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- EMDB-31199: Pyochelin synthetase, a dimeric nonribosomal peptide synthetase e... -

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Basic information

Entry
Database: EMDB / ID: EMD-31199
TitlePyochelin synthetase, a dimeric nonribosomal peptide synthetase elongation module-after-condensation
Map dataPchE-after-condensation
Sample
  • Organelle or cellular component: pyochelin synthetase, a dimeric nonribosomal peptide synthetase elongation module
    • Protein or peptide: Dihydroaeruginoic acid synthetase
  • Ligand: 4'-PHOSPHOPANTETHEINEPhosphopantetheine
  • Ligand: (4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: CYSTEINE
  • Ligand: 2-HYDROXYBENZOIC ACIDSalicylic acid
Keywordsnonribosomal peptide synthetase / BIOSYNTHETIC PROTEIN / LIGASE
Function / homology
Function and homology information


L-cysteine-[L-cysteinyl-carrier protein] ligase / 2,3-dihydroxybenzoate-serine ligase activity / amino acid activation for nonribosomal peptide biosynthetic process / enterobactin synthetase complex / enterobactin biosynthetic process / phosphopantetheine binding / cytoplasm
Similarity search - Function
Condensation domain / Condensation domain / Amino acid adenylation domain / ANL, N-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / Chloramphenicol acetyltransferase-like domain superfamily / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase ...Condensation domain / Condensation domain / Amino acid adenylation domain / ANL, N-terminal domain / AMP-binding, conserved site / Putative AMP-binding domain signature. / Chloramphenicol acetyltransferase-like domain superfamily / Polyketide synthase, phosphopantetheine-binding domain / Phosphopantetheine attachment site / AMP-dependent synthetase/ligase / AMP-binding enzyme, C-terminal domain superfamily / AMP-binding enzyme / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
Pyochelin synthase PchE
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsWang JL / Wang ZJ
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)91753123 China
CitationJournal: Nat Commun / Year: 2022
Title: Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain.
Authors: Jialiang Wang / Dandan Li / Lu Chen / Wei Cao / Liangliang Kong / Wei Zhang / Tristan Croll / Zixin Deng / Jingdan Liang / Zhijun Wang /
Abstract: Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic ...Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique "H"-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and L-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents.
History
DepositionApr 15, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7en1
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31199.png

Downloads & links

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Map

FileDownload / File: emd_31199.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPchE-after-condensation
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.009
Minimum - Maximum-0.023367358 - 0.06380251
Average (Standard dev.)0.000008890555 (±0.0021684973)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 250.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z250.000250.000250.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0230.0640.000

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Supplemental data

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Additional map: postprocess

Fileemd_31199_additional_1.map
Annotationpostprocess
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half1

Fileemd_31199_half_map_1.map
Annotationhalf1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half2

Fileemd_31199_half_map_2.map
Annotationhalf2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : pyochelin synthetase, a dimeric nonribosomal peptide synthetase e...

EntireName: pyochelin synthetase, a dimeric nonribosomal peptide synthetase elongation module
Components
  • Organelle or cellular component: pyochelin synthetase, a dimeric nonribosomal peptide synthetase elongation module
    • Protein or peptide: Dihydroaeruginoic acid synthetase
  • Ligand: 4'-PHOSPHOPANTETHEINEPhosphopantetheine
  • Ligand: (4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: CYSTEINE
  • Ligand: 2-HYDROXYBENZOIC ACIDSalicylic acid

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Supramolecule #1: pyochelin synthetase, a dimeric nonribosomal peptide synthetase e...

SupramoleculeName: pyochelin synthetase, a dimeric nonribosomal peptide synthetase elongation module
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 160 kDa/nm

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Macromolecule #1: Dihydroaeruginoic acid synthetase

MacromoleculeName: Dihydroaeruginoic acid synthetase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 158.762641 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDLPPDSRTA LRDWLTEQLA DLLGEPLADV RALADDDDLL GCGLDSIRLM YLQERLRARG STLDFAQLAQ RPCLGAWLDL LACADRLSA PATVALPTAQ DRDQPFELSS VQQAYWLGRG AGEVLGNVSC HAFLEFRTRD VDPQRLAAAA ECVRQRHPML R ARFLDGRQ ...String:
MDLPPDSRTA LRDWLTEQLA DLLGEPLADV RALADDDDLL GCGLDSIRLM YLQERLRARG STLDFAQLAQ RPCLGAWLDL LACADRLSA PATVALPTAQ DRDQPFELSS VQQAYWLGRG AGEVLGNVSC HAFLEFRTRD VDPQRLAAAA ECVRQRHPML R ARFLDGRQ QILPTPPLSC FDLQDWRTLQ VDEAERDWQA LRDWRAHECL AVERGQVFLL GLVRMPGGED RLWLSLDLLA AD VESLRLL LAELGVAYLA PERLAEPPAL HFADYLAHRA AQRAEAAARA RDYWLERLPR LPDAPALPLA CAPESIRQPR TRR LAFQLS AGESRRLERL AAQHGVTLSS VFGCAFALVL ARWSESAEFL LNVPLFDRHA DDPRIGEVIA DFTTLLLLEC RMQA GVSFA EAVKSFQRNL HGAIDHAAFP ALEVLREARR QGQPRSAPVV FASNLGEEGF VPAAFRDAFG DLHDMLSQTP QVWLD HQLY RVGDGILLAW DSVVGLFPEG LPETMFEAYV GLLQRLCDSA WGQPADLPLP WAQQARRALL NGQPACATAR TLHRDF FLR AAEAPDADAL LYRDQRVTRG ELAERALRIA GGLREAGVRP GDAVEVSLPR GPQQVAAVFG VLAAGACYVP LDIDQPP AR RRLIEEAAGV CLAITEEDDP QALPPRLDVQ RLLRGPALAA PVPLAPQASA YVIYTSGSTG VPKGVEVSHA AAINTIDA L LDLLRVNASD RLLAVSALDF DLSVFDLFGG LGAGASLVLP AQEQARDAAA WAEAIQRHAV SLWNSAPALL EMALSLPAS QADYRSLRAV LLSGDWVALD LPGRLRPRCA EGCRLHVLGG ATEAGIWSNL QSVDTVPPHW RSIPYGRPLP GQAYRVVDTH GRDVPDLVV GELWIGGASL ARGYRNDPEL SARRFVHDAQ GRWYRTGDRG RYWGDGTLEF LGRVDQQVKV RGQRIELGEV E AALCAQAG VESACAAVLG GGVASLGAVL VPRLAPRAEG SMDLPAAQPF AGLAEAEAVL TREILGALLE APLELDDGLR RR WLDWLAD SAASALPSLD EALRRLGWQA AGLTAMGNAL RGLLAGEQAP AALLLDPWLA PQAVAARLPD GREALARLLE ALP TPAAGE RLRVAVLDTR AGLWLDQGMA SLLRPGLELT LFERSRVLLD AAATRLPERI VVQALDDGLL PAEHLGRYDR VISF AALHA YEASREGLAL AAALLRPQGR LLLVDLLCES PLALLGAALL DDRPLRLAEL PSLLADLAAA GLAPRCLWRS ERIAL VEAL APGLGLDAAA LQAGLEQRLP QAMRPERLWC LPSLPLNGNG KVDRRRLAES MTRALGECRH EPSAEEPLEA HEQALA ECW EAVLKRPVRR REASFFSLGG DSLLATRLLA GIRERFGVRL GMADFYRQPT LAGLARHLQV QTVEIEETQL EEGVLHH HH HHLPSWSHPQ FEK

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Macromolecule #2: 4'-PHOSPHOPANTETHEINE

MacromoleculeName: 4'-PHOSPHOPANTETHEINE / type: ligand / ID: 2 / Number of copies: 2 / Formula: PNS
Molecular weightTheoretical: 358.348 Da
Chemical component information

ChemComp-PNS:
4'-PHOSPHOPANTETHEINE / Phosphopantetheine

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Macromolecule #3: (4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid

MacromoleculeName: (4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid
type: ligand / ID: 3 / Number of copies: 1 / Formula: J9F
Molecular weightTheoretical: 223.248 Da
Chemical component information

ChemComp-J9F:
(4S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

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

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Macromolecule #6: CYSTEINE

MacromoleculeName: CYSTEINE / type: ligand / ID: 6 / Number of copies: 2 / Formula: CYS
Molecular weightTheoretical: 121.158 Da
Chemical component information

ChemComp-BTC:
CYSTEINE / Cysteine

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Macromolecule #7: 2-HYDROXYBENZOIC ACID

MacromoleculeName: 2-HYDROXYBENZOIC ACID / type: ligand / ID: 7 / Number of copies: 1 / Formula: SAL
Molecular weightTheoretical: 138.121 Da
Chemical component information

ChemComp-SAL:
2-HYDROXYBENZOIC ACID / Salicylic acid

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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: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was homogenous

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 58112
FSC plot (resolution estimation)

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