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- PDB-8p47: IPNS variant N252A in complex with Fe and ACV under anaerobic con... -

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Basic information

Entry
Database: PDB / ID: 8p47
TitleIPNS variant N252A in complex with Fe and ACV under anaerobic conditions
ComponentsIsopenicillin N synthase
KeywordsOXIDOREDUCTASE / Isopenicillin N synthase / penicillins / biosynthesis / 2OG oxygenases / antibiotic
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: 1.56 Å
AuthorsRabe, P. / Schofield, C.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V001892/1 United Kingdom
CitationJournal: To Be Published
Title: IPNS variant N252A in complex with Fe and ACV under anaerobic conditions
Authors: Rabe, P. / Schofield, C.J.
History
DepositionMay 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 5, 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)38,2286
Polymers37,5211
Non-polymers7075
Water7,674426
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1350 Å2
ΔGint-39 kcal/mol
Surface area14390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.675, 74.730, 101.459
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: 37520.812 Da / Num. of mol.: 1 / Mutation: N252A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus nidulans FGSC A4 (mold) / Gene: ipnA, ips, AN2622 / Plasmid: pCold / Details (production host): pCold_N252A / 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.: 3 / Source method: obtained synthetically / Formula: SO4
#3: 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
#4: 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 426 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.11 Å3/Da / Density % sol: 41.78 % / Description: needle morphology, 4 um x 4 um x 140 um
Crystal growTemperature: 298 K / Method: batch mode / pH: 8.3 / Details: 1.7 M Li2SO4, 0.1 M 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: Mar 9, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.56→50.73 Å / Num. obs: 45988 / % possible obs: 100 % / Redundancy: 13.5 % / Biso Wilson estimate: 17.97 Å2 / CC1/2: 0.997 / Rrim(I) all: 0.219 / Net I/σ(I): 8.2
Reflection shellResolution: 1.56→1.59 Å / Redundancy: 13.9 % / Mean I/σ(I) obs: 0.6 / Num. unique obs: 2254 / CC1/2: 0.536 / Rrim(I) all: 2.554 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.56→50.73 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 22.11 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2094 2186 4.77 %
Rwork0.1812 --
obs0.1826 45876 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.56→50.73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2614 0 40 427 3081
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0042808
X-RAY DIFFRACTIONf_angle_d0.6243842
X-RAY DIFFRACTIONf_dihedral_angle_d12.0981016
X-RAY DIFFRACTIONf_chiral_restr0.047399
X-RAY DIFFRACTIONf_plane_restr0.005513
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.56-1.590.31891290.31632683X-RAY DIFFRACTION100
1.59-1.630.31521260.30422668X-RAY DIFFRACTION100
1.63-1.670.26951370.26752717X-RAY DIFFRACTION100
1.67-1.720.32071140.26172701X-RAY DIFFRACTION100
1.72-1.770.25661290.24542692X-RAY DIFFRACTION100
1.77-1.820.2911320.23682747X-RAY DIFFRACTION100
1.82-1.890.26141470.23432664X-RAY DIFFRACTION100
1.89-1.970.29091360.24772708X-RAY DIFFRACTION100
1.97-2.060.23841470.18432671X-RAY DIFFRACTION100
2.06-2.160.20671340.17742738X-RAY DIFFRACTION100
2.16-2.30.21781380.17812719X-RAY DIFFRACTION100
2.3-2.480.21071360.15742745X-RAY DIFFRACTION100
2.48-2.730.19871550.16392732X-RAY DIFFRACTION100
2.73-3.120.1811380.15852754X-RAY DIFFRACTION100
3.12-3.930.16891360.14462819X-RAY DIFFRACTION100
3.93-50.730.17011520.15292932X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.4564-0.22840.02243.99752.5713.46060.0701-0.0962-0.02450.135-0.06920.06230.1116-0.2370.01690.1312-0.00640.02170.15730.03410.1263-15.5684-4.798414.6211
21.7838-0.3141-0.86740.9569-0.50193.60680.0970.06560.0109-0.01530.06260.085-0.0908-0.3831-0.14620.1108-0.0138-0.02830.1269-0.0150.1434-22.59780.9851-1.6762
37.23031.2756-3.24895.52610.13454.57840.1560.34490.2423-0.0079-0.05590.1039-0.5444-0.1709-0.09520.2049-0.0011-0.00940.125-0.00020.0885-11.01936.6867-24.0425
40.88420.3780.13761.3920.0321.983-0.04070.09110.0919-0.09130.1227-0.0469-0.49710.1092-0.05220.2054-0.01560.02820.12240.01780.1224-9.440210.3528-13.9967
50.9828-0.75010.91241.4489-1.07644.55440.0089-0.02230.0711-0.07580.01160.0787-0.3724-0.2504-0.09980.13690.01510.01660.10280.01190.1288-15.42819.45622.4935
60.4886-0.45140.52851.3394-0.9934.6428-0.0209-0.02690.09960.17070.0082-0.1304-0.72060.40680.03250.1885-0.03910.0090.171-0.02710.1689-4.79259.75588.7716
71.0836-0.0710.36620.95480.13051.89430.0098-0.0383-0.1234-0.04610.00440.02790.1474-0.0168-0.02390.11850.0020.01880.12530.01090.1414-12.5754-8.8689-3.9247
81.17650.3134-0.4581.0465-0.30512.66030.0575-0.3949-0.24660.1775-0.1209-0.24830.02241.0190.07850.2008-0.018-0.02310.48530.03810.23425.57892.020410.1124
90.1308-0.0042-0.4672.07841.43492.6722-0.025-0.0389-0.0684-0.19790.1722-0.1356-0.01860.3175-0.14940.1714-0.00550.0160.27370.02310.21230.6429-1.3035-8.4279
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 2 through 37 )
2X-RAY DIFFRACTION2chain 'A' and (resid 38 through 64 )
3X-RAY DIFFRACTION3chain 'A' and (resid 65 through 82 )
4X-RAY DIFFRACTION4chain 'A' and (resid 83 through 137 )
5X-RAY DIFFRACTION5chain 'A' and (resid 138 through 163 )
6X-RAY DIFFRACTION6chain 'A' and (resid 164 through 183 )
7X-RAY DIFFRACTION7chain 'A' and (resid 184 through 286 )
8X-RAY DIFFRACTION8chain 'A' and (resid 287 through 312 )
9X-RAY DIFFRACTION9chain 'A' and (resid 313 through 331 )

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