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- PDB-4z5p: Crystal structure of the LnmA cytochrome P450 hydroxylase from th... -

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Basic information

Entry
Database: PDB / ID: 4z5p
TitleCrystal structure of the LnmA cytochrome P450 hydroxylase from the leinamycin biosynthetic pathway of Streptomyces atroolivaceus S-140 at 1.9 A resolution
ComponentsCytochrome P450 hydroxylase
KeywordsOXIDOREDUCTASE / hydroxylase / leinamycin / heme / monooxygenase / Structural Genomics / PSI-Biology / Midwest Center for Structural Genomics / MCSG / Enzyme Discovery for Natural Product Biosynthesis / NatPro
Function / homology
Function and homology information


oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen / monooxygenase activity / iron ion binding / heme binding
Similarity search - Function
Cytochrome P450, B-class / Cytochrome p450 / Cytochrome P450 / Cytochrome P450, conserved site / Cytochrome P450 cysteine heme-iron ligand signature. / Cytochrome P450 / Cytochrome P450 superfamily / Cytochrome P450 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / TRIETHYLENE GLYCOL / Cytochrome P450 hydroxylase
Similarity search - Component
Biological speciesStreptomyces atroolivaceus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.9 Å
AuthorsMa, M. / Lohman, J. / Rudolf, J. / Miller, M.D. / Cao, H. / Osipiuk, J. / Babnigg, G. / Phillips Jr., G.N. / Joachimiak, A. / Shen, B. ...Ma, M. / Lohman, J. / Rudolf, J. / Miller, M.D. / Cao, H. / Osipiuk, J. / Babnigg, G. / Phillips Jr., G.N. / Joachimiak, A. / Shen, B. / Midwest Center for Structural Genomics (MCSG) / Enzyme Discovery for Natural Product Biosynthesis (NatPro)
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U01GM098248 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA106150 United States
CitationJournal: To be Published
Title: Crystal structure of the LnmA cytochrome P450 hydroxylase from the leinamycin biosynthetic pathway of Streptomyces atroolivaceus S-140
Authors: Ma, M. / Lohman, J. / Rudolf, J. / Miller, M.D. / Cao, H. / Osipiuk, J. / Babnigg, G. / Phillips Jr., G.N. / Joachimiak, A. / Shen, B.
History
DepositionApr 2, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Author supporting evidence / Derived calculations ...Author supporting evidence / Derived calculations / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support ...entity_src_gen / pdbx_audit_support / pdbx_struct_oper_list / software
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome P450 hydroxylase
B: Cytochrome P450 hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,1525
Polymers88,7682
Non-polymers1,3833
Water6,828379
1
A: Cytochrome P450 hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,0012
Polymers44,3841
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Cytochrome P450 hydroxylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,1513
Polymers44,3841
Non-polymers7672
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)55.759, 75.454, 93.858
Angle α, β, γ (deg.)90.000, 98.910, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Cytochrome P450 hydroxylase


Mass: 44384.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces atroolivaceus (bacteria) / Strain: S-140 / Gene: LnmA / Plasmid: pRSF-TEV/LIC / Production host: Escherichia Coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q8GGQ1
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 379 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHE CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSSHHHHHHSQDPGDENLYFQS. THE TAG ...THE CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSSHHHHHHSQDPGDENLYFQS. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A SERINE (0) FOLLOWED BY THE TARGET SEQUENCE.

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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: 2.2 Å3/Da / Density % sol: 44.02 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 6.77
Details: 0.2 M ammonium acetate, 0.1 M Bis-Tris, pH 6.77, 25% PEG3350, VAPOR DIFFUSION, SITTING DROP
Temp details: room temperature
Experiment crystal grow comp
ConcComp nameComp-IDCrystal-IDSol-ID
17. mg/mlprotein11macromolecule
10. mMTEAOH21macromolecule
20. mMNaCl31macromolecule
10. mMKCl41macromolecule
25. %(w/v)PEG 335051precipitant
0.1 MBis-Tris61precipitant
0.2 Mammonium acetate71precipitant
25. %(w/v)PEG 335081reservoir
0.1 MBis-Tris91reservoir
0.2 Mammonium acetate101reservoir
Experiment crystal grow sol
Crystal-IDSol-IDpHVolume (Å3)
1macromolecule7.51.0 µL
1precipitant6.771.0 µL
1reservoir6.770.5 mL

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Data collection

DiffractionMean temperature: 100 K / Crystal support: 0.2-0.3 mm nylon loop
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.97918 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Apr 10, 2014 / Details: mirrors
RadiationMonochromator: double crystals / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 1.9→46.36 Å / Num. all: 57513 / Num. obs: 57513 / % possible obs: 94.9 % / Redundancy: 3.7 % / CC1/2: 0.997 / Rpim(I) all: 0.056 / Rrim(I) all: 0.108 / Rsym value: 0.092 / Net I/av σ(I): 7.03 / Net I/σ(I): 11.1 / Num. measured all: 210829
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) allRmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allCC1/2Rpim(I) allRrim(I) allRsym valueDiffraction-IDNet I/σ(I) obs% possible all
1.9-1.953.20.9910.8331.61007631150.7330.530.9910.83311.670.1
1.95-23.20.8420.70121255239160.7420.4580.8420.701290.5
2-2.063.40.6810.5742.81407741020.820.3620.6810.5742.897.1
2.06-2.123.80.5690.4883.61518039840.8490.290.5690.4883.697.5
2.12-2.193.80.4650.44.41494739100.8930.2370.4650.44.497.5
2.19-2.273.80.4030.3465.11441137620.9090.2050.4030.3465.197.6
2.27-2.363.80.3290.28261391336460.9350.1680.3290.282697.7
2.36-2.453.80.2780.23871335835030.9490.1420.2780.238798.2
2.45-2.563.80.2320.1997.91282833880.9670.1180.2320.1997.998.3
2.56-2.693.80.1830.1579.41227632400.9780.0940.1830.1579.497.8
2.69-2.833.80.150.12810.81157430600.9840.0770.150.12810.898.2
2.83-33.70.120.10212.81087029050.990.0620.120.10212.897.8
3-3.213.70.0870.07416.41014127110.9950.0450.0870.07416.497.5
3.21-3.473.70.0650.05520.3942725380.9970.0330.0650.05520.396.8
3.47-3.83.70.0490.04225.8842622910.9970.0260.0490.04225.896.1
3.8-4.253.70.0420.03629.8768821000.9980.0220.0420.03629.896.2
4.25-4.913.50.040.03432.3651218360.9980.0210.040.03432.396.4
4.91-6.013.60.0420.03628.9564015780.9980.0220.0420.03628.996.3
6.01-8.53.70.0380.03231.2453312410.9980.020.0380.03231.296.9
8.5-46.363.50.0330.02837.924006870.9980.0170.0330.02837.996

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
PHASERphasing
REFMAC5.8.0107refinement
PDB_EXTRACT3.11data extraction
SBC-Collectdata collection
XDSdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4Z5Q

4z5q


Resolution: 1.9→46.36 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.945 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 9.452 / SU ML: 0.132 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.181 / ESU R Free: 0.148 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. WATERS WERE ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 4. THE HEME IRON IS MODELED AS FE2+ AND IS LIKELY REDUCED BY X-RAY EXPOSURE. 5. A PEG FRAGMENT FROM THE CRYSTLLIZATOION CONDITIONS HAVE BEEN MODELED.
RfactorNum. reflection% reflectionSelection details
Rfree0.2167 2884 5 %RANDOM
Rwork0.1908 54626 --
obs0.1922 57510 94.73 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 114.12 Å2 / Biso mean: 34.8009 Å2 / Biso min: 12.61 Å2
Baniso -1Baniso -2Baniso -3
1-0.8 Å20 Å20.6 Å2
2---0.23 Å20 Å2
3----0.72 Å2
Refinement stepCycle: LAST / Resolution: 1.9→46.36 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6005 0 96 379 6480
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0196359
X-RAY DIFFRACTIONr_bond_other_d0.0040.026061
X-RAY DIFFRACTIONr_angle_refined_deg1.491.9898689
X-RAY DIFFRACTIONr_angle_other_deg1.083313860
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5255804
X-RAY DIFFRACTIONr_dihedral_angle_2_deg26.17422.391297
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.099151001
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.4021575
X-RAY DIFFRACTIONr_chiral_restr0.0930.2958
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0217295
X-RAY DIFFRACTIONr_gen_planes_other0.0050.021504
X-RAY DIFFRACTIONr_mcbond_it2.1953.8053150
X-RAY DIFFRACTIONr_mcbond_other2.1943.8053149
X-RAY DIFFRACTIONr_mcangle_it3.2987.1013946
LS refinement shellResolution: 1.9→1.95 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.332 143 -
Rwork0.316 2968 -
all-3111 -
obs--69.1 %
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)
10.6593-0.0738-0.32420.39410.03420.5746-0.06640.0578-0.0525-0.04720.00830.03440.0342-0.05710.05810.291-0.0046-0.01430.0587-0.01690.012-14.338417.314144.904
21.2432-0.24660.86690.3785-0.03811.1412-0.02050.19550.0379-0.004-0.0397-0.0067-0.08860.15980.06020.2235-0.0171-0.01930.05360.01350.00577.12697.46770.828
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 401
2X-RAY DIFFRACTION2B7 - 401

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