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- PDB-8ali: IPNS H270E variant in complex with Fe and ACV after 30 min O2 exposure -

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Basic information

Entry
Database: PDB / ID: 8ali
TitleIPNS H270E variant in complex with Fe and ACV after 30 min O2 exposure
ComponentsIsopenicillin N synthase
KeywordsOXIDOREDUCTASE / Isopenicillin N synthase / antibiotic production / O2 binding / reactive intermediate
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: 1.6 Å
AuthorsRabe, P. / Schfoeidl, 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 H270E variant in complex with Fe and ACV after 30 min O2 exposure
Authors: Rabe, P. / Schfoeidl, C.J.
History
DepositionAug 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: 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)38,3587
Polymers37,5551
Non-polymers8046
Water5,513306
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1510 Å2
ΔGint-49 kcal/mol
Surface area14240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)41.060, 74.308, 101.020
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Isopenicillin N synthase / IPNS


Mass: 37554.805 Da / Num. of mol.: 1 / Mutation: H270E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus nidulans FGSC A4 (mold) / Strain: FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139 / Gene: ipnA, ips, AN2622 / Plasmid: pCold_IPNS / Details (production host): pCold_IPNS / 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-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.: 306 / 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: 2.05 Å3/Da / Density % sol: 40.05 % / Description: needle morphology, 4 um x 4 um x 180 um
Crystal growTemperature: 298 K / Method: batch mode / pH: 8.3 / Details: 1.7 M Li2So4, O.1 M Tris pH 8.3

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: cryo / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Apr 15, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.6→59.86 Å / Num. obs: 41690 / % possible obs: 100 % / Redundancy: 13.3 % / Biso Wilson estimate: 18.08 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.168 / Rpim(I) all: 0.047 / Rrim(I) all: 0.174 / Net I/σ(I): 9.5 / Num. measured all: 554545 / Scaling rejects: 4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
1.6-1.6313.52.0941.220410.6730.5842.175100
8.76-59.8611.10.043190.9990.0120.04299.9

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Processing

Software
NameVersionClassification
Aimless0.7.8data scaling
PHENIX1.19.2_4158refinement
PDB_EXTRACT3.27data extraction
DIALSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6ZAE

6zae


Resolution: 1.6→59.86 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 25.1 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2151 3792 4.81 %
Rwork0.1934 74974 -
obs0.1945 41624 99.96 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 79.81 Å2 / Biso mean: 26.2178 Å2 / Biso min: 12.46 Å2
Refinement stepCycle: final / Resolution: 1.6→59.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2612 0 69 306 2987
Biso mean--38.85 30.7 -
Num. residues----329
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 27 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
1.6-1.620.35051390.361727492888
1.62-1.640.31511400.341528062946
1.64-1.660.30221410.322527472888
1.66-1.690.33471370.321127832920
1.69-1.710.3391480.322828152963
1.71-1.740.29811410.311827392880
1.74-1.770.2581430.289627922935
1.77-1.80.29381380.272827642902
1.8-1.830.28991390.255927752914
1.83-1.870.29371420.24628032945
1.87-1.90.27021380.219727602898
1.9-1.950.25371410.209827982939
1.95-1.990.23991380.207127282866
1.99-2.040.20741410.197428012942
2.04-2.10.21711380.195428002938
2.1-2.160.20971390.191327582897
2.16-2.230.25091380.179927762914
2.23-2.310.19481410.177628022943
2.31-2.40.21861370.179827692906
2.4-2.510.23131410.184127582899
2.51-2.640.22051390.182928002939
2.64-2.810.18491430.184327672910
2.81-3.020.18571400.180327702910
3.02-3.330.22931430.180627712914
3.33-3.810.16921430.147227812924
3.81-4.80.15571460.137427732919
4.8-59.860.17741380.180127892927
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)
12.32560.1847-0.00152.3065-1.02884.2340.01530.1605-0.0797-0.1074-0.0825-0.00990.09580.19550.07560.18210.02140.02210.1507-0.03120.113315.9439-3.6793-15.0703
20.9114-0.0801-1.02730.54821.52165.99650.1285-0.12720.05980.07730.094-0.1058-0.30170.4551-0.2150.28160.0273-0.02930.13470.02150.179617.98183.559311.4378
31.3636-0.55030.4331.05040.00542.4711-0.0563-0.13610.07340.10860.08190.0487-0.2966-0.0985-0.0130.23340.01130.01710.1346-0.01830.11489.338910.373613.9963
40.7580.31121.01531.0361.20254.57940.01820.05050.09170.062-0.0122-0.0155-0.34260.00730.02510.24710.00890.01270.11860.00910.136810.65719.4739-5.6375
51.08550.17040.86960.9417-0.11392.17610.0362-0.0271-0.12240.04290.0185-0.04860.1304-0.0437-0.06780.18240.00710.01790.1341-0.00330.137812.4908-8.88533.9863
62.0688-0.38941.27561.76250.16724.6195-0.0009-0.1433-0.08080.1810.00220.05870.1285-0.6588-0.01740.2329-0.00250.01720.2804-0.02470.1903-3.58950.853-2.4949
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 3 through 37 )A3 - 37
2X-RAY DIFFRACTION2chain 'A' and (resid 38 through 82 )A38 - 82
3X-RAY DIFFRACTION3chain 'A' and (resid 83 through 137 )A83 - 137
4X-RAY DIFFRACTION4chain 'A' and (resid 138 through 183 )A138 - 183
5X-RAY DIFFRACTION5chain 'A' and (resid 184 through 286 )A184 - 286
6X-RAY DIFFRACTION6chain 'A' and (resid 287 through 331 )A287 - 331

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