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- PDB-8rdb: Isopenicillin N synthase N252E variant in complex with Fe and ACV... -

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Basic information

Entry
Database: PDB / ID: 8rdb
TitleIsopenicillin N synthase N252E variant in complex with Fe and ACV under anaerobic conditions
ComponentsIsopenicillin N synthase
KeywordsOXIDOREDUCTASE / Isopenicillin N synthase / IPNS / ACV / anaerobic / penicillin biosynthesis
Function / homology
Function and homology information


isopenicillin-N synthase / isopenicillin-N synthase activity / penicillin biosynthetic process / L-ascorbic acid binding / iron ion binding / cytosol
Similarity search - Function
Isopenicillin N synthase signature 1. / Isopenicillin N synthase, conserved site / Isopenicillin N synthase signature 2. / Non-haem dioxygenase N-terminal domain / non-haem dioxygenase in morphine synthesis N-terminal / Isopenicillin N synthase-like, Fe(2+) 2OG dioxygenase domain / 2OG-Fe(II) oxygenase superfamily / Isopenicillin N synthase-like superfamily / Oxoglutarate/iron-dependent dioxygenase / Fe(2+) 2-oxoglutarate dioxygenase domain profile.
Similarity search - Domain/homology
L-D-(A-AMINOADIPOYL)-L-CYSTEINYL-D-VALINE / : / Isopenicillin N synthase
Similarity search - Component
Biological speciesAspergillus nidulans FGSC A4 (mold)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.37 Å
AuthorsStead, A. / Rabe, P. / Schofield, C.J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V001892/1 United Kingdom
Wellcome Trust227298/Z/23/Z United Kingdom
CitationJournal: To Be Published
Title: Isopenicillin N synthase N252E variant in complex with Fe and ACV under anaerobic conditions
Authors: Stead, A. / Rabe, P. / Schofield, C.J.
History
DepositionDec 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isopenicillin N synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,3827
Polymers37,5791
Non-polymers8046
Water7,206400
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1510 Å2
ΔGint-51 kcal/mol
Surface area14280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.270, 74.560, 101.070
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Isopenicillin N synthase / IPNS


Mass: 37578.848 Da / Num. of mol.: 1 / Mutation: N252E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus nidulans FGSC A4 (mold) / Gene: ipnA, ips, AN2622 / Plasmid: pCold_IPNS_N252E / Details (production host): pCold / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P05326, isopenicillin-N synthase
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ACV / L-D-(A-AMINOADIPOYL)-L-CYSTEINYL-D-VALINE


Mass: 363.430 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H25N3O6S / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 400 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.07 Å3/Da / Density % sol: 40.59 % / Description: Needle morphology
Crystal growTemperature: 298 K / Method: batch mode / pH: 8.3 / Details: 1.7M Li2SO4, 0.1M Tris pH 8.3

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryogenic / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Nov 12, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.37→41.83 Å / Num. obs: 66337 / % possible obs: 99.9 % / Redundancy: 13.4 % / CC1/2: 0.999 / Rmerge(I) obs: 0.122 / Rpim(I) all: 0.034 / Rrim(I) all: 0.127 / Χ2: 0.98 / Net I/σ(I): 12.5 / Num. measured all: 885861
Reflection shellResolution: 1.37→1.41 Å / % possible obs: 99.1 % / Redundancy: 13 % / Rmerge(I) obs: 2.699 / Num. measured all: 62522 / Num. unique obs: 4804 / CC1/2: 0.554 / Rpim(I) all: 0.768 / Rrim(I) all: 2.809 / Χ2: 0.9 / Net I/σ(I) obs: 1

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
DIALSdata scaling
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.37→41.83 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 20.86 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1929 3237 4.89 %
Rwork0.1555 --
obs0.1573 66205 99.84 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.37→41.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2613 0 45 402 3060
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0082804
X-RAY DIFFRACTIONf_angle_d0.9353836
X-RAY DIFFRACTIONf_dihedral_angle_d6.454392
X-RAY DIFFRACTIONf_chiral_restr0.089399
X-RAY DIFFRACTIONf_plane_restr0.008510
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.37-1.390.32151220.30222661X-RAY DIFFRACTION98
1.39-1.410.29441490.27242671X-RAY DIFFRACTION100
1.41-1.440.28081310.25932735X-RAY DIFFRACTION100
1.44-1.460.31811480.24862678X-RAY DIFFRACTION100
1.46-1.490.30771360.22542718X-RAY DIFFRACTION100
1.49-1.520.24761360.19872713X-RAY DIFFRACTION100
1.52-1.550.22281500.18672693X-RAY DIFFRACTION100
1.55-1.580.22861260.16312728X-RAY DIFFRACTION100
1.58-1.620.21681260.16192701X-RAY DIFFRACTION100
1.62-1.660.21981550.15832725X-RAY DIFFRACTION100
1.66-1.70.22761550.14892687X-RAY DIFFRACTION100
1.7-1.750.21271340.14022704X-RAY DIFFRACTION100
1.75-1.810.20311380.14042745X-RAY DIFFRACTION100
1.81-1.870.19741370.13332740X-RAY DIFFRACTION100
1.87-1.950.18821440.1342727X-RAY DIFFRACTION100
1.95-2.040.18671430.12942731X-RAY DIFFRACTION100
2.04-2.140.19331420.13232737X-RAY DIFFRACTION100
2.14-2.280.18461380.13792778X-RAY DIFFRACTION100
2.28-2.460.18111350.14562747X-RAY DIFFRACTION100
2.46-2.70.1821570.15442744X-RAY DIFFRACTION100
2.7-3.090.17911390.16552809X-RAY DIFFRACTION100
3.09-3.90.17771490.14412826X-RAY DIFFRACTION100
3.9-41.830.15881470.15012970X-RAY DIFFRACTION100

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