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- EMDB-4470: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4470
TitleSpiral structure of E. coli RavA in the RavA-LdcI cage-like complex
Map dataCryo-EM map of Class 2 of the E. coli RavA-LdcI cage-like complex after symmetry expansion and masked 3D classification.
Sample
  • Complex: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
    • Protein or peptide: Inducible lysine decarboxylase
    • Protein or peptide: ATPase RavA
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
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 / 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, LARA domain ...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 / 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
ATPase RavA / Inducible lysine decarboxylase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsArragain B / Felix J / Malet H / Gutsche I / Jessop M
Funding support France, 4 items
OrganizationGrant numberCountry
European Union647784 France
French Infrastructure for Integrated Structural BiologyANR-10-INSB-05-02 France
Grenoble Instruct-ERIC CenterUMS 3518 CNRS-CEA-UJF-EMBL 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, 2018-
Header (metadata) releaseOct 2, 2019-
Map releaseFeb 12, 2020-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6q7m
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4470.png

Downloads & links

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Map

FileDownload / File: emd_4470.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Class 2 of the E. coli RavA-LdcI cage-like complex after symmetry expansion and masked 3D classification.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.42 Å/pix.
x 200 pix.
= 484.002 Å		2.42 Å/pix.
x 200 pix.
= 484. Å		2.42 Å/pix.
x 200 pix.
= 484. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX: 2.42 Å / Y: 2.42 Å / Z: 2.42001 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.101965666 - 0.19264637
Average (Standard dev.)0.00012397133 (±0.006753406)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA: 484.0002 Å / B: 484.0002 Å / C: 484.002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.422.422.42001
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z484.000484.000484.002
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1020.1930.000

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Supplemental data

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Mask #1

Fileemd_4470_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map 1.

Fileemd_4470_half_map_1.map
AnnotationUnfiltered half-map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map 1.

Fileemd_4470_half_map_2.map
AnnotationUnfiltered half-map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric ...

EntireName: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
Components
  • Complex: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
    • Protein or peptide: Inducible lysine decarboxylase
    • Protein or peptide: ATPase RavA
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric ...

SupramoleculeName: Complex of the hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: MG1655 delta_relA delta_spoT
Molecular weightTheoretical: 3.3 MDa

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Macromolecule #1: Inducible lysine decarboxylase

MacromoleculeName: Inducible lysine decarboxylase / type: protein_or_peptide / ID: 1 / Number of copies: 20 / Enantiomer: LEVO / EC number: lysine decarboxylase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 81.357008 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNVIAILNHM GVYFKEEPIR ELHRALERLN FQIVYPNDRD DLLKLIENNA RLCGVIFDWD KYNLELCEEI SKMNENLPLY AFANTYSTL DVSLNDLRLQ ISFFEYALGA AEDIANKIKQ TTDEYINTIL PPLTKALFKY VREGKYTFCT PGHMGGTAFQ K SPVGSLFY ...String:
MNVIAILNHM GVYFKEEPIR ELHRALERLN FQIVYPNDRD DLLKLIENNA RLCGVIFDWD KYNLELCEEI SKMNENLPLY AFANTYSTL DVSLNDLRLQ ISFFEYALGA AEDIANKIKQ TTDEYINTIL PPLTKALFKY VREGKYTFCT PGHMGGTAFQ K SPVGSLFY DFFGPNTMKS DISISVSELG SLLDHSGPHK EAEQYIARVF NADRSYMVTN GTSTANKIVG MYSAPAGSTI LI DRNCHKS LTHLMMMSDV TPIYFRPTRN AYGILGGIPQ SEFQHATIAK RVKETPNATW PVHAVITNST YDGLLYNTDF IKK TLDVKS IHFDSAWVPY TNFSPIYEGK CGMSGGRVEG KVIYETQSTH KLLAAFSQAS MIHVKGDVNE ETFNEAYMMH TTTS PHYGI VASTETAAAM MKGNAGKRLI NGSIERAIKF RKEIKRLRTE SDGWFFDVWQ PDHIDTTECW PLRSDSTWHG FKNID NEHM YLDPIKVTLL TPGMEKDGTM SDFGIPASIV AKYLDEHGIV VEKTGPYNLL FLFSIGIDKT KALSLLRALT DFKRAF DLN LRVKNMLPSL YREDPEFYEN MRIQELAQNI HKLIVHHNLP DLMYRAFEVL PTMVMTPYAA FQKELHGMTE EVYLDEM VG RINANMILPY PPGVPLVMPG EMITEESRPV LEFLQMLCEI GAHYPGFETD IHGAYRQADG RYTVKVLKEE SKK

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Macromolecule #2: ATPase RavA

MacromoleculeName: ATPase RavA / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 56.351445 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHPHLLAER ISRLSSSLEK GLYERSHAIR LCLLAALSGE SVFLLGPPGI AKSLIARRLK FAFQNARAFE YLMTRFSTPE EVFGPLSIQ ALKDEGRYER LTSGYLPEAE IVFLDEIWKA GPAILNTLLT AINERQFRNG AHVEKIPMRL LVAASNELPE A DSSLEALY ...String:
MAHPHLLAER ISRLSSSLEK GLYERSHAIR LCLLAALSGE SVFLLGPPGI AKSLIARRLK FAFQNARAFE YLMTRFSTPE EVFGPLSIQ ALKDEGRYER LTSGYLPEAE IVFLDEIWKA GPAILNTLLT AINERQFRNG AHVEKIPMRL LVAASNELPE A DSSLEALY DRMLIRLWLD KVQDKANFRS MLTSQQDEND NPVPDALQVT DEEYERWQKE IGEITLPDHV FELIFMLRQQ LD KLPDAPY VSDRRWKKAI RLLQASAFFS GRSAVAPVDL ILLKDCLWYD AQSLNLIQQQ IDVLMTGHAW QQQGMLTRLG AIV QRHLQL QQQQSDKTAL TVIRLGGIFS RRQQYQLPVN VTASTLTLLL QKPLKLHDME VVHISFERSA LEQWLSKGGE IRGK LNGIG FAQKLNLEVD SAQHLVVRDV SLQGSTLALP GSSAEGLPGE IKQQLEELES DWRKQHALFS EQQKCLFIPG DWLGR IEAS LQDVGAQIRQ AQQ

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Macromolecule #3: PYRIDOXAL-5'-PHOSPHATE

MacromoleculeName: PYRIDOXAL-5'-PHOSPHATE / type: ligand / ID: 3 / Number of copies: 20 / Formula: PLP
Molecular weightTheoretical: 247.142 Da
Chemical component information

ChemComp-PLP:
PYRIDOXAL-5'-PHOSPHATE

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

Concentration0.98 mg/mL
BufferpH: 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
VitrificationCryogen name: ETHANE
DetailsConcentrations of LdcI and RavA were 0.38 mg/ml (4.67 microM) and 0.6 mg/ml (10.64 microM) respectively.

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Electron microscopy

MicroscopeFEI POLARA 300
DetailsData 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.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 3-40 / Number real images: 1819 / Average exposure time: 8.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41270 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 18902
Details: The cleanded dataset contains 11866 particles. Dataset expansion (C5) resulted in a dataset containing 59330 particles.
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL
In silico model: The initial 3D model based on 2D class averages was calculated with sxviper (SPARX) (Hohn et al., 2007) imposing D5 symmetry.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 16513
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 2 / Avg.num./class: 17867 / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

3n75

,

3nbx

)
DetailsLocal 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.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6q7m:
Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex

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