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- EMDB-23862: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23862
TitleCryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
Map data
Sample
  • Complex: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
    • Complex: SidJ-SdeA
      • Protein or peptide: Calmodulin-dependent glutamylase SidJ
      • Protein or peptide: Ubiquitinating/deubiquitinating enzyme SdeA
    • Complex: Calmodulin
      • Protein or peptide: Calmodulin-2
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


Ligases / NAD+-protein-arginine ADP-ribosyltransferase / negative regulation of calcium ion transmembrane transporter activity / deNEDDylase activity / NAD+-protein-arginine ADP-ribosyltransferase activity / protein deneddylation / Transferases; Acyltransferases; Aminoacyltransferases / host cell / K63-linked deubiquitinase activity / negative regulation of calcium ion export across plasma membrane ...Ligases / NAD+-protein-arginine ADP-ribosyltransferase / negative regulation of calcium ion transmembrane transporter activity / deNEDDylase activity / NAD+-protein-arginine ADP-ribosyltransferase activity / protein deneddylation / Transferases; Acyltransferases; Aminoacyltransferases / host cell / K63-linked deubiquitinase 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 / protein deubiquitination / protein phosphatase activator activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / ligase activity / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / voltage-gated potassium channel complex / cysteine-type peptidase activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / positive regulation of protein autophosphorylation / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / regulation of heart rate / nucleotidyltransferase activity / protein serine/threonine kinase activator activity / sarcomere / positive regulation of peptidyl-threonine phosphorylation / regulation of cytokinesis / 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 / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / protein ubiquitination / G protein-coupled receptor signaling pathway / nucleotide binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / proteolysis / extracellular region / membrane / metal ion binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
SidE, DUB domain / SidE, mono-ADP-ribosyltransferase domain / SidE mono-ADP-ribosyltransferase domain / SidE DUB domain / SidE, PDE domain / SidE phosphodiesterase (PDE) domain / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. ...SidE, DUB domain / SidE, mono-ADP-ribosyltransferase domain / SidE mono-ADP-ribosyltransferase domain / SidE DUB domain / SidE, PDE domain / SidE phosphodiesterase (PDE) domain / 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 / Ubiquitinating/deubiquitinating enzyme SdeA / Calmodulin-dependent glutamylase SidJ
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsOsinski A / Black MH / Pawlowski K / Chen Z / Li Y / Tagliabracci VS
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM137419 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F30HL143859 United States
Welch FoundationI-1911 United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural and mechanistic basis for protein glutamylation by the kinase fold.
Authors: Adam Osinski / Miles H Black / Krzysztof Pawłowski / Zhe Chen / Yang Li / Vincent S Tagliabracci /
Abstract: The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ...The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.
History
DepositionApr 17, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mir
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23862.png

Downloads & links

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Map

FileDownload / File: emd_23862.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.08341905 - 0.19145602
Average (Standard dev.)5.467521e-06 (±0.0040399507)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0830.1910.000

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex

EntireName: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
Components
  • Complex: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
    • Complex: SidJ-SdeA
      • Protein or peptide: Calmodulin-dependent glutamylase SidJ
      • Protein or peptide: Ubiquitinating/deubiquitinating enzyme SdeA
    • Complex: Calmodulin
      • Protein or peptide: Calmodulin-2
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex

SupramoleculeName: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: SidJ-SdeA

SupramoleculeName: SidJ-SdeA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Source (natural)Organism: Legionella pneumophila (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Calmodulin

SupramoleculeName: Calmodulin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Calmodulin-dependent glutamylase SidJ

MacromoleculeName: Calmodulin-dependent glutamylase SidJ / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Ligases
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 87.374031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SGHVKQYYFA RRGETSTHDT SLPPPVKVLS GRSIPLKEIP FEATRNELVQ IYLTSIDKLI KSNKLNSIPS QQIASHYLFL RSLANSETD GIKKNQILSL AKPLGTYLAS KEPHVWKMIN ELIEKSEYPI IHYLKNNRAH SNFMLALIHE YHKEPLTKNQ S AFVQKFRD ...String:
SGHVKQYYFA RRGETSTHDT SLPPPVKVLS GRSIPLKEIP FEATRNELVQ IYLTSIDKLI KSNKLNSIPS QQIASHYLFL RSLANSETD GIKKNQILSL AKPLGTYLAS KEPHVWKMIN ELIEKSEYPI IHYLKNNRAH SNFMLALIHE YHKEPLTKNQ S AFVQKFRD SSVFLFPNPI YTAWLAHSYD EDSSFNPMFR ERLSTNFYHS TLTDNLLLRT EPKEVTLSSE HHYKKEKGPI DS SFRYQMS SDRLLRIQGR TLLFSTPQND VVAVKVQKKG EPKSTLEEEF EMADYLLKHQ RRLDVHSKLP QPLGQYSVKK SEI LEISRG SLDFERFKTL IDDSKDLEVY VYKAPQSYFT YLHDKNQDLE DLTASVKTNV HDLFVLLREG IVFPQLADIF HTHF GEDER EDKGRYQALV QLLNVLQFQL GRIDKWQKAV EYVNLRSSGL ADLGDSLPIT SLFTSSDFTK HYFSELLTGG YHPTF FDKS SGTANSLFTG KRRLFGNYLY LNTIAEYLLV IQLTLGSYGD KVTRDMMDKP KKEAVWRELA NVMFTSCAEA IHIMTG IPQ SRALTLLKQR ANIEKHFRQT QFWMTPDYSK LDEDTLQMEQ YSIYSGEPEY EFTDKLVSGV GLSVDGVHQD LGGYNRE SP LRELEKLLYA TVTLIEGTMQ LDKEFFKQLE QVEKILSGEI KTDANSCFEA VAQLLDLARP GCHFQKRLVL SYYEEAKL K YPSAPTDAYD SRFQVVARTN AAITIQRFWR EARKN

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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: 16.939623 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMADQLTEEQ IAEFKEAFSL FDKDGDGTIT TKELGTVMRS LGQNPTEAEL QDMINEVDAD GNGTIDFPEF LTMMARKMKD TDSEEEIRE AFRVFDKDGN GYISAAELRH VMTNLGEKLT DEEVDEMIRE ADIDGDGQVN YEEFVQMMTA K

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Macromolecule #3: Ubiquitinating/deubiquitinating enzyme SdeA

MacromoleculeName: Ubiquitinating/deubiquitinating enzyme SdeA / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 109.120219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMGSGFSLY TDDTVKAAAQ YAYDNYLGKP YTGSVESAPA NFGGRMVYRQ HHGLSHTLRT MAYAELIVEE ARKAKLRGET LGKFKDGRT IADVTPQELK KIMIAQAFFV AGRDDEASDA KNYQKYHEQS RDAFLKYVKD NESTLIPDVF KDQEDVNFYA R VIEDKSHD ...String:
GAMGSGFSLY TDDTVKAAAQ YAYDNYLGKP YTGSVESAPA NFGGRMVYRQ HHGLSHTLRT MAYAELIVEE ARKAKLRGET LGKFKDGRT IADVTPQELK KIMIAQAFFV AGRDDEASDA KNYQKYHEQS RDAFLKYVKD NESTLIPDVF KDQEDVNFYA R VIEDKSHD WESTPAHVLI NQGHMVDLVR VKQPPESFLQ RYFSSMQRWI GSQATEAVFG IQRQFFHATY EVVAGFDSDN KE PHLVVSG LGRYVIGEDG QPIREAPKKG QKEGDLKVFP QTYKLKENER LMRVDEFLKL PEIQNTFPGS GKHLQGGMPG MNE MDYWNR LNSLNRARCE NDVDFCLKQL QTAHDKAKIE PIKQAFQSSK GKERRQPNVD EIAAARIIQQ ILANPDCIHD DHVL INGQK LEQQFFRDLL AKCEMAVVGS LLNDTDIGNI DTLMRHEKDT EFHSTNPEAV PVKIGEYWIN DQRINNSSGN ITQKK HDLI FLMQNDAWYF SRVNAIAQNR DKGSTFKEVL ITTLMTPLTS KALVDTSQAK PPTRLFRGLN LSEEFTKGLI DQANAM IAN TTERLFTDHS PEAFKQIKLN DLSKMSGRTN ASTTTEIKLV KETWDSNVIF EMLDPDGLLH SKQVGRHGEG TESEFSV YL PEDVALVPVK VTLDGKTQKG ENRYVFTFVA VKSPDFTPRH ESGYAVEPFL RMQAAKLAEV KSSIEKAQRA PDLETIFN L QNEVEAVQYS HLSTGYKNFL KNTVGPVLEN SLSGLMESDT DTLSKALAAF PSDTQWSAFN FEEARQAKRQ MDAIKQMVG NKVVLDALTQ CQDALEKQNI AGALDALKKI PSEKEMGTIR RELREQIQSA RQELESLQRA VVTPVVTDEK KVRERYDALI ENTSKKITE LETGKLPNLD AVKKGISNLS NLKQEVTVLR NEKIRMHVGT DKVDFSDVEK LEQQIQVIDT KLADAYLLEV T KQISA

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Macromolecule #4: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM / Adenosine monophosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #7: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.35 mg/mL
BufferpH: 6.5
Details: 25 mM Bis-tris pH 6.5, 100 mM NaCl, 1 mM TCEP, 2 mM MgCl2, 1 mM ATP
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 310154

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