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- PDB-4upf: Assembly principles of the unique cage formed by the ATPase RavA ... -

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Basic information

Entry
Database: PDB / ID: 4upf
TitleAssembly principles of the unique cage formed by the ATPase RavA hexamer and the lysine decarboxylase LdcI decamer
Components
  • ATPASE RAVA
  • LYSINE DECARBOXYLASE, INDUCIBLE
KeywordsLYASE/HYDROLASE / LYASE-HYDROLASE COMPLEX / LYSINE DECARBOXYLASE / AAA+ ATPASE / ACID STRESS RESPONSE
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, 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
Inducible lysine decarboxylase / ATPase RavA
Similarity search - Component
Biological speciesESCHERICHIA COLI STR. K-12 SUBSTR. MG1655 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 Å
Model type detailsCA ATOMS ONLY, CHAIN A, D
AuthorsMalet, H. / Liu, K. / El Bakkouri, M. / Chan, S.W.S. / Effantin, G. / Bacia, M. / Houry, W.A. / 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 16, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 20, 2014Provider: repository / Type: Initial release
Revision 1.1Sep 10, 2014Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Assembly

Deposited unit
A: LYSINE DECARBOXYLASE, INDUCIBLE
D: ATPASE RAVA


Theoretical massNumber of molelcules
Total (without water)93,9762
Polymers93,9762
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein LYSINE DECARBOXYLASE, INDUCIBLE / / LDC / INDUCIBLE LYSINE DECARBOXYLASE / Coordinate model: Cα atoms only


Mass: 81357.008 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI STR. K-12 SUBSTR. MG1655 (bacteria)
Production host: ESCHERICHIA COLI (E. coli) / Variant (production host): CF1693 SOURCE 7 / References: UniProt: P0A9H3, lysine decarboxylase
#2: Protein ATPASE RAVA / REGULATORY ATPASE VARIANT A / Coordinate model: Cα atoms only


Mass: 12619.461 Da / Num. of mol.: 1 / Fragment: LARA DOMAIN RESIDUES 329-440
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI STR. K-12 SUBSTR. MG1655 (bacteria)
Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): GOLD PLYSS / References: UniProt: P31473, EC: 3.6.3.1
Sequence detailsTHE LARA DOMAIN (FROM GLN329 TO GLU440) WAS PCR AMPLIFIED FROM THE P11-RAVA PLASMID. THE PCR ...THE LARA DOMAIN (FROM GLN329 TO GLU440) WAS PCR AMPLIFIED FROM THE P11-RAVA PLASMID. THE PCR PRODUCT WAS DIGESTED WITH NDEI AND BAMHI (NEB) AND LIGATED INTO AN EMPTY P11 VECTOR TO PRODUCE P11-LARA. THE RESULTING CONSTRUCT HAS AN N-TERMINAL HIS6-TAG FOLLOWED BY A TOBACCO ETCH VIRUS (TEV) CUT SITE THAT LEAVES THE THREE RESIDUES GHM AT THE TERMINUS OF THE CONSTRUCT AFTER TEV CLEAVAGE.

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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 BETWEEN A DECAMER OF INDUCIBLE LYSINE DECARBOXYLASE LDCI AND TEN LARA DOMAINS OF THE ATPASE RAVA
Type: COMPLEX
Buffer solutionName: 50MM MES PH 6.5, 100MM NACL, 0.2MM PLP, 1MM DTT, 0.01% GLUTARALDEHYDE
pH: 6.5
Details: 50MM MES PH 6.5, 100MM NACL, 0.2MM PLP, 1MM DTT, 0.01% GLUTARALDEHYDE
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: OTHER
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 91, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT FOR 2 SECONDS BEFORE PLUNGING

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30 / Date: Sep 14, 2012 / Details: AUTOMATIC DATA ACQUISITION WITH FEI EPU SOFTWARE
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 51660 X / Calibrated magnification: 51660 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Specimen holderTemperature: 91 K / Tilt angle max: 0 ° / Tilt angle min: -0.1 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 911
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategoryDetails
1CTFFIND3CTF correction
2Flex-EMmodel fitting
3Situsmodel fitting
4EMANparticle selectionBOXER
5EMAN3D reconstruction
6FPM3D reconstruction
7IMAGIC3D reconstruction
8SPIDER3D reconstruction
CTF correctionDetails: PHASE FLIPPING
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionMethod: CROSS-COMMON LINE, PROJECTION MATCHING / Resolution: 7.5 Å / Num. of particles: 23540 / Nominal pixel size: 1.464 Å / Actual pixel size: 1.464 Å
Details: CROSS-COMMON LINE, PROJECTION MATCHING SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2681. (DEPOSITION ID: 12595).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--FLEXIBLE FOR 3N75, RIGID FOR 3NBX REFINEMENT PROTOCOL--X-RAY
Atomic model building
IDPDB-ID 3D fitting-ID
13N751
23NBX1
RefinementHighest resolution: 7.5 Å
Refinement stepCycle: LAST / Highest resolution: 7.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms784 0 0 0 784

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