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

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Entry
Database: EMDB / ID: 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
SampleComplex between the E.coli inducible lysine decarboxylase LdcI and the E. coli AAA+ ATPase RavA:
Inducible lysine decarboxylase / AAA+ ATPase RavA
KeywordsLysine decarboxylase / AAA+ ATPase / bacterial acid stress
Function / homologyOrnithine/lysine/arginine decarboxylase / Orn/Lys/Arg decarboxylase, major domain / ATPase RavA / ATPase, RavA, C-terminal / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / ATPase, dynein-related, AAA domain / Orn/Lys/Arg decarboxylase, C-terminal / Orn/Lys/Arg decarboxylase, N-terminal ...Ornithine/lysine/arginine decarboxylase / Orn/Lys/Arg decarboxylase, major domain / ATPase RavA / ATPase, RavA, C-terminal / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / ATPase, dynein-related, AAA domain / Orn/Lys/Arg decarboxylase, C-terminal / Orn/Lys/Arg decarboxylase, N-terminal / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Orn/Lys/Arg decarboxylase, C-terminal domain superfamily / Orn/Lys/Arg decarboxylase, major domain / Orn/Lys/Arg decarboxylase, N-terminal domain / Orn/Lys/Arg decarboxylase, C-terminal domain / AAA domain (dynein-related subfamily) / Protein of unknown function (DUF3763) / Orn/Lys/Arg decarboxylases family 1 pyridoxal-P attachment site. / ATPase RavA / Ornithine/lysine/arginine decarboxylase / lysine decarboxylase / lysine decarboxylase activity / guanosine tetraphosphate binding / lysine catabolic process / Hydrolases, Acting on acid anhydrides, Acting on acid anhydrides to catalyse transmembrane movement of substances / ATPase activity, coupled to transmembrane movement of substances / ATPase activity / ATP binding / identical protein binding / cytosol / cytoplasm / Inducible lysine decarboxylase / ATPase RavA
Function and homology information
SourceEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / 11 Å resolution
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.
Validation ReportPDB-ID: 4upb

SummaryFull reportAbout validation report
DateDeposition: Jun 15, 2014 / Header (metadata) release: Jul 2, 2014 / Map release: Aug 20, 2014 / Last update: Aug 12, 2015

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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
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-4upb
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2679.map.gz (map file in CCP4 format, 31251 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
200 pix
2.37 Å/pix.
= 474. Å
200 pix
2.37 Å/pix.
= 474. Å
200 pix
2.37 Å/pix.
= 474. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.37 Å
Density
Contour Level:0.0012 (by author), 0.0012 (movie #1):
Minimum - Maximum-0.00785532 - 0.01041579
Average (Standard dev.)0.00000517 (0.00087319)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions200200200
Origin-100-100-100
Limit999999
Spacing200200200
CellA=B=C: 473.99997 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
Number of components: 2
Oligomeric State: Two homodecamers of LdcI bind to five homohexamers of RavA
MassTheoretical: 3.3 MDa / Experimental: 3.3 MDa
Measured by: Analytical ultracentrifugation Size exclusion chromatography

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Component #1: protein, Inducible lysine decarboxylase

ProteinName: Inducible lysine decarboxylase / a.k.a: LdcI / Oligomeric Details: Two decamers / Number of Copies: 20 / Recombinant expression: Yes
MassTheoretical: 81.2 kDa / Experimental: 81.2 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: MG1655
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET22b / Strain: CF1693
Source (natural)Location in cell: cytoplasm / Cell: bacteria
External referencesGene Ontology: lysine catabolic process / UniProt: Inducible lysine decarboxylase / InterPro: Ornithine/lysine/arginine decarboxylase

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Component #2: protein, AAA+ ATPase RavA

ProteinName: AAA+ ATPase RavA / a.k.a: RavA / Oligomeric Details: Five homohexamers / Recombinant expression: Yes / Number of Copies: 30
MassTheoretical: 56.39 kDa / Experimental: 56.39 kDa
SourceSpecies: Escherichia coli K-12 (bacteria) / Strain: MG1655
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: p11 / Strain: Gold pLysS
Source (natural)Location in cell: cytoplasm
External referencesInterPro: ATPase RavA / UniProt: ATPase RavA / Gene Ontology: ATPase activity

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.94 mg/ml
Buffer solution: 25 mM MES pH 6.5, 200 mM NaCl, 3mM ADP, 0.8 mM PLP, 1mM DTT
pH: 6.5
Support film400 mesh 2/1 1.2/1.3 quantifoil grids Glow discharged
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 91 K / Humidity: 100 % / Method: Blot 2 or 3 s before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Apr 8, 2012 / Details: Weak beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Electron beam tilt params: 0 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 59000 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1300 - 3300 nm
Specimen HolderHolder: Nitrogen cooled / Model: GATAN HELIUM / Temperature: 91 K ( 90 - 92 K)
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionScanner: ZEISS SCAI / Sampling size: 7 microns / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D5 (2*5 fold dihedral) / Number of projections: 21265
Details: Initial 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.
3D reconstructionAlgorithm: Projection matching / Software: IMOD, PEET, CTFFIND3D, EMAN, (boxer), FPM, SPIDER / CTF correction: Phase flipping / Resolution: 11 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Flex-EM / Refinement protocol: flexible / Target criteria: Cross-correlation, energy / Refinement space: REAL
Details: 3 rigid bodies, linkers between rigid bodies left flexible
Input PDB model: 3N75
Chain ID: A
Modeling #2Software: Flex-EM / Refinement protocol: flexible / Target criteria: Cross-correlation, energy / Refinement space: REAL
Details: 3 rigid bodies, linkers between rigid bodies left flexible
Input PDB model: 3NBX
Chain ID: X
Output model

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