PDB-4is2: Crystal structure of the apo form of a 3alpha-hydroxysteroid dehy...

Basic information

• Entry: Database: PDB / ID: 4is2
• Title: Crystal structure of the apo form of a 3alpha-hydroxysteroid dehydrogenase (BaiA2) associated with secondary bile acid synthesis from Clostridium scindens VPI12708 at 1.90 A resolution
• Components: Bile acid 3-alpha hydroxysteroid dehydrogenase
• Keywords: OXIDOREDUCTASE / NAD(P)-binding Rossmann-fold domains / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-BIOLOGY

Function / homology

Function:

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

Domain/homology:

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

Component:

3alpha-hydroxy bile acid-CoA-ester 3-dehydrogenase 2

• Biological species: Clostridium scindens (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.9 Å
• Authors: Joint Center for Structural Genomics (JCSG)
• Citation: Journal: To be published; Title: Crystal structure of the apo form of a 3alpha-hydroxysteroid dehydrogenase (BaiA2) associated with secondary bile acid synthesis from Clostridium scindens VPI12708 at 1.90 A resolution; Authors: Joint Center for Structural Genomics (JCSG)

History

Deposition	Jan 16, 2013	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Apr 3, 2013	Provider: repository / Type: Initial release
Revision 1.1	Sep 4, 2013	Group: Structure summary
Revision 1.2	Nov 15, 2017	Group: Refinement description / Category: software / Item: _software.classification / _software.name
Revision 1.3	Jan 24, 2018	Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.4	Feb 1, 2023	Group: Database references / Derived calculations Category: database_2 / struct_conn / struct_ref_seq_dif / struct_site Item: _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

Assembly

Deposited unit

A: Bile acid 3-alpha hydroxysteroid dehydrogenase

hetero molecules

Summary:

• 29.4 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	29,380	2
Polymers	29,345	1
Non-polymers	35	1
Water	1,820	101

1

A: Bile acid 3-alpha hydroxysteroid dehydrogenase

hetero molecules

A: Bile acid 3-alpha hydroxysteroid dehydrogenase

hetero molecules

A: Bile acid 3-alpha hydroxysteroid dehydrogenase

hetero molecules

A: Bile acid 3-alpha hydroxysteroid dehydrogenase

hetero molecules

Summary:

• defined by author&software
• 118 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	117,521	8
Polymers	117,380	4
Non-polymers	142	4
Water	72	4

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_665	-x+1,-y+1,z	1
crystal symmetry operation	3_656	-x+1,y,-z+1	1
crystal symmetry operation	4_566	x,-y+1,-z+1	1

Calculated values:

Buried area	9140 Å2
ΔGint	-106 kcal/mol
Surface area	31000 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	54.041, 93.178, 105.443
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	23
Space group name H-M	I222

Components

#1: Protein

A Bile acid 3-alpha hydroxysteroid dehydrogenase

Details:

Mass: 29344.883 Da / Num. of mol.: 1
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

A-301 ChemComp-CL / CHLORIDE ION

Details:

Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl

#3: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 101 / Source method: isolated from a natural source / Formula: H₂O

• Sequence details: THIS CONSTRUCT (RESIDUES 1-249) WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG.

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 2.26 ų/Da / Density % sol: 45.62 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.5 ; Details: 0.1M sodium acetate pH 4.5, 2.5M sodium chloride, 0.2M lithium sulfate, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.91162, 0.97934, 0.97915
• Detector: Type: MARMOSAIC 325 mm CCD / Detector: CCD / Date: May 13, 2010 / Details: double crystal monochromator
• Radiation: Monochromator: double crystal / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	0.91162	1
2	0.97934	1
3	0.97915	1

• Reflection: Resolution: 1.9→29.794 Å / Num. all: 21347 / Num. obs: 21347 / % possible obs: 99.9 % / Redundancy: 3.7 % / R_pim(I) all: 0.037 / R_rim(I) all: 0.074 / R_sym value: 0.063 / Net I/av σ(I): 7.675 / Net I/σ(I): 9.9 / Num. measured all: 78306

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	Rmerge(I) all	Rmerge(I) obs	Mean I/σ(I) obs	Num. measured all	Num. unique all	Rpim(I) all	Rrim(I) all	Rsym value	Net I/σ(I) obs	% possible all
1.9-1.95	3.7	0.887	0.758	1.7	5806	1573	0.452	0.887	0.758	1.7	100
1.95-2	3.7	0.708	0.606	2	5592	1505	0.359	0.708	0.606	2	100
2-2.06	3.7	0.535	0.457	2.7	5423	1479	0.272	0.535	0.457	2.7	100
2.06-2.12	3.7	0.365	0.312	3.6	5269	1432	0.186	0.365	0.312	3.6	100
2.12-2.19	3.7	0.298	0.255	4.4	5170	1409	0.152	0.298	0.255	4.4	100
2.19-2.27	3.7	0.237	0.203	5.3	5020	1358	0.12	0.237	0.203	5.2	100
2.27-2.36	3.7	0.194	0.166	6.1	4846	1308	0.099	0.194	0.166	6.1	100
2.36-2.45	3.7	0.155	0.133	7.2	4644	1261	0.078	0.155	0.133	7.2	100
2.45-2.56	3.7	0.136	0.117	8.5	4424	1199	0.068	0.136	0.117	8.5	100
2.56-2.69	3.7	0.119	0.102	10.3	4304	1167	0.059	0.119	0.102	10.3	100
2.69-2.83	3.7	0.108	0.093	11.4	4032	1097	0.054	0.108	0.093	11.4	100
2.83-3	3.7	0.091	0.079	14	3870	1049	0.045	0.091	0.079	14	100
3-3.21	3.7	0.079	0.067	15.9	3560	975	0.04	0.079	0.067	15.9	100
3.21-3.47	3.7	0.065	0.056	19	3403	928	0.032	0.065	0.056	19	99.9
3.47-3.8	3.7	0.052	0.045	21.7	3126	851	0.025	0.052	0.045	21.7	99.9
3.8-4.25	3.6	0.043	0.037	23.3	2774	766	0.022	0.043	0.037	23.3	99.7
4.25-4.91	3.6	0.044	0.038	24.8	2524	696	0.022	0.044	0.038	24.8	99.8
4.91-6.01	3.6	0.048	0.042	23.5	2063	579	0.024	0.048	0.042	23.5	99.5
6.01-8.5	3.5	0.048	0.041	23.7	1630	462	0.024	0.048	0.041	23.7	98.9
8.5-29.794	3.3	0.042	0.035	25.4	826	253	0.022	0.042	0.035	25.4	93.6

Phasing

• Phasing: Method: MAD

Processing

Software

Name	Version	Classification	NB
MolProbity	3beta29	model building
PDB_EXTRACT	3.1	data extraction
SHELX		phasing
SHARP		phasing
SCALA	3.3.15	data scaling
REFMAC	5.7.0032	refinement
MOSFLM		data reduction
SHELXD		phasing

Refinement

Method to determine structure: MAD / Resolution: 1.9→29.794 Å / Cor.coef. F_o:F_c: 0.973 / Cor.coef. F_o:F_c free: 0.963 / Occupancy max: 1 / Occupancy min: 0.25 / SU B: 5.463 / SU ML: 0.076 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.114 / ESU R Free: 0.107
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. 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. 3. CHLORIDE (CL) FROM THE CRYSTALLIZATION SOLUTION HAS BEEN MODELED INTO THE STRUCTURE. 4. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 5. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 6. THE SCATTERING FACTORS FOR SULFUR, CHLORINE AND SELENIUM ATOMS WERE ADJUSTED BY REFMAC 5.7.0032 TO ACCOUNT FOR ANOMALOUS DISPERSION BASED ON THE WAVELENGTH 0.91162 A (S f'= 0.16, Cl f'= 0.19, Se f'= -1.81). THE CROMER MANN c VALUES LISTED IN THE CIF VERSION OF THE FILE INCLUDE THIS CORRECTION.

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.1873	1097	5.1 %	RANDOM
Rwork	0.1616	20248	-	-
obs	0.1629	21345	99.63 %	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK

Displacement parameters

B_iso max: 132.19 Ų / B_iso mean: 44.9133 Ų / B_iso min: 21.46 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-0.87 Å2	-0 Å2	0 Å2
2-	-	2.64 Å2	0 Å2
3-	-	-	-1.77 Å2

Refinement step

Cycle: LAST / Resolution: 1.9→29.794 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1592	0	1	101	1694

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.011	0.019	1686
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.02	1671
X-RAY DIFFRACTION	r_angle_refined_deg	1.404	1.971	2297
X-RAY DIFFRACTION	r_angle_other_deg	0.729	3	3845
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.483	5	236
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	39.26	25.072	69
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.245	15	277
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	14.652	15	9
X-RAY DIFFRACTION	r_chiral_restr	0.07	0.2	278
X-RAY DIFFRACTION	r_gen_planes_refined	0.005	0.02	1946
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	367
X-RAY DIFFRACTION	r_mcbond_it	4.167	5.938	878
X-RAY DIFFRACTION	r_mcbond_other	4.165	5.941	879
X-RAY DIFFRACTION	r_mcangle_it	5.652	11	1099
X-RAY DIFFRACTION	r_scbond_it	5.983	7.094	808

LS refinement shell

Resolution: 1.9→1.949 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.296	71	-
Rwork	0.291	1495	-
all	-	1566	-
obs	-	-	99.81 %

Refinement TLS params.

Method: refined / Origin x: 27.1884 Å / Origin y: 63.267 Å / Origin z: 68.8789 Å

	11	12	13	21	22	23	31	32	33
T	0.0714 Å2	0.0001 Å2	0.0056 Å2	-	0.0788 Å2	-0.0148 Å2	-	-	0.0896 Å2
L	1.1577 °2	0.1051 °2	0.1795 °2	-	1.1567 °2	-0.2648 °2	-	-	1.281 °2
S	0.009 Å °	0.0973 Å °	-0.0998 Å °	-0.0305 Å °	-0.0357 Å °	-0.0844 Å °	0.0582 Å °	0.0771 Å °	0.0268 Å °