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- PDB-8qpo: E. coli NfsB with the unnatural amino acid p-nitrophenylalanine a... -

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Basic information

Entry
Database: PDB / ID: 8qpo
TitleE. coli NfsB with the unnatural amino acid p-nitrophenylalanine at position 124.
ComponentsOxygen-insensitive NAD(P)H nitroreductase
KeywordsOXIDOREDUCTASE / Nitroreductase / unnatural aminoacid
Function / homology
Function and homology information


oxidoreductase activity, acting on other nitrogenous compounds as donors, with NAD or NADP as acceptor / 6,7-dihydropteridine reductase / 2,4,6-trinitrotoluene catabolic process / 6,7-dihydropteridine reductase activity / NAD(P)H dehydrogenase (quinone) activity / Oxidoreductases / FMN binding / protein homodimerization activity / identical protein binding / membrane / cytosol
Similarity search - Function
: / Oxygen-insensitive NAD(P)H nitroreductase NfsB-like / Nitroreductase / Nitroreductase family / Nitroreductase-like
Similarity search - Domain/homology
ACETATE ION / FLAVIN MONONUCLEOTIDE / Oxygen-insensitive NAD(P)H nitroreductase
Similarity search - Component
Biological speciesEscherichia coli DH5[alpha] (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.74 Å
AuthorsDay, M.A. / White, S.A. / Hyde, E.I.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: To Be Published
Title: E. coli NfsB with unnatural amino acids at position 124.
Authors: Day, M.A. / White, S.A. / Searle, P.F. / Hyde, E.I.
History
DepositionOct 2, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 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: Oxygen-insensitive NAD(P)H nitroreductase
B: Oxygen-insensitive NAD(P)H nitroreductase
C: Oxygen-insensitive NAD(P)H nitroreductase
D: Oxygen-insensitive NAD(P)H nitroreductase
E: Oxygen-insensitive NAD(P)H nitroreductase
F: Oxygen-insensitive NAD(P)H nitroreductase
G: Oxygen-insensitive NAD(P)H nitroreductase
H: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,69323
Polymers195,6298
Non-polymers4,06415
Water4,990277
1
A: Oxygen-insensitive NAD(P)H nitroreductase
B: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,9386
Polymers48,9072
Non-polymers1,0314
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9190 Å2
ΔGint-43 kcal/mol
Surface area17310 Å2
2
C: Oxygen-insensitive NAD(P)H nitroreductase
G: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,8795
Polymers48,9072
Non-polymers9723
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9030 Å2
ΔGint-43 kcal/mol
Surface area17180 Å2
3
D: Oxygen-insensitive NAD(P)H nitroreductase
E: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,9386
Polymers48,9072
Non-polymers1,0314
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9160 Å2
ΔGint-43 kcal/mol
Surface area17280 Å2
4
F: Oxygen-insensitive NAD(P)H nitroreductase
H: Oxygen-insensitive NAD(P)H nitroreductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,9386
Polymers48,9072
Non-polymers1,0314
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9200 Å2
ΔGint-43 kcal/mol
Surface area17270 Å2
Unit cell
Length a, b, c (Å)58.231, 138.552, 121.609
Angle α, β, γ (deg.)90.00, 90.20, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Oxygen-insensitive NAD(P)H nitroreductase / Dihydropteridine reductase / FMN-dependent nitroreductase


Mass: 24453.607 Da / Num. of mol.: 8 / Mutation: F124(PPN), 5 aa tag
Source method: isolated from a genetically manipulated source
Details: Unnatural aminoacid p-nitrophenylalanine at position 124
Source: (gene. exp.) Escherichia coli DH5[alpha] (bacteria) / Strain: DH5a / Gene: nfsB, dprA, nfnB, nfsI, ntr, b0578, JW0567 / Plasmid: pBAD-NTR-124TAG, pDule-PPN
Details (production host): pBAD-NTR-124TAG expresses NfsB with an amber codon at position 124, pDule-PPN contains a uppressor tRNA/aminoacyl-tRNA synthetase pair incorporating PPN at the stop codon
Production host: Escherichia coli (strain K12 / DH10B) (bacteria)
Strain (production host): DH10B
References: UniProt: P38489, Oxidoreductases, 6,7-dihydropteridine reductase
#2: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C17H21N4O9P
#3: Chemical
ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C2H3O2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 277 / 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: 2.51 Å3/Da / Density % sol: 50.95 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 4.6 / Details: 100 mM sodium acetate, pH 4.6, 15% ethylene glycol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Dec 18, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.74→29.71 Å / Num. obs: 47766 / % possible obs: 98.21 % / Redundancy: 3.72 % / Rsym value: 0.146 / Net I/σ(I): 10.5
Reflection shellResolution: 2.741→2.812 Å / Redundancy: 3.68 % / Num. unique obs: 3160 / Rsym value: 0.562 / % possible all: 88.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0411refinement
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.74→29.71 Å / SU B: 22.544 / SU ML: 0.227 / Cross valid method: THROUGHOUT / ESU R Free: 0.305 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.20078 2002 4 %RANDOM
Rwork0.17045 ---
obs0.17169 47766 98.18 %-
Displacement parametersBiso mean: 10.69 Å2
Baniso -1Baniso -2Baniso -3
1--0.88 Å20 Å20.19 Å2
2--0.33 Å20 Å2
3---0.55 Å2
Refinement stepCycle: LAST / Resolution: 2.74→29.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13440 0 276 279 13995
LS refinement shellResolution: 2.741→2.812 Å
RfactorNum. reflection% reflection
Rfree0.307 132 -
Rwork0.268 3160 -
obs--88.8 %
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)
11.87590.12170.33531.66880.27762.596-0.042500.15190.0624-0.0056-0.0284-0.53390.20870.04810.3274-0.0373-0.05330.1190.00860.15711.478-21.33123.27
21.55630.20290.07141.36510.08992.7803-0.00280.0580.0991-0.0195-0.02260.1453-0.3148-0.20220.02540.20570.01-0.03660.13790.01130.1389-8.437-27.53412.363
31.8682-0.09050.06611.50180.21732.3191-0.06160.05580.186-0.10950.04380.0484-0.3117-0.13570.01770.2801-0.0024-0.04190.19280.02640.159614.521-29.12363.547
41.7505-0.19350.01141.7616-0.49592.37580.03710.0037-0.111-0.1125-0.0554-0.07940.27130.14690.01830.2372-0.0317-0.01440.16280.00060.133314.18115.40825.553
51.7224-0.235-0.00322.1696-0.57091.9941-0.0463-0.0754-0.1360.08020.12410.27110.2454-0.2414-0.07780.2806-0.0465-0.00750.19640.02720.18723.65913.06337.359
62.1905-0.1656-0.64171.44290.14572.8421-0.17380.1209-0.32840.0063-0.02990.13210.4269-0.30490.20360.2678-0.04550.06230.1529-0.01960.216120.772-55.25825.528
72.0244-0.1366-0.08762.18130.17911.7961-0.0462-0.01460.2490.09670.0744-0.2563-0.2710.2087-0.02830.3157-0.005-0.05690.1956-0.00950.225924.931-26.84675.518
82.7484-0.2479-0.3331.9540.28282.5106-0.11630.2538-0.4347-0.1478-0.0213-0.08440.5970.03830.13760.3686-0.03210.10520.1405-0.05040.232730.735-61.44714.695
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 217
2X-RAY DIFFRACTION2B2 - 217
3X-RAY DIFFRACTION3C2 - 217
4X-RAY DIFFRACTION4D2 - 217
5X-RAY DIFFRACTION5E2 - 217
6X-RAY DIFFRACTION6F2 - 217
7X-RAY DIFFRACTION7G2 - 217
8X-RAY DIFFRACTION8H2 - 217

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