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- EMDB-4469: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4469
TitleSpiral structure of E. coli RavA in the RavA-LdcI cage-like complex
Map data
SampleComplex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.:
Inducible lysine decarboxylase / (ATPase RavA) x 2 / (ligand) x 2
Function / homology
Function and homology information


lysine decarboxylase activity / lysine decarboxylase / Hydrolases, Acting on acid anhydrides, Acting on acid anhydrides to catalyse transmembrane movement of substances / cellular amino acid metabolic process / ATPase-coupled transmembrane transporter activity / ATPase activity / ATP binding / identical protein binding / cytosol / cytoplasm
Orn/Lys/Arg decarboxylase, C-terminal / Pyridoxal phosphate-dependent transferase, major domain / ATPase RavA-like, AAA lid domain / Orn/Lys/Arg decarboxylase, C-terminal domain superfamily / P-loop containing nucleoside triphosphate hydrolase / ATPase RavA / ATPase, RavA, C-terminal / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Orn/Lys/Arg decarboxylase, major domain ...Orn/Lys/Arg decarboxylase, C-terminal / Pyridoxal phosphate-dependent transferase, major domain / ATPase RavA-like, AAA lid domain / Orn/Lys/Arg decarboxylase, C-terminal domain superfamily / P-loop containing nucleoside triphosphate hydrolase / ATPase RavA / ATPase, RavA, C-terminal / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Orn/Lys/Arg decarboxylase, major domain / AAA+ ATPase domain / Orn/Lys/Arg decarboxylase, N-terminal / Ornithine/lysine/arginine decarboxylase / ATPase, dynein-related, AAA domain
ATPase RavA / Inducible lysine decarboxylase / ATPase RavA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsArragain B / Felix J / Malet H / Gutsche I / Jessop M
Funding support France, 4 items
OrganizationGrant numberCountry
European Union647784 France
French Infrastructure for Integrated Structural BiologyANR-10-INSB-05-02 France
Grenoble Instruct-ERIC CenterUMS 3518 CNRS-CEA-UJF-EMBL France
European Molecular Biology OrganizationALTF441-2017 France
CitationJournal: Commun Biol / Year: 2020
Title: Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex.
Authors: Matthew Jessop / Benoit Arragain / Roger Miras / Angélique Fraudeau / Karine Huard / Maria Bacia-Verloop / Patrice Catty / Jan Felix / Hélène Malet / Irina Gutsche /
Abstract: The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that ...The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.
Validation ReportPDB-ID: 6q7l

SummaryFull reportAbout validation report
History
DepositionDec 13, 2018-
Header (metadata) releaseOct 2, 2019-
Map releaseFeb 12, 2020-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6q7l
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4469.png

Downloads & links

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Map

FileDownload / File: emd_4469.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.42 Å/pix.
x 200 pix.
= 484.002 Å		2.42 Å/pix.
x 200 pix.
= 484. Å		2.42 Å/pix.
x 200 pix.
= 484. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX: 2.42 Å / Y: 2.42 Å / Z: 2.42001 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.11787947 - 0.1848557
Average (Standard dev.)0.00013527564 (±0.0068504186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA: 484.0002 Å / B: 484.0002 Å / C: 484.002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.422.422.42001
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z484.000484.000484.002
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1180.1850.000

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Supplemental data

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Sample components

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Entire Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric ...

EntireName: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
Number of components: 6

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Component #1: protein, Complex of the hexameric MoxR AAA+ ATPase RavA and the d...

ProteinName: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
Recombinant expression: No
MassTheoretical: 3.3 MDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: MG1655 delta_relA delta_spoT

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Component #2: protein, Inducible lysine decarboxylase

ProteinName: Inducible lysine decarboxylase / Number of Copies: 20 / Recombinant expression: No
MassTheoretical: 81.357008 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, ATPase RavA

ProteinName: ATPase RavA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 56.454586 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, ATPase RavA

ProteinName: ATPase RavA / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 56.351445 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: ligand, PYRIDOXAL-5'-PHOSPHATE

LigandName: PYRIDOXAL-5'-PHOSPHATEPyridoxal phosphate / Number of Copies: 20 / Recombinant expression: No
MassTheoretical: 0.247142 kDa

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Component #6: ligand, ADENOSINE-5'-DIPHOSPHATE

LigandName: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 0.427201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.98 mg/mL
Buffer solution: 20 mM Tris pH 7.9, 300 mM NaCl, 2 mM ADP, 10 mM MgCl 2 , 0.1 mM PLP and 1 mM DTT
pH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Details: Data collection was performed on an FEI Polara microscope operated at 300 kV. Movies of 40 frames were collected with a total exposure time of 8s and a total dose of 40e-/Angstrom^2 on a K2 summit direct electron detector (Gatan) at a magnification of 41270x, corresponding to 1.21 Angstrom/pixel at the specimen level.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 41270 X (calibrated) / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1819

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 19221
3D reconstructionSoftware: RELION / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Details: Local resolution estimation and subsequent filtering of maps were performed in Relion3.0. For fitting of atomic models in the resulting filtered maps, we used the previously-determined X-ray structures of LdcI (PDB ID: 3N75) (Kanjee et al., 2011) and RavA (PDB ID: 3NBX) (El Bakkouri et al., 2010). In each map, two decameric LdcI molecules extracted from PDB 3N75 and one spiral RavA hexamer extracted from a continuous RavA helix generated from PDB 3NBX were fitted separately using iMODFIT (Lopez-Blanco and Chacon, 2013), followed by a single round of B-factor (ADP) refinement in Phenix.
Input PDB model: 3N75, 3NBX

3n75

3nbx

Output model

6q7l

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