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- EMDB-10850: Inducible lysine decarboxylase LdcI stacks, pH 5.7 -

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Basic information

Entry
Database: EMDB / ID: EMD-10850
TitleInducible lysine decarboxylase LdcI stacks, pH 5.7
Map dataLdcI stack, post-processed map.
Sample
  • Organelle or cellular component: E. coli inducible lysine decarboxylase at pH 5.7
    • Protein or peptide: Inducible lysine decarboxylase
Function / homology
Function and homology information


lysine catabolic process / lysine decarboxylase / 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 K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsFelix J / Jessop M / Desfosses A / Effantin G / Gutsche I
Funding support3 items
OrganizationGrant numberCountry
European Research Council (ERC)Chap4Resp
Marie Sklodowska-Curie Actions, FragNET ITNRespViRALI
European Molecular Biology Organization (EMBO)ALTF441-2017
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, 2020-
Header (metadata) releaseJan 13, 2021-
Map releaseJan 13, 2021-
UpdateJan 13, 2021-
Current statusJan 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yn6
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6yn6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10850.png

Downloads & links

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Map

FileDownload / File: emd_10850.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLdcI stack, post-processed map.
Voxel sizeX=Y=Z: 1.052 Å
Density
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-1.7729001 - 2.9629934
Average (Standard dev.)0.024371587 (±0.19197333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 336.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0521.0521.052
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z336.640336.640336.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-1.7732.9630.024

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Supplemental data

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Mask #1

Fileemd_10850_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LdcI stack, half map 2.

Fileemd_10850_half_map_1.map
AnnotationLdcI stack, half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: LdcI stack, half map 1.

Fileemd_10850_half_map_2.map
AnnotationLdcI stack, 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 : E. coli inducible lysine decarboxylase at pH 5.7

EntireName: E. coli inducible lysine decarboxylase at pH 5.7
Components
  • Organelle or cellular component: E. coli inducible lysine decarboxylase at pH 5.7
    • Protein or peptide: Inducible lysine decarboxylase

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Supramolecule #1: E. coli inducible lysine decarboxylase at pH 5.7

SupramoleculeName: E. coli inducible lysine decarboxylase at pH 5.7 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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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 (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 81.11057 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
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 (LLP)LLAAFSQAS MIHVKGDVNE ETFNEAYMMH TTTSPHYGI VASTETAAAM MKGNAGKRLI NGSIERAIKF RKEIKRLRTE SDGWFFDVWQ PDHIDTTECW PLRSDSTWHG F KNIDNEHM YLDPIKVTLL TPGMEKDGTM SDFGIPASIV AKYLDEHGIV VEKTGPYNLL FLFSIGIDKT KALSLLRALT DF KRAFDLN LRVKNMLPSL YREDPEFYEN MRIQELAQNI HKLIVHHNLP DLMYRAFEVL PTMVMTPYAA FQKELHGMTE EVY LDEMVG RINANMILPY PPGVPLVMPG EMITEESRPV LEFLQMLCEI GAHYPGFETD IHGAYRQADG RYTVKVLKE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 5.7
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2564 / Average exposure time: 6.0 sec. / Average electron dose: 29.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15165
CTF correctionSoftware: (Name: CTFFIND (ver. 4), Gctf)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3n75


Details: Initial model generated by manually stacking three LdcI decamers (PDB ID: 3N75) in Chimera and low-pass filtering the resulting LdcI-stack to 40 A.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 15157
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3n75

RefinementSpace: REAL / Overall B value: 96
Output model

PDB-6yn6:
Inducible lysine decarboxylase LdcI stacks, pH 5.7

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