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- PDB-3eo8: Crystal structure of BluB-like flavoprotein (YP_001089088.1) from... -

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Basic information

Entry
Database: PDB / ID: 3eo8
TitleCrystal structure of BluB-like flavoprotein (YP_001089088.1) from CLOSTRIDIUM DIFFICILE 630 at 1.74 A resolution
ComponentsBluB-like flavoprotein
KeywordsFLAVOPROTEIN / YP_001089088.1 / BluB-like flavoprotein / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-2 / Nitroreductase family
Function / homology
Function and homology information


oxidoreductase activity / nucleotide binding
Similarity search - Function
NADH Oxidase / NADH Oxidase / Nitroreductase / Nitroreductase family / Nitroreductase-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / FLAVIN MONONUCLEOTIDE / Putative nitroreductase
Similarity search - Component
Biological speciesClostridium difficile 630 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.74 Å
AuthorsJoint Center for Structural Genomics (JCSG)
CitationJournal: To be published
Title: Crystal structure of BluB-like flavoprotein (YP_001089088.1) from CLOSTRIDIUM DIFFICILE 630 at 1.74 A resolution
Authors: Joint Center for Structural Genomics (JCSG)
History
DepositionSep 26, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Refinement description / Version format compliance
Revision 1.2Oct 25, 2017Group: Author supporting evidence / Refinement description / Category: pdbx_struct_assembly_auth_evidence / software / Item: _software.classification / _software.name
Revision 1.3Jul 24, 2019Group: Data collection / Derived calculations / Refinement description
Category: software / struct_conn
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.language / _software.location / _software.name / _software.type / _software.version / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Feb 1, 2023Group: Database references / Derived calculations / Category: database_2 / 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

Downloads & links

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Assembly

Deposited unit
A: BluB-like flavoprotein
B: BluB-like flavoprotein
C: BluB-like flavoprotein
D: BluB-like flavoprotein
E: BluB-like flavoprotein
F: BluB-like flavoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,07337
Polymers150,7696
Non-polymers4,30431
Water36,8412045
1
A: BluB-like flavoprotein
B: BluB-like flavoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,66012
Polymers50,2562
Non-polymers1,40410
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11490 Å2
ΔGint-88 kcal/mol
Surface area17070 Å2
MethodPISA
2
C: BluB-like flavoprotein
D: BluB-like flavoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,69312
Polymers50,2562
Non-polymers1,43710
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11480 Å2
ΔGint-90 kcal/mol
Surface area16930 Å2
MethodPISA
3
E: BluB-like flavoprotein
F: BluB-like flavoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,71913
Polymers50,2562
Non-polymers1,46311
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11290 Å2
ΔGint-89 kcal/mol
Surface area17070 Å2
MethodPISA
Unit cell
Length a, b, c (Å)51.843, 183.265, 74.393
Angle α, β, γ (deg.)90.000, 104.890, 90.000
Int Tables number4
Space group name H-MP1211
DetailsAUTHORS STATE THAT SIZE EXCLUSION CHROMATOGRAPHY SUPPORTS THE ASSIGNMENT OF A DIMER AS THE SIGNIFICANT OLIGOMERIZATION STATE.

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Components

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Protein , 1 types, 6 molecules ABCDEF

#1: Protein
BluB-like flavoprotein


Mass: 25128.119 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium difficile 630 (bacteria) / Gene: YP_001089088.1, CD2572 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q182R2

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Non-polymers , 5 types, 2076 molecules

#2: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C17H21N4O9P
#3: Chemical
ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C2H3O2
#4: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2045 / Source method: isolated from a natural source / Formula: H2O

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Details

Sequence detailsTHE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (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: 2.27 Å3/Da / Density % sol: 45.7 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 15.0000% Glycerol, 0.1700M NH4OAc, 25.5000% PEG-4000, 0.1M Acetate pH 4.6, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.91837,0.97918,0.97934
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Aug 4, 2008 / Details: Flat collimating mirror, toroid focusing mirror
RadiationMonochromator: Double crystal monochromator / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.918371
20.979181
30.979341
ReflectionResolution: 1.74→48.337 Å / Num. obs: 133857 / % possible obs: 97.8 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 14.167 Å2 / Rmerge(I) obs: 0.12 / Net I/σ(I): 8.64
Reflection shell
Resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obsDiffraction-ID% possible all
1.74-1.80.7111.94933212773196.8
1.8-1.870.5352.54987312943197.1
1.87-1.960.3953.55413314037197.2
1.96-2.060.2934.54994312956197.5
2.06-2.190.21565203813496197.7
2.19-2.360.1617.75197613476197.8
2.36-2.60.1398.85241213570198.2
2.6-2.970.10211.55120913286198.5
2.97-3.740.05717.35206713584198.4
3.74-48.3370.04321.95223713693198.5

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
REFMAC5.2.0019refinement
PHENIXrefinement
SHELXAUTOSHARPphasing
MolProbity3beta29model building
XSCALEdata scaling
PDB_EXTRACT3.006data extraction
XDSdata reduction
RefinementMethod to determine structure: MAD / Resolution: 1.74→48.337 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.941 / Occupancy max: 1 / Occupancy min: 0.45 / SU B: 4.587 / SU ML: 0.076 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.115 / ESU R Free: 0.113
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORDS CONTAIN RESIDUAL B FACTORS ONLY. 3. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORDS CONTAIN RESIDUAL B FACTORS ONLY. 3. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 4. ACETATE (ACT) AND CHLORIDE ANIONS (CL) FROM THE CRYSTALLIZATION BUFFER WERE MODELED INTO THE STRUCTURE. 5.GLYCEROL MOLECULES (GOL) FROM THE CRYOPROTECTANT SOLUTION WERE MODELED INTO THE STRUCTURE. 6. ASN 135 ON ALL SIX SUBUNITS IN THE ASYMMETRIC UNIT ARE RAMACHANDRAN OUTLIERS; HOWEVER ELECTRON DENSITY SUPPORTS THEIR POSITIONING IN THE MODEL. 7. A FLAVIN MONONUCLEOTIDE (FMN) IS BOUND TO EACH OF THE TWO ACTIVE SITES WITHIN THE DIMER.
RfactorNum. reflection% reflectionSelection details
Rfree0.198 6747 5 %RANDOM
Rwork0.156 ---
obs0.159 133813 97.86 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 62.82 Å2 / Biso mean: 27.441 Å2 / Biso min: 9.27 Å2
Baniso -1Baniso -2Baniso -3
1-0.55 Å20 Å2-1.2 Å2
2---0.55 Å20 Å2
3----0.61 Å2
Refinement stepCycle: LAST / Resolution: 1.74→48.337 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10336 0 282 2045 12663
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0140.02211034
X-RAY DIFFRACTIONr_bond_other_d0.0010.027492
X-RAY DIFFRACTIONr_angle_refined_deg1.3341.99914966
X-RAY DIFFRACTIONr_angle_other_deg0.898318452
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5151385
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.7625.319470
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.347152008
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.7591541
X-RAY DIFFRACTIONr_chiral_restr0.0810.21679
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0212049
X-RAY DIFFRACTIONr_gen_planes_other0.0010.022094
X-RAY DIFFRACTIONr_nbd_refined0.2250.22434
X-RAY DIFFRACTIONr_nbd_other0.190.27950
X-RAY DIFFRACTIONr_nbtor_refined0.1870.25511
X-RAY DIFFRACTIONr_nbtor_other0.0870.25246
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1550.21498
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2290.227
X-RAY DIFFRACTIONr_symmetry_vdw_other0.240.283
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.180.2110
X-RAY DIFFRACTIONr_mcbond_it0.7671.57309
X-RAY DIFFRACTIONr_mcbond_other0.2061.52686
X-RAY DIFFRACTIONr_mcangle_it1.061210833
X-RAY DIFFRACTIONr_scbond_it1.96535014
X-RAY DIFFRACTIONr_scangle_it2.864.54093
LS refinement shellResolution: 1.74→1.785 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.295 491 -
Rwork0.218 9315 -
all-9806 -
obs--96.8 %
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)
10.26580.02980.08190.6722-0.07250.3304-0.02610.0126-0.00380.040.0051-0.0162-0.0196-0.0110.021-0.1865-0.00430.0167-0.1326-0.0033-0.166769.036149.972-18.369
20.25440.00670.05690.52010.03540.36930.0158-0.0003-0.00710.0117-0.00540.0081-0.0065-0.0051-0.0104-0.2049-0.00490.0195-0.1367-0.0096-0.163966.72489.76-16.691
30.25180.05690.03160.5470.0390.35720.00280.01840.01790.0222-0.0018-0.0138-0.0070.0024-0.001-0.2087-0.0030.0203-0.13350.0011-0.160333.636121.19817.341
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A0 - 218
2X-RAY DIFFRACTION1B0 - 218
3X-RAY DIFFRACTION2C0 - 218
4X-RAY DIFFRACTION2D0 - 218
5X-RAY DIFFRACTION3E0 - 218
6X-RAY DIFFRACTION3F0 - 218

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