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- PDB-6rkd: Molybdenum storage protein under turnover conditions -

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Basic information

Entry
Database: PDB / ID: 6rkd
TitleMolybdenum storage protein under turnover conditions
Components(Molybdenum storage protein subunit ...) x 2
KeywordsMETAL BINDING PROTEIN / molybdenum storage protein / ATPase
Function / homology
Function and homology information


nutrient reservoir activity / molybdenum ion binding / cytoplasm
Similarity search - Function
Molybdenum storage protein subunit alpha/beta / Carbamate kinase / Acetylglutamate kinase-like / Aspartate/glutamate/uridylate kinase / Acetylglutamate kinase-like superfamily / Amino acid kinase family / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-8M0 / ADENOSINE-5'-TRIPHOSPHATE / Chem-J8E / MOLYBDATE ION / MO(VI)(=O)(OH)2 CLUSTER / Molybdenum storage protein subunit beta / Molybdenum storage protein subunit alpha
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBruenle, S. / Mills, D.J. / Vonck, J. / Ermler, U.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism.
Authors: Steffen Brünle / Martin L Eisinger / Juliane Poppe / Deryck J Mills / Julian D Langer / Janet Vonck / Ulrich Ermler /
Abstract: The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αβ) cage-like protein complex under ATP consumption. Here, we suggest ...The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αβ) cage-like protein complex under ATP consumption. Here, we suggest a unique mechanism for the ATP-powered molybdate pumping process based on X-ray crystallography, cryoelectron microscopy, hydrogen-deuterium exchange mass spectrometry, and mutational studies of MoSto from . First, we show that molybdate, ATP, and Mg consecutively bind into the open ATP-binding groove of the β-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the β-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of β-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Metβ149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.
History
DepositionApr 30, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Molybdenum storage protein subunit alpha
B: Molybdenum storage protein subunit beta
C: Molybdenum storage protein subunit alpha
D: Molybdenum storage protein subunit beta
E: Molybdenum storage protein subunit alpha
F: Molybdenum storage protein subunit beta
G: Molybdenum storage protein subunit alpha
H: Molybdenum storage protein subunit beta
I: Molybdenum storage protein subunit alpha
J: Molybdenum storage protein subunit beta
K: Molybdenum storage protein subunit alpha
L: Molybdenum storage protein subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)381,494102
Polymers346,53312
Non-polymers34,96090
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, The dimeric assembly is also seen in crystal structures, but is not distinguishable from crystal contacts.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area98340 Å2
ΔGint-577 kcal/mol
Surface area91140 Å2
MethodPISA

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Components

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Molybdenum storage protein subunit ... , 2 types, 12 molecules ACEGIKBDFHJL

#1: Protein
Molybdenum storage protein subunit alpha / Mo storage protein subunit alpha / MoSto subunit alpha


Mass: 29376.773 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Gene: mosA, Avin_43200 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84308
#2: Protein
Molybdenum storage protein subunit beta / MoSto subunit beta


Mass: 28378.775 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Gene: mosB, Avin_43210 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84253

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Non-polymers , 6 types, 90 molecules

#3: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-8M0 / bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octamolybdenum (VI) / Octamolybdate [Mo(VI)8O28]8-


Mass: 1215.503 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mo8O28
#6: Chemical
ChemComp-J8E / oxidanyl-[[2,2,4,4,4-pentakis($l^{1}-oxidanyl)-1-(oxidanylmolybdenio)-1$l^{3},3-dioxa-2$l^{5},4$l^{5}-dimolybdacyclobut-2-yl]oxy]molybdenum


Mass: 545.770 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: H2Mo4O10
#7: Chemical...
ChemComp-MOO / MOLYBDATE ION / MOLYBDATE


Mass: 159.938 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: MoO4
#8: Chemical
ChemComp-OMO / MO(VI)(=O)(OH)2 CLUSTER


Mass: 145.954 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: H2MoO3

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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: Dimer of A3B3 heterohexamer of molybdenum storage protein
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.38 MDa / Experimental value: NO
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.5
Details: 1 mM molybdate and 1 mM mg-ATP were added before vitrification.
Buffer componentConc.: 50 mM / Name: MOPS/NaOH
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 283 K

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 30000 X / Calibrated magnification: 45045 X / Calibrated defocus min: 900 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL
Image recordingAverage exposure time: 8 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1238
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1RELION2particle selection
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 174681
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137558 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: Phenix_real_space_refine
Atomic model building

3D fitting-ID: 1 / Accession code: 4F6T / Initial refinement model-ID: 1 / PDB-ID: 4F6T

4f6t

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B

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