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- PDB-6yn5: Inducible lysine decarboxylase LdcI decamer, pH 7.0 -

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Basic information

Entry
Database: PDB / ID: 6yn5
TitleInducible lysine decarboxylase LdcI decamer, pH 7.0
ComponentsInducible lysine decarboxylase
KeywordsLYASE / Acid stress-inducible / decarboxylase / LAOdc
Function / homology
Function and homology information


lysine decarboxylase / lysine catabolic process / lysine decarboxylase activity / guanosine tetraphosphate binding / identical protein binding / cytoplasm
Similarity search - Function
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 ...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
Similarity search - Domain/homology
Inducible lysine decarboxylase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsJessop, M. / Felix, J. / Desfosses, A. / Effantin, G. / Gutsche, I.
Funding support3items
OrganizationGrant numberCountry
European Research Council (ERC)Chap4Resp
European Molecular Biology Organization (EMBO)ALTF441-2017
Marie Sklodowska-Curie Actions, FragNET ITNRespViRALI
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Supramolecular assembly of the LdcI upon acid stress.
Authors: Matthew Jessop / Clarissa Liesche / Jan Felix / Ambroise Desfosses / Megghane Baulard / Virgile Adam / Angélique Fraudeau / Karine Huard / Grégory Effantin / Jean-Philippe Kleman / Maria ...Authors: Matthew Jessop / Clarissa Liesche / Jan Felix / Ambroise Desfosses / Megghane Baulard / Virgile Adam / Angélique Fraudeau / Karine Huard / Grégory Effantin / Jean-Philippe Kleman / Maria Bacia-Verloop / Dominique Bourgeois / Irina Gutsche /
Abstract: Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ...Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.
History
DepositionApr 10, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release

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Structure visualization

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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


Theoretical massNumber of molelcules
Total (without water)811,10610
Polymers811,10610
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area76640 Å2
ΔGint-420 kcal/mol
Surface area232960 Å2
MethodPISA

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Components

#1: Protein
Inducible lysine decarboxylase / LDCI


Mass: 81110.570 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: cadA, ldcI, b4131, JW4092
Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Variant (production host): delta_relA delta_spoT / References: UniProt: P0A9H3, lysine decarboxylase
Has ligand of interestY

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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: E. coli inducible lysine decarboxylase at pH 7.0 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.82 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Buffer solutionpH: 7
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 45 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 2772
Image scansMovie frames/image: 29 / Used frames/image: 3-29

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.13model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 796000
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229000 / Symmetry type: POINT
Atomic model buildingB value: 173 / Space: REAL
Atomic model buildingPDB-ID: 3N75

3n75

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.65 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006758418
ELECTRON MICROSCOPYf_angle_d0.805979214
ELECTRON MICROSCOPYf_chiral_restr0.05238674
ELECTRON MICROSCOPYf_plane_restr0.006510158
ELECTRON MICROSCOPYf_dihedral_angle_d14.52717838

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