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- PDB-3ept: Structure of the rebeccamycin biosynthetic enzyme RebC with reduc... -

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Basic information

Entry
Database: PDB / ID: 3ept
TitleStructure of the rebeccamycin biosynthetic enzyme RebC with reduced flavin
ComponentsRebC
KeywordsOXIDOREDUCTASE / flavin / Monooxygenase
Function / homology
Function and homology information


oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen / FAD binding / methyltransferase activity / methylation
Similarity search - Function
Glutaredoxin - #120 / Aromatic-ring hydroxylase, C-terminal / : / D-Amino Acid Oxidase, subunit A, domain 2 / D-Amino Acid Oxidase; Chain A, domain 2 / FAD-binding domain / FAD binding domain / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich ...Glutaredoxin - #120 / Aromatic-ring hydroxylase, C-terminal / : / D-Amino Acid Oxidase, subunit A, domain 2 / D-Amino Acid Oxidase; Chain A, domain 2 / FAD-binding domain / FAD binding domain / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich / Glutaredoxin / FAD/NAD(P)-binding domain superfamily / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / Putative FAD-monooxygenase
Similarity search - Component
Biological speciesLechevalieria aerocolonigenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / RIGID BODY / Resolution: 2.97 Å
AuthorsRyan, K.S. / Drennan, C.L.
CitationJournal: Biochemistry / Year: 2008
Title: The FAD cofactor of RebC shifts to an IN conformation upon flavin reduction
Authors: Ryan, K.S. / Chakraborty, S. / Howard-Jones, A.R. / Walsh, C.T. / Ballou, D.P. / Drennan, C.L.
History
DepositionSep 30, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Sep 6, 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_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RebC
B: RebC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,4535
Polymers119,8552
Non-polymers1,5983
Water1,17165
1
A: RebC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,7383
Polymers59,9281
Non-polymers8112
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: RebC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,7152
Polymers59,9281
Non-polymers7881
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)63.261, 78.223, 123.063
Angle α, β, γ (deg.)90.00, 98.66, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein RebC / Putative monooxygenase / Putative FAD-monooxygenase


Mass: 59927.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lechevalieria aerocolonigenes (bacteria)
Gene: rbmD, rebC / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q8KI25
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H35N9O15P2
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 65 / Source method: isolated from a natural source / Formula: H2O

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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: 51.02 %
Crystal growTemperature: 298 K / pH: 7.4
Details: 1.5 microliters of RebC (9 mg/mL in 150 mM NaCl, 10% glycerol, 25 mM HEPES pH 7.5) were incubated with 0.35 microliters of guanidine-HCl for 30 seconds, followed by addition of 1.5 ...Details: 1.5 microliters of RebC (9 mg/mL in 150 mM NaCl, 10% glycerol, 25 mM HEPES pH 7.5) were incubated with 0.35 microliters of guanidine-HCl for 30 seconds, followed by addition of 1.5 microliters of precipitant solution (19% PEG-8000, 0.1 M HEPES pH 7.4), without mixing, at room temperature and sealed over a precipitant well solution. Immediately after set up, crystal trays were placed on a gel shaker and then, after 12 hours, transferred to a storage space in vibration-isolation. Reduced-RebC was generated by incubating a RebC crystal in a cryogenic solution (19% PEG-8000, 0.1 M HEPES pH 7.4, 20% glycerol) and adding an amount of solid sodium dithionite that is in slight excess of the point at which the crystal became completely clear, indicating that all FAD was reduced. The crystal was then flash-frozen in liquid nitrogen, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.9797
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jul 22, 2007
Details: FLAT COLLIMATING MIRROR, DOUBLE CRYSTAL MONOCHROMATOR, TOROID FOCUSING MIRROR
RadiationMonochromator: DOUBLE CRYSTAL MONOCHROMATOR / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9797 Å / Relative weight: 1
ReflectionResolution: 2.97→40 Å / Num. obs: 22485 / % possible obs: 90.4 % / Redundancy: 4.1 % / Rsym value: 0.149
Reflection shellResolution: 2.97→3.08 Å / Redundancy: 3.1 % / Mean I/σ(I) obs: 3 / Rsym value: 0.27 / % possible all: 50.7

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Processing

Software
NameClassification
HKL-2000data collection
CNSrefinement
HKL-2000data reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: RIGID BODY
Starting model: PDB ENTRY 2R0G

2r0g


Resolution: 2.97→40 Å / Cross valid method: THROUGHOUT / Stereochemistry target values: ENGH & HUBER
RfactorNum. reflection% reflectionSelection details
Rfree0.276 1140 -RANDOM
Rwork0.225 ---
obs0.225 21868 88.4 %-
all-24731 --
Solvent computationBsol: 10 Å2 / ksol: 0.299767 e/Å3
Displacement parameters
Baniso -1Baniso -2Baniso -3
1-9.287 Å20 Å20.108 Å2
2---12.425 Å20 Å2
3---3.137 Å2
Refinement stepCycle: LAST / Resolution: 2.97→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7913 0 107 65 8085
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.34
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.191.5
X-RAY DIFFRACTIONc_mcangle_it1.5452
X-RAY DIFFRACTIONc_scbond_it2.0832
X-RAY DIFFRACTIONc_scangle_it2.4852.5
LS refinement shellResolution: 2.97→3.02 Å /
RfactorNum. reflection
Rfree0.4695 35
Rwork0.3587 -
Xplor file
Refine-IDSerial noParam file
X-RAY DIFFRACTION1protein_rep.param
X-RAY DIFFRACTION2fad-1N4v.par
X-RAY DIFFRACTION3water_rep.param
X-RAY DIFFRACTION4ion.param

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