PDB-7a2b: X-ray structure of Lactobacillus brevis alcohol dehydrogenase mut...

Basic information

• Entry: Database: PDB / ID: 7a2b
• Title: X-ray structure of Lactobacillus brevis alcohol dehydrogenase mutant Q207D
• Components: R-specific alcohol dehydrogenase
• Keywords: OXIDOREDUCTASE / Nucleotide Binding Oxidoreductase Activity
• Function / homology: Short-chain dehydrogenase/reductase, conserved site / Short-chain dehydrogenases/reductases family signature. / Enoyl-(Acyl carrier protein) reductase / Short-chain dehydrogenase/reductase SDR / oxidoreductase activity / NAD(P)-binding domain superfamily / nucleotide binding / metal ion binding / R-specific alcohol dehydrogenase
• Biological species: Lactobacillus brevis (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.399 Å
• Authors: Bischoff, D. / Hermann, J. / Janowski, R. / Niessing, D. / Grob, P. / Hekmat, D. / Weuster-Botz, D.

Funding support

Germany, 1items

Organization	Grant number	Country
German Research Foundation (DFG)	SPP 1934	Germany

• Citation: Journal: Crystals / Year: 2021; Title: Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts; Authors: Hermann, J. / Bischoff, D. / Grob, P. / Janowski, R. / Hekmat, D. / Niessing, D. / Zacharias, M. / Weuster-Botz, D.

History

Deposition	Aug 17, 2020	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Oct 28, 2020	Provider: repository / Type: Initial release
Revision 1.1	Jun 9, 2021	Group: Database references / Category: citation / citation_author Item: _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2	Jan 31, 2024	Group: Data collection / Database references / Derived calculations / Refinement description Category: atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Assembly

Deposited unit

A: R-specific alcohol dehydrogenase

hetero molecules

Summary:

• 26.7 kDa, 1 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	26,667	2
Polymers	26,643	1
Non-polymers	24	1
Water	5,729	318

1

A: R-specific alcohol dehydrogenase

hetero molecules

A: R-specific alcohol dehydrogenase

hetero molecules

A: R-specific alcohol dehydrogenase

hetero molecules

A: R-specific alcohol dehydrogenase

hetero molecules

Summary:

• defined by author&software
• Evidence: gel filtration
• 107 kDa, 4 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	106,669	8
Polymers	106,572	4
Non-polymers	97	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_655	-x+1,y,-z	1
crystal symmetry operation	4_565	x,-y+1,-z	1

Calculated values:

Buried area	14110 Å2
ΔGint	-94 kcal/mol
Surface area	32700 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	55.820, 80.520, 115.100
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	23
Space group name H-M	I222

Components on special symmetry positions

ID	Model	Components
1	1	A-301- MG
2	1	A-460- HOH
3	1	A-520- HOH
4	1	A-581- HOH
5	1	A-590- HOH
6	1	A-591- HOH

Components

#1: Protein

A R-specific alcohol dehydrogenase

Details:

Mass: 26643.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus brevis (bacteria) / Gene: radh / Production host: Escherichia coli (E. coli) / References: UniProt: Q84EX5

#2: Chemical

A-301 ChemComp-MG / MAGNESIUM ION

Details:

Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

#3: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 318 / Source method: isolated from a natural source / Formula: H₂O

• Has ligand of interest: N

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 2.44 ų/Da / Density % sol: 49.6 %
• Crystal grow: Temperature: 293 K / Method: microbatch; Details: Protein solution (10 g/L LbADH, 20mM HEPES/NaOH pH7.0, 1 mM MgCl2) and precipitation buffer (1 mM Tris/HCl pH 7.0, 50 mM MgCl2 and 100 g/L PEG 550 MME)

Data collection

• Diffraction: Mean temperature: 100 K / Serial crystal experiment: N
• Diffraction source: Source: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
• Detector: Type: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Nov 28, 2019
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1 Å / Relative weight: 1
• Reflection: Resolution: 1.399→46.83 Å / Num. obs: 98425 / % possible obs: 99.6 % / Redundancy: 4.07 % / CC_1/2: 0.999 / R_rim(I) all: 0.05 / Net I/σ(I): 17.4
• Reflection shell: Resolution: 1.4→1.49 Å / Redundancy: 3.91 % / Mean I/σ(I) obs: 5.43 / Num. unique obs: 15718 / CC_1/2: 0.9969 / R_rim(I) all: 0.174 / % possible all: 98.6

Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
XDS	Jan 31, 2020	data reduction
XSCALE	Jan 31, 2020	data scaling
PHASER	5.8.0258	phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT
Starting model: 6h07

6h07

Resolution: 1.399→40.26 Å / Cor.coef. F_o:F_c: 0.975 / Cor.coef. F_o:F_c free: 0.969 / SU B: 2.462 / SU ML: 0.042 / Cross valid method: FREE R-VALUE / ESU R: 0.059 / ESU R Free: 0.055

	Rfactor	Num. reflection	% reflection
Rfree	0.175	2567	5.001 %
Rwork	0.144	48763	-
all	0.146	-	-
obs	-	51330	99.724 %

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT

Displacement parameters

B_iso mean: 16.542 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	-0.977 Å2	-0 Å2	-0 Å2
2-	-	2.31 Å2	0 Å2
3-	-	-	-1.333 Å2

Refinement step

Cycle: LAST / Resolution: 1.399→40.26 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	1870	0	1	318	2189

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.006	0.013	2057
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.017	1940
X-RAY DIFFRACTION	r_angle_refined_deg	1.29	1.639	2801
X-RAY DIFFRACTION	r_angle_other_deg	1.401	1.59	4504
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.5	5	282
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.087	23.895	95
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	11.555	15	361
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	20.013	15	9
X-RAY DIFFRACTION	r_chiral_restr	0.062	0.2	273
X-RAY DIFFRACTION	r_gen_planes_refined	0.006	0.02	2450
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	444
X-RAY DIFFRACTION	r_nbd_refined	0.208	0.2	428
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.166	0.2	1758
X-RAY DIFFRACTION	r_nbtor_refined	0.156	0.2	1024
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.076	0.2	826
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.145	0.2	230
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.197	0.2	13
X-RAY DIFFRACTION	r_nbd_other	0.206	0.2	70
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.107	0.2	30
X-RAY DIFFRACTION	r_mcbond_it	1.223	1.551	1083
X-RAY DIFFRACTION	r_mcbond_other	1.223	1.552	1082
X-RAY DIFFRACTION	r_mcangle_it	1.478	2.341	1380
X-RAY DIFFRACTION	r_mcangle_other	1.478	2.34	1381
X-RAY DIFFRACTION	r_scbond_it	1.378	1.746	974
X-RAY DIFFRACTION	r_scbond_other	1.378	1.747	974
X-RAY DIFFRACTION	r_scangle_it	1.788	2.548	1421
X-RAY DIFFRACTION	r_scangle_other	1.788	2.548	1421
X-RAY DIFFRACTION	r_lrange_it	2.277	20.524	2459
X-RAY DIFFRACTION	r_lrange_other	2.072	19.639	2364
X-RAY DIFFRACTION	r_rigid_bond_restr	0.959	3	3995

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 20

Resolution (Å)	Rfactor Rfree	Num. reflection Rfree	Rfactor Rwork	Num. reflection Rwork	Rfactor all	Num. reflection all	Fsc free	Fsc work	% reflection obs (%)	WRfactor Rwork
1.399-1.436	0.26	182	0.195	3449	0.198	3753	0.909	0.936	96.7493	0.171
1.436-1.475	0.232	183	0.187	3481	0.189	3664	0.929	0.936	100	0.167
1.475-1.518	0.247	178	0.185	3379	0.188	3557	0.893	0.933	100	0.167
1.518-1.564	0.223	173	0.171	3293	0.174	3466	0.931	0.947	100	0.155
1.564-1.615	0.192	167	0.154	3179	0.156	3346	0.948	0.96	100	0.141
1.615-1.672	0.206	163	0.147	3099	0.15	3262	0.949	0.965	100	0.135
1.672-1.735	0.213	158	0.147	2997	0.15	3156	0.931	0.96	99.9683	0.138
1.735-1.806	0.205	152	0.155	2880	0.157	3032	0.942	0.958	100	0.148
1.806-1.886	0.185	146	0.145	2787	0.147	2933	0.955	0.964	100	0.141
1.886-1.978	0.184	139	0.144	2627	0.146	2767	0.957	0.964	99.9639	0.142
1.978-2.084	0.177	132	0.135	2521	0.137	2653	0.961	0.973	100	0.136
2.084-2.21	0.156	126	0.122	2392	0.123	2519	0.97	0.977	99.9603	0.125
2.21-2.362	0.151	119	0.122	2263	0.123	2384	0.977	0.977	99.9161	0.13
2.362-2.551	0.18	110	0.132	2084	0.134	2194	0.963	0.972	100	0.143
2.551-2.793	0.16	104	0.135	1967	0.136	2071	0.971	0.974	100	0.151
2.793-3.121	0.172	92	0.138	1759	0.139	1852	0.966	0.973	99.946	0.159
3.121-3.6	0.17	83	0.14	1566	0.142	1652	0.97	0.977	99.8184	0.161
3.6-4.401	0.131	70	0.137	1341	0.136	1413	0.983	0.98	99.8585	0.165
4.401-6.187	0.134	56	0.15	1066	0.149	1122	0.979	0.98	100	0.192
6.187-40.26	0.166	34	0.162	633	0.163	672	0.977	0.971	99.256	0.2