- EMDB-4470: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-4470
Title
Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex
Map data
Sample
Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.: Inducible lysine decarboxylase / ATPase RavA / (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 / cytoplasm
French Infrastructure for Integrated Structural Biology
ANR-10-INSB-05-02
France
Grenoble Instruct-ERIC Center
UMS 3518 CNRS-CEA-UJF-EMBL
France
European Molecular Biology Organization
ALTF441-2017
France
Citation
Journal: 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.
Name: Inducible lysine decarboxylase / Number of Copies: 20 / Recombinant expression: No
Mass
Theoretical: 81.357008 kDa
Source
Species: Escherichia coli (E. coli)
Source (engineered)
Expression System: Escherichia coli (E. coli)
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Component #3: protein, ATPase RavA
Protein
Name: ATPase RavA / Number of Copies: 6 / Recombinant expression: No
Mass
Theoretical: 56.351445 kDa
Source
Species: Escherichia coli (E. coli)
Source (engineered)
Expression System: Escherichia coli (E. coli)
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Component #4: ligand, PYRIDOXAL-5'-PHOSPHATE
Ligand
Name: PYRIDOXAL-5'-PHOSPHATEPyridoxal phosphate / Number of Copies: 20 / Recombinant expression: No
Mass
Theoretical: 0.247142 kDa
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Component #5: ligand, ADENOSINE-5'-DIPHOSPHATE
Ligand
Name: ADENOSINE-5'-DIPHOSPHATE / Number of Copies: 5 / Recombinant expression: No
Mass
Theoretical: 0.427201 kDa
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Experimental details
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Sample preparation
Specimen
Specimen state: Particle / Method: cryo EM
Sample solution
Specimen 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
Vitrification
Cryogen name: ETHANE
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Electron microscopy imaging
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
Imaging
Microscope: 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 ...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 gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Magnification: 41270 X (calibrated) / Imaging mode: BRIGHT FIELD
Specimen Holder
Model: OTHER
Camera
Detector: GATAN K2 SUMMIT (4k x 4k)
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Image acquisition
Image acquisition
Number of digital images: 1819
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Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 16513
Refinement 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 ...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
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