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- EMDB-10100: Legionella pneumophila SidJ-Human calmodulin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-10100
TitleLegionella pneumophila SidJ-Human calmodulin complex
Map dataSidJ_Cam map
Sample
  • Complex: Legionella pneumophila SidJ - human calmodulin complex
    • Complex: SidJ
      • Protein or peptide: SidJ
    • Complex: calmodulin
      • Protein or peptide: Calmodulin-2
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsBacterial glutamylase / pseudo kinase / calmodulin-dependent / Legionella / SidJ / SdeA / serine ubiquitination / transferase
Function / homology
Function and homology information


Ligases / negative regulation of calcium ion transmembrane transporter activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / negative regulation of calcium ion export across plasma membrane / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / negative regulation of ryanodine-sensitive calcium-release channel activity / ligase activity / protein phosphatase activator activity ...Ligases / negative regulation of calcium ion transmembrane transporter activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / negative regulation of calcium ion export across plasma membrane / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / negative regulation of ryanodine-sensitive calcium-release channel activity / ligase activity / protein phosphatase activator activity / : / adenylate cyclase binding / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / voltage-gated potassium channel complex / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / : / titin binding / positive regulation of protein autophosphorylation / cysteine-type peptidase activity / regulation of calcium-mediated signaling / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / regulation of heart rate / sarcomere / protein serine/threonine kinase activator activity / regulation of cytokinesis / positive regulation of peptidyl-threonine phosphorylation / spindle microtubule / positive regulation of protein serine/threonine kinase activity / spindle pole / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / transferase activity / vesicle / transmembrane transporter binding / G protein-coupled receptor signaling pathway / nucleotide binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / proteolysis / membrane / nucleus / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
: / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Calmodulin-2 / Calmodulin-dependent glutamylase SidJ
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsPfleiderer MM / Galej WP / Adams M / Bhogaraju S
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research FoundationSFB 1177 Germany
German Research FoundationEXC115 Germany
European Research Council742720
CitationJournal: Nature / Year: 2019
Title: Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation.
Authors: Sagar Bhogaraju / Florian Bonn / Rukmini Mukherjee / Michael Adams / Moritz M Pfleiderer / Wojciech P Galej / Vigor Matkovic / Jaime Lopez-Mosqueda / Sissy Kalayil / Donghyuk Shin / Ivan Dikic /
Abstract: The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease. ...The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.
History
DepositionJul 2, 2019-
Header (metadata) releaseJul 17, 2019-
Map releaseJul 24, 2019-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s5t
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10100.png

Downloads & links

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Map

FileDownload / File: emd_10100.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSidJ_Cam map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 160 pix.
= 153.6 Å		0.96 Å/pix.
x 160 pix.
= 153.6 Å		0.96 Å/pix.
x 160 pix.
= 153.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.50874287 - 0.80865717
Average (Standard dev.)0.00041713717 (±0.026325678)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 153.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.960.960.96
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z153.600153.600153.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.5090.8090.000

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Supplemental data

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Half map: #2

Fileemd_10100_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10100_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Legionella pneumophila SidJ - human calmodulin complex

EntireName: Legionella pneumophila SidJ - human calmodulin complex
Components
  • Complex: Legionella pneumophila SidJ - human calmodulin complex
    • Complex: SidJ
      • Protein or peptide: SidJ
    • Complex: calmodulin
      • Protein or peptide: Calmodulin-2
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Legionella pneumophila SidJ - human calmodulin complex

SupramoleculeName: Legionella pneumophila SidJ - human calmodulin complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 120 KDa

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Supramolecule #2: SidJ

SupramoleculeName: SidJ / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Legionella pneumophila (bacteria)

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Supramolecule #3: calmodulin

SupramoleculeName: calmodulin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: SidJ

MacromoleculeName: SidJ / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 100.338469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFGFIKKVLD FFGVDQSEDN PSETAVETTD VSTKIKTTDT TQEESSVKTK TVVPTQPGGS VKPETIAPDQ QKKHQIKTET TTSTTKQKG PKVTLMDGHV KQYYFARRGE TSTHDTSLPP PVKVLSGRSI PLKEIPFEAT RNELVQIYLT SIDKLIKSNK L NSIPSQQI ...String:
MFGFIKKVLD FFGVDQSEDN PSETAVETTD VSTKIKTTDT TQEESSVKTK TVVPTQPGGS VKPETIAPDQ QKKHQIKTET TTSTTKQKG PKVTLMDGHV KQYYFARRGE TSTHDTSLPP PVKVLSGRSI PLKEIPFEAT RNELVQIYLT SIDKLIKSNK L NSIPSQQI ASHYLFLRSL ANSETDGIKK NQILSLAKPL GTYLASKEPH VWKMINELIE KSEYPIIHYL KNNRAHSNFM LA LIHEYHK EPLTKNQSAF VQKFRDSSVF LFPNPIYTAW LAHSYDEDSS FNPMFRERLS TNFYHSTLTD NLLLRTEPKE VTL SSEHHY KKEKGPIDSS FRYQMSSDRL LRIQGRTLLF STPQNDVVAV KVQKKGEPKS TLEEEFEMAD YLLKHQRRLD VHSK LPQPL GQYSVKKSEI LEISRGSLDF ERFKTLIDDS KDLEVYVYKA PQSYFTYLHD KNQDLEDLTA SVKTNVHDLF VLLRE GIVF PQLADIFHTH FGEDEREDKG RYQALVQLLN VLQFQLGRID KWQKAVEYVN LRSSGLADLG DSLPITSLFT SSDFTK HYF SELLTGGYHP TFFDKSSGTA NSLFTGKRRL FGNYLYLNTI AEYLLVIQLT LGSYGDKVTR DMMDKPKKEA VWRELAN VM FTSCAEAIHI MTGIPQSRAL TLLKQRANIE KHFRQTQFWM TPDYSKLDED TLQMEQYSIY SGEPEYEFTD KLVSGVGL S VDGVHQDLGG YNRESPLREL EKLLYATVTL IEGTMQLDKE FFKQLEQVEK ILSGEIKTDA NSCFEAVAQL LDLARPGCH FQKRLVLSYY EEAKLKYPSA PTDAYDSRFQ VVARTNAAIT IQRFWREARK NLSEKSDIDS EKPESERTTD KRL

UniProtKB: Calmodulin-dependent glutamylase SidJ

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Macromolecule #2: Calmodulin-2

MacromoleculeName: Calmodulin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.852545 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREA FRVFDKDGNG YISAAELRHV MTNLGEKLTD EEVDEMIREA DIDGDGQVNY EEFVQMMTAK

UniProtKB: Calmodulin-2

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*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

BufferpH: 7.5
VitrificationCryogen name: ETHANE
DetailsRecombinant Legionella pneumophila SidJ -human calmodulin complex

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 19851
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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