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- PDB-8h7e: Crystal structure of a de novo enzyme, ferric enterobactin estera... -

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Basic information

Entry
Database: PDB / ID: 8h7e
TitleCrystal structure of a de novo enzyme, ferric enterobactin esterase Syn-F4 (K4T) at 2.0 angstrom resolution
ComponentsDe novo ferric enterobactin esterase Syn-F4
KeywordsDE NOVO PROTEIN / Binary patterned library / De novo enzyme / Dimeric 4-helix bundle / Ferric enterobactin esterase
Function / homologyACETATE ION
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsKurihara, K. / Umezawa, K. / Donnelly, A.E. / Hecht, M.H. / Arai, R.
Funding support Japan, United States, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19H02522, JP17KK0104, JP16K05841, JP16H00761, JP24780097 Japan
Japan Society for the Promotion of Science (JSPS)Invitational Fellowship S18035 Japan
National Science Foundation (NSF, United States)MCB-1947720 United States
Citation
Journal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.
Authors: Kurihara, K. / Umezawa, K. / Donnelly, A.E. / Sperling, B. / Liao, G. / Hecht, M.H. / Arai, R.
#1: Journal: Nat Chem Biol / Year: 2018
Title: A de novo enzyme catalyzes a life-sustaining reaction in Escherichia coli.
Authors: Donnelly, A.E. / Murphy, G.S. / Digianantonio, K.M. / Hecht, M.H.
#2: Journal: Protein Sci / Year: 2015
Title: Divergent evolution of a bifunctional de novo protein.
Authors: Smith, B.A. / Mularz, A.E. / Hecht, M.H.
History
DepositionOct 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: De novo ferric enterobactin esterase Syn-F4
B: De novo ferric enterobactin esterase Syn-F4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,0213
Polymers24,9622
Non-polymers591
Water1,35175
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, Dimer, light scattering, Dimer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3510 Å2
ΔGint-30 kcal/mol
Surface area11350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.342, 61.391, 135.729
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11A-201-

ACT

21A-337-

HOH

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Components

#1: Protein De novo ferric enterobactin esterase Syn-F4


Mass: 12481.063 Da / Num. of mol.: 2 / Mutation: K4T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Gene: synF4 / Plasmid: pCA24N / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star / References: iron(III)-enterobactin esterase
#2: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 75 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.98 Å3/Da / Density % sol: 37.73 % / Description: Plate-like crystal
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.9
Details: 0.1M Tris, 0.2M ammonium acetate, 15% PEG 3350 (After crystallization, ferric enterobactin was added.)

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Data collection

DiffractionMean temperature: 95 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Feb 23, 2019 / Details: Focusing Mirror
RadiationMonochromator: Numerical link type Si(111) double crystal monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2→50 Å / Num. obs: 12201 / % possible obs: 88.3 % / Redundancy: 11.6 % / Rmerge(I) obs: 0.104 / Rpim(I) all: 0.031 / Rrim(I) all: 0.109 / Χ2: 0.955 / Net I/σ(I): 5.4 / Num. measured all: 141588
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2-2.039.60.6644140.8390.2180.7010.71859.9
2.03-2.079.40.6033900.8760.1980.6360.75859.2
2.07-2.119.70.574420.8870.1820.60.7765.2
2.11-2.1510.50.4294940.9740.1330.4510.84469.5
2.15-2.29.90.4264720.960.1360.4480.80572.6
2.2-2.2510.40.3635310.9620.1140.3820.77878.4
2.25-2.3110.70.325490.9840.0980.3350.84980.6
2.31-2.3710.80.3285950.9750.10.3440.87187.1
2.37-2.4411.30.2756490.9870.0830.2880.95693.4
2.44-2.5211.30.2726530.9880.0810.2840.9298
2.52-2.6111.60.2396890.9910.0710.2490.96199.4
2.61-2.7111.70.2146800.9920.0650.2240.94399.6
2.71-2.8412.40.1786720.9940.0520.1850.99899.6
2.84-2.9913.20.1617110.9970.0460.1671.11399.7
2.99-3.1713.30.1456740.9970.0410.1511.067100
3.17-3.4212.70.1166940.9980.0340.1211.166100
3.42-3.7612.20.1016940.9970.030.1051.157100
3.76-4.3113.20.0897200.9980.0250.0931.14100
4.31-5.4312.20.0777070.9970.0220.081.035100
5.43-5011.80.05377110.0160.0560.64100

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Processing

Software
NameVersionClassification
REFMAC5.8refinement
HKL-2000data scaling
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8H7C

8h7c


Resolution: 2→50 Å / Cor.coef. Fo:Fc: 0.925 / Cor.coef. Fo:Fc free: 0.877 / Cross valid method: THROUGHOUT / ESU R: 0.341 / ESU R Free: 0.242 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26624 538 5.1 %RANDOM
Rwork0.21714 ---
obs0.21967 9988 76.38 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 35.875 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å20 Å20 Å2
2---0.45 Å20 Å2
3---0.5 Å2
Refinement stepCycle: 1 / Resolution: 2→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1567 0 4 75 1646
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0191622
X-RAY DIFFRACTIONr_bond_other_d00.021567
X-RAY DIFFRACTIONr_angle_refined_deg1.2351.8992188
X-RAY DIFFRACTIONr_angle_other_deg3.52333556
X-RAY DIFFRACTIONr_dihedral_angle_1_deg4.7245181
X-RAY DIFFRACTIONr_dihedral_angle_2_deg38.73524.22109
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.69615300
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.6361514
X-RAY DIFFRACTIONr_chiral_restr0.0740.2237
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.021867
X-RAY DIFFRACTIONr_gen_planes_other0.0090.02451
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.9833.268728
X-RAY DIFFRACTIONr_mcbond_other1.9683.263727
X-RAY DIFFRACTIONr_mcangle_it3.2044.852904
X-RAY DIFFRACTIONr_mcangle_other3.2094.857905
X-RAY DIFFRACTIONr_scbond_it2.5143.741893
X-RAY DIFFRACTIONr_scbond_other2.5133.741893
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other4.2555.4861283
X-RAY DIFFRACTIONr_long_range_B_refined6.55326.1441847
X-RAY DIFFRACTIONr_long_range_B_other6.54426.1421844
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2→2.052 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.182 27 -
Rwork0.186 379 -
obs--39.34 %

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