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- PDB-6q7m: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex -
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Open data
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Basic information
Entry | Database: PDB / ID: 6q7m | |||||||||||||||
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Title | Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex | |||||||||||||||
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![]() | HYDROLASE / Complex / MoxR ATPase / Lysine decarboxylase | |||||||||||||||
Function / homology | ![]() lysine decarboxylase / lysine decarboxylase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / amino acid metabolic process / ATP hydrolysis activity / ATP binding / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | ![]() ![]() | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.8 Å | |||||||||||||||
![]() | Arragain, B. / Felix, J. / Malet, H. / Gutsche, I. / Jessop, M. | |||||||||||||||
Funding support | ![]()
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![]() | ![]() 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. | |||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 2.9 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 4470MC ![]() 4469C ![]() 6q7lC ![]() 6szaC ![]() 6szbC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 81357.008 Da / Num. of mol.: 20 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 56351.445 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: J7QAN2, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances #3: Chemical | ChemComp-PLP / #4: Chemical | ChemComp-ADP / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI. Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT |
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Molecular weight | Value: 3.3 MDa |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.9 Details: 20 mM Tris pH 7.9, 300 mM NaCl, 2 mM ADP, 10 mM MgCl 2 , 0.1 mM PLP and 1 mM DTT |
Specimen | Conc.: 0.98 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Concentrations of LdcI and RavA were 0.38 mg/ml (4.67 microM) and 0.6 mg/ml (10.64 microM) respectively. |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: 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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 41270 X |
Image recording | Average exposure time: 8 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1819 |
Image scans | Movie frames/image: 40 / Used frames/image: 3-40 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 18902 Details: The cleanded dataset contains 11866 particles. Dataset expansion (C5) resulted in a dataset containing 59330 particles. | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16513 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT 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. | ||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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