276
ELECTRON MICROSCOPYf_plane_restr0.0042450

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