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- PDB-6q7l: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex -

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Basic information

Entry
Database: PDB / ID: 6q7l
TitleSpiral structure of E. coli RavA in the RavA-LdcI cage-like complex
Components
  • (ATPase RavA) x 2
  • Inducible lysine decarboxylase
KeywordsHYDROLASE / Complex / MoxR ATPase / Lysine decarboxylase
Function / homology
Function and homology information


arginine decarboxylase activity / lysine decarboxylase / lysine decarboxylase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / arginine catabolic process / pyridoxal phosphate binding / ATP hydrolysis activity / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
: / ATPase, RavA, C-terminal / ATPase RavA / ATPase RavA-like, AAA lid domain / MoxR domain / ATPase RavA, LARA domain / ATPase RavA, LARA domain superfamily / ATPase, RavA, C-terminal / AAA lid domain / MoxR domain in the MoxR-vWA-beta-propeller ternary systems ...: / ATPase, RavA, C-terminal / ATPase RavA / ATPase RavA-like, AAA lid domain / MoxR domain / ATPase RavA, LARA domain / ATPase RavA, LARA domain superfamily / ATPase, RavA, C-terminal / AAA lid domain / MoxR domain in the MoxR-vWA-beta-propeller ternary systems / ATPase, RavA, LARA domain / Orn/Lys/Arg decarboxylase, N-terminal / Ornithine/lysine/arginine decarboxylase / Orn/Lys/Arg decarboxylase, N-terminal domain / Orn/Lys/Arg decarboxylases family 1 pyridoxal-P attachment site. / Orn/Lys/Arg decarboxylase, major domain / Orn/Lys/Arg decarboxylase, C-terminal / Orn/Lys/Arg decarboxylase, C-terminal domain superfamily / Orn/Lys/Arg decarboxylase, major domain / Orn/Lys/Arg decarboxylase, C-terminal domain / AAA domain (dynein-related subfamily) / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PYRIDOXAL-5'-PHOSPHATE / ATPase RavA / Inducible lysine decarboxylase / ATPase RavA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsArragain, B. / Felix, J. / Malet, H. / Gutsche, I. / Jessop, M.
Funding support France, 4items
OrganizationGrant numberCountry
European Union647784 France
Grenoble Instruct-ERIC CenterUMS 3518 CNRS-CEA-UJF-EMBL France
French Infrastructure for Integrated Structural BiologyANR-10-INSB-05-02 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.
History
DepositionDec 13, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4469
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Assembly

Deposited unit
A: Inducible lysine decarboxylase
B: Inducible lysine decarboxylase
C: Inducible lysine decarboxylase
D: Inducible lysine decarboxylase
E: Inducible lysine decarboxylase
F: Inducible lysine decarboxylase
G: Inducible lysine decarboxylase
H: Inducible lysine decarboxylase
I: Inducible lysine decarboxylase
J: Inducible lysine decarboxylase
K: Inducible lysine decarboxylase
L: Inducible lysine decarboxylase
M: Inducible lysine decarboxylase
N: Inducible lysine decarboxylase
O: Inducible lysine decarboxylase
P: Inducible lysine decarboxylase
Q: Inducible lysine decarboxylase
R: Inducible lysine decarboxylase
S: Inducible lysine decarboxylase
T: Inducible lysine decarboxylase
U: ATPase RavA
V: ATPase RavA
W: ATPase RavA
X: ATPase RavA
Y: ATPase RavA
Z: ATPase RavA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,972,43151
Polymers1,965,35226
Non-polymers7,07925
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area211250 Å2
ΔGint-895 kcal/mol
Surface area633700 Å2
MethodPISA

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Components

#1: Protein
Inducible lysine decarboxylase / LDC


Mass: 81357.008 Da / Num. of mol.: 20
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cadA, ldcI, Z5734, ECs5113 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A9H4, lysine decarboxylase
#2: Protein ATPase RavA / Regulatory ATPase variant A


Mass: 56454.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ravA, yieN, b3746, JW3725 / Production host: Escherichia coli (E. coli)
References: UniProt: P31473, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
#3: Protein
ATPase RavA / Regulatory ATPase variant A


Mass: 56351.445 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: yieN, ravA, BN17_37001 / Production host: Escherichia coli (E. coli)
References: UniProt: J7QAN2, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
#4: Chemical
ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C8H10NO6P
#5: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 3.3 MDa
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: MG1655 delta_relA delta_spoT
Buffer solutionpH: 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
SpecimenConc.: 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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 41270 X
Image recordingAverage 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 scansMovie frames/image: 40 / Used frames/image: 3-40

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2Latitudeimage acquisition
4GctfCTF correction
7iMODFITmodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 18902
Details: The cleanded dataset contains 11866 particles. Dataset expansion (C5) resulted in a dataset containing 59330 particles.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19221 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: 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
IDPDB-ID 3D fitting-ID
13N75

3n75

1
23NBX

3nbx

1

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