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- PDB-4is3: Crystal structure of a 3alpha-hydroxysteroid dehydrogenase (BaiA2... -

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Basic information

Entry
Database: PDB / ID: 4is3
TitleCrystal structure of a 3alpha-hydroxysteroid dehydrogenase (BaiA2) associated with secondary bile acid synthesis from Clostridium scindens VPI12708 in complex with a putative NAD(+)-OH- adduct at 2.0 A resolution
ComponentsBile acid 3-alpha hydroxysteroid dehydrogenase
KeywordsOXIDOREDUCTASE / NAD(P)-binding Rossmann-fold domains / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-BIOLOGY
Function / homology
Function and homology information


3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase / 3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase activity / steroid dehydrogenase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / bile acid catabolic process / response to bile acid / bile acid biosynthetic process / bile acid metabolic process / bile acid binding / oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / NAD+ binding ...3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase / 3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase activity / steroid dehydrogenase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / bile acid catabolic process / response to bile acid / bile acid biosynthetic process / bile acid metabolic process / bile acid binding / oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor / NAD+ binding / protein homotetramerization / cytoplasm
Similarity search - Function
short chain dehydrogenase / Short-chain dehydrogenase/reductase, conserved site / Short-chain dehydrogenases/reductases family signature. / Short-chain dehydrogenase/reductase SDR / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Unknown ligand / 3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase 2
Similarity search - Component
Biological speciesClostridium scindens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2 Å
AuthorsJoint Center for Structural Genomics (JCSG)
CitationJournal: To be published
Title: Crystal structure of a 3alpha-hydroxysteroid dehydrogenase (BaiA2) associated with secondary bile acid synthesis from Clostridium scindens VPI12708 in complex with a putative NAD(+)-OH- adduct at 2.0 A resolution
Authors: Joint Center for Structural Genomics (JCSG)
History
DepositionJan 16, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2013Group: Structure summary
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software
Revision 1.3Jan 24, 2018Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.4Feb 1, 2023Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / 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_conn.pdbx_leaving_atom_flag / _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: Bile acid 3-alpha hydroxysteroid dehydrogenase
B: Bile acid 3-alpha hydroxysteroid dehydrogenase
C: Bile acid 3-alpha hydroxysteroid dehydrogenase
D: Bile acid 3-alpha hydroxysteroid dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,15114
Polymers117,3804
Non-polymers2,77210
Water13,529751
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17800 Å2
ΔGint-113 kcal/mol
Surface area33460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.512, 92.891, 82.915
Angle α, β, γ (deg.)90.00, 90.78, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Bile acid 3-alpha hydroxysteroid dehydrogenase


Mass: 29344.883 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium scindens (bacteria) / Strain: VPI 12708
Description: The source organism was previously designated Eubacterium sp. (strain VPI 12708).
Gene: baiA, BAIA2 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: P19337
#2: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#3: Chemical
ChemComp-UNL / UNKNOWN LIGAND


Num. of mol.: 4 / Source method: obtained synthetically
#4: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 751 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHIS CONSTRUCT (RESIDUES 1-249) WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG.

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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: 2.25 Å3/Da / Density % sol: 45.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.1M tris hydrochloride pH 8.5, 30% polyethylene glycol 4000, 0.2M sodium acetate, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.91162, 0.97936, 0.97919
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: May 14, 2010 / Details: double crystal monochromator
RadiationMonochromator: double crystal / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.911621
20.979361
30.979191
ReflectionResolution: 2→29.007 Å / Num. obs: 68376 / % possible obs: 97.2 % / Observed criterion σ(I): -3 / Redundancy: 3.8 % / Biso Wilson estimate: 25.317 Å2 / Rmerge(I) obs: 0.091 / Net I/σ(I): 12.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obs% possible all
2-2.053.60.6492.316415460688.9
2.05-2.113.80.5153.118414488696.9
2.11-2.173.80.4243.618140479797.3
2.17-2.233.80.374.217593462897.3
2.23-2.313.80.3025.117317453397.4
2.31-2.393.80.2525.916546433697.4
2.39-2.483.80.2087.116173422697.7
2.48-2.583.80.178815587406597.7
2.58-2.693.80.1449.814980390197.9
2.69-2.823.80.11611.514408377098.2
2.82-2.983.80.09314.113707358098.3
2.98-3.163.80.07816.312869337198.5
3.16-3.383.80.06519.412173320298.7
3.38-3.653.80.04924.311254296698.4
3.65-3.993.80.0442810359273598.8
3.99-4.473.80.04131.79304247798.1
4.47-5.163.80.03933.68295218998.6
5.16-6.323.80.04431.37086187198.9
6.32-8.933.80.04434.35450145299
8.93-29.013.60.03539.7284678595.4

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SHELXphasing
SHARPphasing
XSCALEJanuary 30, 2009data scaling
BUSTER-TNT2.10.0refinement
XDSdata reduction
SHELXDphasing
BUSTER2.10.0refinement
RefinementMethod to determine structure: MAD / Resolution: 2→29.007 Å / Cor.coef. Fo:Fc: 0.9531 / Cor.coef. Fo:Fc free: 0.9376 / Occupancy max: 1 / Occupancy min: 0.33 / Cross valid method: THROUGHOUT / σ(F): 0
Details: 1. 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 ...Details: 1. 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. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. THE REFINEMENT WAS RESTRAINED AGAINST THE MAD PHASES. 4. NCS RESTRAINTS WERE APPLIED USING BUSTER'S LSSR RESTRAINT (AUTONCS). 5. ACETATE (ACT) FROM THE CRYSTALLIZATION SOLUTION WAS MODELED INTO THE STRUCTURE. 6. ADDITIONAL ELECTRON DENSITY ADJACENT TO THE C6N ATOM OF THE NAD NICOTINAMIDE RING WAS MODELED AS AN UNKNOWN LIGAND (UNL). QUANTUM MECHANICAL CALCULATIONS SUGGEST THAT THE NAD AND UNL MAY COMPRISE AN NAD(+)-HYDROXIDE ADDUCT. FOR SIMPLICITY, THE UNL WAS MODELED AT FULL OCCUPANCY. THE PLANARITY RESTRAINTS ON THE NAD NICOTINAMIDE RING WERE RELAXED TO MODEL THE DISTORTION OF THE NICOTINAMIDE RING NEAR THE UNL EVIDENT IN THE ELECTRON DENSITY MAPS. THE BOND LENGTH AND ANGLE RESTRAINTS WERE NOT ADJUSTED IN THIS MODEL.
RfactorNum. reflection% reflectionSelection details
Rfree0.182 3444 5.04 %RANDOM
Rwork0.1519 ---
obs0.1534 68331 97.36 %-
Displacement parametersBiso max: 113.08 Å2 / Biso mean: 30.2578 Å2 / Biso min: 13.22 Å2
Baniso -1Baniso -2Baniso -3
1-7.151 Å20 Å2-2.0607 Å2
2---0.1745 Å20 Å2
3----6.9766 Å2
Refine analyzeLuzzati coordinate error obs: 0.216 Å
Refinement stepCycle: LAST / Resolution: 2→29.007 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7440 0 188 751 8379
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d3753SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes206HARMONIC2
X-RAY DIFFRACTIONt_gen_planes1232HARMONIC5
X-RAY DIFFRACTIONt_it7947HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion1099SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact10016SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d7947HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg10841HARMONIC21
X-RAY DIFFRACTIONt_omega_torsion3.09
X-RAY DIFFRACTIONt_other_torsion2.73
LS refinement shellResolution: 2→2.05 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.2384 238 4.95 %
Rwork0.2095 4569 -
all0.211 4807 -
obs--97.36 %
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)
10.6193-0.0609-0.10550.8809-0.20310.5392-0.0364-0.032-0.1507-0.02830.07240.16810.0557-0.0727-0.0359-0.0904-0.01860.0076-0.04920.00730.0355-0.67175.027319.9
20.8002-0.13010.06310.6614-0.04320.3818-0.0478-0.02050.08380.03060.02980.0985-0.0388-0.03710.018-0.0620.00910.0075-0.0262-0.0043-0.01070.711637.436221.8646
31.03720.22220.01550.5938-0.09120.2572-0.02110.02150.0455-0.02590.0322-0.088-0.04640.0119-0.011-0.05880.00050.0076-0.02380.0035-0.022330.692635.425615.1882
41.21640.09660.00121.04150.05890.6049-0.0256-0.0831-0.35940.07920.0117-0.24450.06530.0650.0139-0.12750.0083-0.0073-0.08710.04290.061629.60754.314725.7596
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{A|1 - 301}A1 - 301
2X-RAY DIFFRACTION2{B|1 - 301}B1 - 301
3X-RAY DIFFRACTION3{C|0 - 300}C0 - 300
4X-RAY DIFFRACTION4{D|1 - 300}D1 - 300

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