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- EMDB-4907: Molybdenum storage protein under turnover conditions -

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Basic information

Entry
Database: EMDB / ID: EMD-4907
TitleMolybdenum storage protein under turnover conditions
Map datamolybdenum storage protein under turnover conditions
Sample
  • Complex: Dimer of A3B3 heterohexamer of molybdenum storage protein
    • Protein or peptide: Molybdenum storage protein subunit alpha
    • Protein or peptide: Molybdenum storage protein subunit beta
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octamolybdenum (VI)
  • Ligand: 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
  • Ligand: MOLYBDATE ION
  • Ligand: MO(VI)(=O)(OH)2 CLUSTER
Function / homologyMolybdenum storage protein subunit alpha/beta / nutrient reservoir activity / molybdenum ion binding / Aspartate/glutamate/uridylate kinase / Amino acid kinase family / Acetylglutamate kinase-like superfamily / cytoplasm / Molybdenum storage protein subunit beta / Molybdenum storage protein subunit alpha
Function and homology information
Biological speciesAzotobacter vinelandii DJ (bacteria) / Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBruenle S / Mills DJ / 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, 2019-
Header (metadata) releaseMay 15, 2019-
Map releaseDec 18, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.068
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.068
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rkd
  • Surface level: 0.068
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4907.png

Downloads & links

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Map

FileDownload / File: emd_4907.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmolybdenum storage protein under turnover conditions
Voxel sizeX=Y=Z: 1.11 Å
Density
Contour LevelBy AUTHOR: 0.068 / Movie #1: 0.068
Minimum - Maximum-0.24090053 - 0.61791456
Average (Standard dev.)-0.0001297631 (±0.02060231)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 222.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.111.111.11
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z222.000222.000222.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2410.618-0.000

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Supplemental data

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Mask #1

Fileemd_4907_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: MoSto half map 1

Fileemd_4907_half_map_1.map
AnnotationMoSto half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: MoSto half map 2

Fileemd_4907_half_map_2.map
AnnotationMoSto half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer of A3B3 heterohexamer of molybdenum storage protein

EntireName: Dimer of A3B3 heterohexamer of molybdenum storage protein
Components
  • Complex: Dimer of A3B3 heterohexamer of molybdenum storage protein
    • Protein or peptide: Molybdenum storage protein subunit alpha
    • Protein or peptide: Molybdenum storage protein subunit beta
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octamolybdenum (VI)
  • Ligand: 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
  • Ligand: MOLYBDATE ION
  • Ligand: MO(VI)(=O)(OH)2 CLUSTER

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Supramolecule #1: Dimer of A3B3 heterohexamer of molybdenum storage protein

SupramoleculeName: Dimer of A3B3 heterohexamer of molybdenum storage protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Azotobacter vinelandii DJ (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 380 KDa

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Macromolecule #1: Molybdenum storage protein subunit alpha

MacromoleculeName: Molybdenum storage protein subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Molecular weightTheoretical: 29.376773 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTDTTNSIKH VISPLARQTL QDRDLTRPVA GKRPIRLLPW LQVVKIGGRV MDRGADAILP LVEELRKLLP EHRLLILTGA GVRARHVFS VGLDLGLPVG SLAPLAASEA GQNGHILAAM LASEGVSYVE HPTVADQLAI HLSATRAVVG SAFPPYHHHE F PGSRIPPH ...String:
MTDTTNSIKH VISPLARQTL QDRDLTRPVA GKRPIRLLPW LQVVKIGGRV MDRGADAILP LVEELRKLLP EHRLLILTGA GVRARHVFS VGLDLGLPVG SLAPLAASEA GQNGHILAAM LASEGVSYVE HPTVADQLAI HLSATRAVVG SAFPPYHHHE F PGSRIPPH RADTGAFLLA DAFGAAGLTI VENVDGIYTA DPNGPDRGQA RFLPETSATD LAKSEGPLPV DRALLDVMAT AR HIERVQV VNGLVPGRLT AALRGEHVGT LIRTGVRPA

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Macromolecule #2: Molybdenum storage protein subunit beta

MacromoleculeName: Molybdenum storage protein subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Molecular weightTheoretical: 28.378775 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MANSTAELEE LLMQRSLTDP QLQAAAAAAA DFRILPDATV IKIGGQSVID RGRAAVYPLV DEIVAARKNH KLLIGTGAGT RARHLYSIA AGLGLPAGVL AQLGSSVADQ NAAMLGQLLA KHGIPVVGGA GLSAVPLSLA EVNAVVFSGM PPYKLWMRPA A EGVIPPYR ...String:
MANSTAELEE LLMQRSLTDP QLQAAAAAAA DFRILPDATV IKIGGQSVID RGRAAVYPLV DEIVAARKNH KLLIGTGAGT RARHLYSIA AGLGLPAGVL AQLGSSVADQ NAAMLGQLLA KHGIPVVGGA GLSAVPLSLA EVNAVVFSGM PPYKLWMRPA A EGVIPPYR TDAGCFLLAE QFGCKQMIFV KDEDGLYTAN PKTSKDATFI PRISVDEMKA KGLHDSILEF PVLDLLQSAQ HV REVQVVN GLVPGNLTRA LAGEHVGTII TAS

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octam...

MacromoleculeName: bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octamolybdenum (VI)
type: ligand / ID: 5 / Number of copies: 12 / Formula: 8M0
Molecular weightTheoretical: 1.215503 KDa
Chemical component information

ChemComp-8M0:
bis(mu4-oxo)-tetrakis(mu3-oxo)-hexakis(mu2-oxo)-hexadecaoxo-octamolybdenum (VI)

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Macromolecule #6: oxidanyl-[[2,2,4,4,4-pentakis($l^{1}-oxidanyl)-1-(oxidanylmolybde...

MacromoleculeName: 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
type: ligand / ID: 6 / Number of copies: 12 / Formula: J8E
Molecular weightTheoretical: 545.77 Da
Chemical component information

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

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Macromolecule #7: MOLYBDATE ION

MacromoleculeName: MOLYBDATE ION / type: ligand / ID: 7 / Number of copies: 42 / Formula: MOO
Molecular weightTheoretical: 159.938 Da
Chemical component information

ChemComp-MOO:
MOLYBDATE ION / Molybdate

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Macromolecule #8: MO(VI)(=O)(OH)2 CLUSTER

MacromoleculeName: MO(VI)(=O)(OH)2 CLUSTER / type: ligand / ID: 8 / Number of copies: 6 / Formula: OMO
Molecular weightTheoretical: 145.954 Da
Chemical component information

ChemComp-OMO:
MO(VI)(=O)(OH)2 CLUSTER

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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 mg/mL
BufferpH: 6.5 / Component - Concentration: 50.0 mM / Component - Name: MOPS/NaOH
Details: 1 mM molybdate and 1 mM mg-ATP were added before vitrification.
GridModel: C-flat / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 0.9 µm / Calibrated magnification: 45045 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 30000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: JEOL / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1238 / Average exposure time: 8.0 sec. / Average electron dose: 54.0 e/Å2

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Image processing

Particle selectionNumber selected: 174681
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4f6t

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 137558
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4f6t

chain_id: A

4f6t

chain_id: B
DetailsPhenix_real_space_refine
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6rkd:
Molybdenum storage protein under turnover conditions

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