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- PDB-3uzy: Crystal structure of 5beta-reductase (AKR1D1) E120H mutant in com... -

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Basic information

Entry
Database: PDB / ID: 3uzy
TitleCrystal structure of 5beta-reductase (AKR1D1) E120H mutant in complex with NADP+ and 5beta-dihydrotestosterone
Components3-oxo-5-beta-steroid 4-dehydrogenase
KeywordsOXIDOREDUCTASE / ALDO-KETO REDUCTASE / STEROID AND BILE ACID METABOLISM / CATALYTIC TETRAD MUTANT / Tim Barrel
Function / homology
Function and homology information


Delta4-3-oxosteroid 5beta-reductase / C21-steroid hormone metabolic process / steroid dehydrogenase activity / bile acid catabolic process / delta4-3-oxosteroid 5beta-reductase activity / bile acid biosynthetic process / ketosteroid monooxygenase activity / Synthesis of bile acids and bile salts via 24-hydroxycholesterol / aldo-keto reductase (NADPH) activity / cholesterol catabolic process ...Delta4-3-oxosteroid 5beta-reductase / C21-steroid hormone metabolic process / steroid dehydrogenase activity / bile acid catabolic process / delta4-3-oxosteroid 5beta-reductase activity / bile acid biosynthetic process / ketosteroid monooxygenase activity / Synthesis of bile acids and bile salts via 24-hydroxycholesterol / aldo-keto reductase (NADPH) activity / cholesterol catabolic process / aldose reductase (NADPH) activity / androgen metabolic process / Synthesis of bile acids and bile salts via 27-hydroxycholesterol / Synthesis of bile acids and bile salts via 7alpha-hydroxycholesterol / digestion / steroid binding / cytosol
Similarity search - Function
Aldo-keto reductase family 1 member D1 / Aldo/keto reductase family putative active site signature. / Aldo/keto reductase family signature 1. / NADP-dependent oxidoreductase domain / Aldo/keto reductase family signature 2. / Aldo/keto reductase, conserved site / Aldo-keto reductase / NADP-dependent oxidoreductase domain / Aldo/keto reductase family / NADP-dependent oxidoreductase domain superfamily ...Aldo-keto reductase family 1 member D1 / Aldo/keto reductase family putative active site signature. / Aldo/keto reductase family signature 1. / NADP-dependent oxidoreductase domain / Aldo/keto reductase family signature 2. / Aldo/keto reductase, conserved site / Aldo-keto reductase / NADP-dependent oxidoreductase domain / Aldo/keto reductase family / NADP-dependent oxidoreductase domain superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
5-beta-DIHYDROTESTOSTERONE / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Aldo-keto reductase family 1 member D1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.832 Å
AuthorsChen, M. / Christianson, D.W. / Penning, T.M.
CitationJournal: J.Biol.Chem. / Year: 2012
Title: Conversion of Human Steroid 5beta-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: EXAMPLE OF PERFECT ENZYME ENGINEERING.
Authors: Chen, M. / Drury, J.E. / Christianson, D.W. / Penning, T.M.
History
DepositionDec 7, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 21, 2012Provider: repository / Type: Initial release
Revision 1.1Jun 27, 2012Group: Database references
Revision 1.2Nov 8, 2017Group: Refinement description / Category: software
Item: _software.classification / _software.contact_author ..._software.classification / _software.contact_author / _software.contact_author_email / _software.date / _software.language / _software.location / _software.name / _software.type / _software.version
Revision 1.3Sep 13, 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

Downloads & links

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Assembly

Deposited unit
A: 3-oxo-5-beta-steroid 4-dehydrogenase
B: 3-oxo-5-beta-steroid 4-dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,3558
Polymers79,2172
Non-polymers2,1396
Water9,908550
1
A: 3-oxo-5-beta-steroid 4-dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,6784
Polymers39,6081
Non-polymers1,0693
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: 3-oxo-5-beta-steroid 4-dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,6784
Polymers39,6081
Non-polymers1,0693
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)50.015, 110.224, 129.497
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein 3-oxo-5-beta-steroid 4-dehydrogenase / Aldo-keto reductase family 1 member D1 / Delta(4)-3-ketosteroid 5-beta-reductase / Delta(4)-3- ...Aldo-keto reductase family 1 member D1 / Delta(4)-3-ketosteroid 5-beta-reductase / Delta(4)-3-oxosteroid 5-beta-reductase


Mass: 39608.289 Da / Num. of mol.: 2 / Mutation: E120H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AKR1D1, SRD5B1 / Plasmid: PET28A-AKR1D1 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): C41(DE3)
References: UniProt: P51857, Delta4-3-oxosteroid 5beta-reductase
#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H28N7O17P3
#3: Chemical ChemComp-BDT / 5-beta-DIHYDROTESTOSTERONE / (5beta,8alpha,17beta)-17-hydroxyandrostan-3-one


Mass: 290.440 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H30O2
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 550 / 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: 2.25 Å3/Da / Density % sol: 45.41 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1 M Tris-HCl, 14-20% polyethylene glycol 4000, 10% isopropanol, pH 7.5, vapor diffusion, hanging drop, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9795 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 11, 2010
RadiationMonochromator: Cryo-Cooled double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.83→50 Å / Num. all: 63671 / Num. obs: 58578 / % possible obs: 92 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 5 % / Biso Wilson estimate: 25.1 Å2 / Rmerge(I) obs: 0.087 / Χ2: 1.004 / Net I/σ(I): 10.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
1.83-1.94.70.40559850.923195.7
1.9-1.974.70.29459780.93195.2
1.97-2.064.80.20859660.997194.7
2.06-2.174.90.15959311.062194.2
2.17-2.3150.12958930.993193.6
2.31-2.485.10.10358851.006192.7
2.48-2.735.20.08558131.046191.7
2.73-3.135.30.07557821.027190.4
3.13-3.945.30.07157051.023188.4
3.94-505.40.05256401.024183.7

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHENIX1.5_2refinement
PDB_EXTRACT3.1data extraction
HKL-2000data collection
HKL-2000data reduction
CNSphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 3UZW

3uzw


Resolution: 1.832→42.966 Å / Occupancy max: 1 / Occupancy min: 0.27 / FOM work R set: 0.8672 / SU ML: 0.22 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0.08 / Phase error: 20.48 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2181 2892 5.09 %RANDOM
Rwork0.1726 ---
all0.187 58538 --
obs0.1749 56863 89.04 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 30.272 Å2 / ksol: 0.314 e/Å3
Displacement parametersBiso max: 101.39 Å2 / Biso mean: 20.8956 Å2 / Biso min: 4.32 Å2
Baniso -1Baniso -2Baniso -3
1-0.966 Å2-0 Å2-0 Å2
2---0.862 Å20 Å2
3----0.0433 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.24 Å0.19 Å
Luzzati sigma a0.2 Å0.13 Å
Refinement stepCycle: LAST / Resolution: 1.832→42.966 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5256 0 140 550 5946
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0075615
X-RAY DIFFRACTIONf_angle_d1.1177664
X-RAY DIFFRACTIONf_chiral_restr0.07835
X-RAY DIFFRACTIONf_plane_restr0.006975
X-RAY DIFFRACTIONf_dihedral_angle_d19.6522204
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 21

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8317-1.86170.25341220.20732291241380
1.8617-1.89380.28531420.20282396253886
1.8938-1.92820.27471300.19952553268388
1.9282-1.96530.2621270.18922513264089
1.9653-2.00540.23341500.19252582273290
2.0054-2.04910.24791520.18712559271191
2.0491-2.09670.2761110.19112652276392
2.0967-2.14910.23841530.19422590274392
2.1491-2.20730.28231250.18512639276492
2.2073-2.27220.24211420.18682632277491
2.2722-2.34550.22191520.18572570272291
2.3455-2.42940.22311440.17732638278292
2.4294-2.52660.22891360.1762639277591
2.5266-2.64160.25081530.17782619277291
2.6416-2.78080.21221370.18042620275791
2.7808-2.9550.2321500.18112613276390
2.955-3.18310.22381390.17872587272690
3.1831-3.50330.20061370.16962574271189
3.5033-4.00990.18371460.1462571271788
4.0099-5.05080.14431250.13242577270286
5.0508-42.97730.15481190.14062556267582

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