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- EMDB-2679: Electron cryo-microscopy of the complex formed between the hexame... -

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Basic information

Entry
Database: EMDB / ID: EMD-2679
TitleElectron cryo-microscopy of the complex formed between the hexameric AAA+ ATPase RavA and the decameric inducible decarboxylase LdcI
Map dataCryo-EM reconstruction of the E. coli LdcI-RavA complex
Sample
  • Sample: Complex between the E.coli inducible lysine decarboxylase LdcI and the E. coli AAA+ ATPase RavA
  • Protein or peptide: Inducible lysine decarboxylase
  • Protein or peptide: AAA+ ATPase RavA
KeywordsLysine decarboxylase / AAA+ ATPase / bacterial acid stress
Function / homology
Function and homology information


lysine catabolic process / lysine decarboxylase / lysine decarboxylase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / guanosine tetraphosphate binding / ATP hydrolysis activity / ATP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
ATPase RavA / 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 / 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 / Ornithine/lysine/arginine decarboxylase / 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 / 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
Inducible lysine decarboxylase / ATPase RavA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsMalet H / Liu K / El Bakkouri M / Chan SWS / Effantin G / Bacia M / Houry WA / Gutsche I
CitationJournal: Elife / Year: 2014
Title: Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.
Authors: Hélène Malet / Kaiyin Liu / Majida El Bakkouri / Sze Wah Samuel Chan / Gregory Effantin / Maria Bacia / Walid A Houry / Irina Gutsche /
Abstract: A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is ...A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily.
History
DepositionJun 15, 2014-
Header (metadata) releaseJul 2, 2014-
Map releaseAug 20, 2014-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4upb
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4upb
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4upb
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2679.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the E. coli LdcI-RavA complex
Voxel sizeX=Y=Z: 2.37 Å
Density
Contour LevelBy AUTHOR: 0.0012 / Movie #1: 0.0012
Minimum - Maximum-0.00785532 - 0.01041579
Average (Standard dev.)0.00000517 (±0.00087319)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 473.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.372.372.37
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z474.000474.000474.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.0080.0100.000

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Supplemental data

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Sample components

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Entire : Complex between the E.coli inducible lysine decarboxylase LdcI an...

EntireName: Complex between the E.coli inducible lysine decarboxylase LdcI and the E. coli AAA+ ATPase RavA
Components
  • Sample: Complex between the E.coli inducible lysine decarboxylase LdcI and the E. coli AAA+ ATPase RavA
  • Protein or peptide: Inducible lysine decarboxylase
  • Protein or peptide: AAA+ ATPase RavA

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Supramolecule #1000: Complex between the E.coli inducible lysine decarboxylase LdcI an...

SupramoleculeName: Complex between the E.coli inducible lysine decarboxylase LdcI and the E. coli AAA+ ATPase RavA
type: sample / ID: 1000
Oligomeric state: Two homodecamers of LdcI bind to five homohexamers of RavA
Number unique components: 2
Molecular weightExperimental: 3.3 MDa / Theoretical: 3.3 MDa
Method: Analytical ultracentrifugation Size exclusion chromatography

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Macromolecule #1: Inducible lysine decarboxylase

MacromoleculeName: Inducible lysine decarboxylase / type: protein_or_peptide / ID: 1 / Name.synonym: LdcI / Number of copies: 20 / Oligomeric state: Two decamers / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: MG1655 / Cell: bacteria / Location in cell: cytoplasm
Molecular weightExperimental: 81.2 KDa / Theoretical: 81.2 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: CF1693 / Recombinant plasmid: pET22b
SequenceUniProtKB: Inducible lysine decarboxylase / GO: lysine catabolic process / InterPro: Ornithine/lysine/arginine decarboxylase

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Macromolecule #2: AAA+ ATPase RavA

MacromoleculeName: AAA+ ATPase RavA / type: protein_or_peptide / ID: 2 / Name.synonym: RavA / Number of copies: 30 / Oligomeric state: Five homohexamers / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: MG1655 / Location in cell: cytoplasm
Molecular weightExperimental: 56.39 KDa / Theoretical: 56.39 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Gold pLysS / Recombinant plasmid: p11
SequenceUniProtKB: ATPase RavA / GO: ATP hydrolysis activity / InterPro: ATPase RavA

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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

Concentration0.94 mg/mL
BufferpH: 6.5
Details: 25 mM MES pH 6.5, 200 mM NaCl, 3mM ADP, 0.8 mM PLP, 1mM DTT
GridDetails: 400 mesh 2/1 1.2/1.3 quantifoil grids Glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 91 K / Instrument: FEI VITROBOT MARK III / Method: Blot 2 or 3 s before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.3 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: GATAN HELIUM
TemperatureMin: 90 K / Max: 92 K / Average: 91 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Legacy - Electron beam tilt params: 0
DetailsWeak beam illumination
DateApr 8, 2012
Image recordingCategory: FILM / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Average electron dose: 20 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER
Software - Name: IMOD, PEET, CTFFIND3D, EMAN, (boxer), FPM, SPIDER
Number images used: 21265
DetailsInitial model determined by cryo-ET and sub-tomogram averaging using IMOD and PEET. Projections of the initial model used for automated picking using the Fast Projection Matching algorithm. Model refined using SPIDER.

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Flex-EM
Details3 rigid bodies, linkers between rigid bodies left flexible
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4upb:
Electron cryo-microscopy of the complex formed between the hexameric ATPase RavA and the decameric inducible decarboxylase LdcI

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Atomic model buiding 2

Initial modelPDB ID:

Chain - Chain ID: X
SoftwareName: Flex-EM
Details3 rigid bodies, linkers between rigid bodies left flexible
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy
Output model

PDB-4upb:
Electron cryo-microscopy of the complex formed between the hexameric ATPase RavA and the decameric inducible decarboxylase LdcI

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