PDB-2gft: Crystal structure of the E263A nucleophile mutant of Bacillus lic...

ID or keywords:

Entry

Database: PDB / ID: 2gft

Title

Crystal structure of the E263A nucleophile mutant of Bacillus licheniformis endo-beta-1,4-galactanase in complex with galactotriose

Components

Glycosyl Hydrolase Family 53

Keywords

HYDROLASE / beta-alpha-8-barrel / protein-oligosaccharide complex / nucleophile inactive mutant

Function / homology

Function and homology information

arabinogalactan endo-beta-1,4-galactanase / arabinogalactan endo-1,4-beta-galactosidase activity / glucosidase activity / pectin catabolic process / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / metal ion binding
Similarity search - Function

Biological species

Bacillus licheniformis (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

FOURIER SYNTHESIS / Resolution: 2.3 Å

Authors

Welner, D. / Le Nours, J. / De Maria, L. / Jorgensen, C.T. / Christensen, L.L.H. / Larsen, S. / Lo Leggio, L.

Citation

Journal: To be Published
Title: Oligosaccharide binding of Bacillus licheniformis endo-beta-1,4-galactanase
Authors: Le Nours, J. / De Maria, L. / Welner, D. / Jorgensen, C.T. / Christensen, L.L.H. / Larsen, S. / Lo Leggio, L.

History

Deposition	Mar 23, 2006	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jun 13, 2006	Provider: repository / Type: Initial release
Revision 1.1	May 1, 2008	Group: Version format compliance
Revision 1.2	Jul 13, 2011	Group: Version format compliance
Revision 1.3	Jan 24, 2018	Group: Data collection / Structure summary / Category: audit_author / diffrn_source Item: _audit_author.name / _diffrn_source.pdbx_synchrotron_site
Revision 2.0	Jul 29, 2020	Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Structure summary Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / struct_ref_seq_dif / struct_site / struct_site_gen Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1	Oct 20, 2021	Group: Database references / Structure summary / Category: chem_comp / database_2 / struct_ref_seq_dif Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 2.2	Feb 14, 2024	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / struct_ncs_dom_lim Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.end_auth_comp_id

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	2gft.cif.gz	167.1 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	2gft_validation.pdf.gz	1 MB	Display	wwPDB validaton report
Full document	2gft_full_validation.pdf.gz	1 MB	Display
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Data in CIF	2gft_validation.cif.gz	45.3 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/gf/2gft ftp://data.pdbj.org/pub/pdb/validation_reports/gf/2gft	HTTPS FTP

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Related structure data

Related structure data	1r8l 1ur0 1ur4 2ccr
Similar structure data	1ur0-1 1ur4-1 1ur0-2 1ur4-2 2j74-2 2ccr-2 6b10-1 3pz9-d 2j74-1 4ooz-1 Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Glycosyl Hydrolase Family 53

B: Glycosyl Hydrolase Family 53

hetero molecules

88.5 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Glycosyl Hydrolase Family 53

hetero molecules

defined by author
44.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Glycosyl Hydrolase Family 53

hetero molecules

defined by author
44.3 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	50.273, 79.354, 103.906
Angle α, β, γ (deg.)	90.00, 100.56, 90.00
Int Tables number	4
Space group name H-M	P12₁1

Noncrystallographic symmetry (NCS)

NCS domain:
NCS domain segments:

Ens-ID: 1

Dom-ID	Component-ID	Beg auth comp-ID	Beg label comp-ID	End auth comp-ID	End label comp-ID	Refine code	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	GLY	GLY	LEU	LEU	5	A	A	11 - 12	11 - 12
2	1	GLY	GLY	LEU	LEU	5	B	B	11 - 12	11 - 12
3	2	TYR	TYR	TYR	TYR	3	A	A	13	13
4	2	TYR	TYR	TYR	TYR	3	B	B	13	13
5	3	VAL	VAL	LEU	LEU	1	A	A	14 - 20	14 - 20
6	3	VAL	VAL	LEU	LEU	1	B	B	14 - 20	14 - 20
7	4	ARG	ARG	LYS	LYS	3	A	A	21 - 22	21 - 22
8	4	ARG	ARG	LYS	LYS	3	B	B	21 - 22	21 - 22
9	5	ASP	ASP	LEU	LEU	1	A	A	23 - 88	23 - 88
10	5	ASP	ASP	LEU	LEU	1	B	B	23 - 88	23 - 88
11	6	GLU	GLU	GLU	GLU	3	A	A	89	89
12	6	GLU	GLU	GLU	GLU	3	B	B	89	89
13	7	LYS	LYS	PHE	PHE	1	A	A	90 - 132	90 - 132
14	7	LYS	LYS	PHE	PHE	1	B	B	90 - 132	90 - 132
15	8	GLU	GLU	GLU	GLU	3	A	A	133	133
16	8	GLU	GLU	GLU	GLU	3	B	B	133	133
17	9	ASP	ASP	LYS	LYS	1	A	A	134 - 144	134 - 144
18	9	ASP	ASP	LYS	LYS	1	B	B	134 - 144	134 - 144
19	10	GLN	GLN	GLN	GLN	3	A	A	145	145
20	10	GLN	GLN	GLN	GLN	3	B	B	145	145
21	11	SER	SER	SER	SER	1	A	A	146 - 187	146 - 187
22	11	SER	SER	SER	SER	1	B	B	146 - 187	146 - 187
23	12	GLN	GLN	GLN	GLN	3	A	A	188	188
24	12	GLN	GLN	GLN	GLN	3	B	B	188	188
25	13	ALA	ALA	VAL	VAL	1	A	A	189 - 190	189 - 190
26	13	ALA	ALA	VAL	VAL	1	B	B	189 - 190	189 - 190
27	14	ARG	ARG	ARG	ARG	3	A	A	191	191
28	14	ARG	ARG	ARG	ARG	3	B	B	191	191
29	15	GLU	GLU	GLU	GLU	5	A	A	192	192
30	15	GLU	GLU	GLU	GLU	5	B	B	192	192
31	16	THR	THR	LEU	LEU	1	A	A	193 - 219	193 - 219
32	16	THR	THR	LEU	LEU	1	B	B	193 - 219	193 - 219
33	17	HIS	HIS	ARG	ARG	3	A	A	220 - 221	220 - 221
34	17	HIS	HIS	ARG	ARG	3	B	B	220 - 221	220 - 221
35	18	HIS	HIS	HIS	HIS	1	A	A	222	222
36	18	HIS	HIS	HIS	HIS	1	B	B	222	222
37	19	HIS	HIS	HIS	HIS	3	A	A	223	223
38	19	HIS	HIS	HIS	HIS	3	B	B	223	223
39	20	VAL	VAL	GLY	GLY	1	A	A	224 - 311	224 - 311
40	20	VAL	VAL	GLY	GLY	1	B	B	224 - 311	224 - 311
41	21	GLU	GLU	GLU	GLU	3	A	A	312	312
42	21	GLU	GLU	GLU	GLU	3	B	B	312	312
43	22	ALA	ALA	HIS	HIS	3	A	A	313 - 331	313 - 331
44	22	ALA	ALA	HIS	HIS	3	B	B	313 - 331	313 - 331
45	23	ARG	ARG	ARG	ARG	3	A	A	332	332
46	23	ARG	ARG	ARG	ARG	3	B	B	332	332
47	24	LEU	LEU	PRO	PRO	1	A	A	333 - 357	333 - 357
48	24	LEU	LEU	PRO	PRO	1	B	B	333 - 357	333 - 357
49	25	GLU	GLU	GLU	GLU	3	A	A	358	358
50	25	GLU	GLU	GLU	GLU	3	B	B	358	358
51	26	ASP	ASP	LYS	LYS	1	A	A	359 - 362	359 - 362
52	26	ASP	ASP	LYS	LYS	1	B	B	359 - 362	359 - 362
53	27	TRP	TRP	TRP	TRP	5	A	A	363	363
54	27	TRP	TRP	TRP	TRP	5	B	B	363	363
55	28	PHE	PHE	PHE	PHE	1	A	A	364 - 377	364 - 377
56	28	PHE	PHE	PHE	PHE	1	B	B	364 - 377	364 - 377
57	29	LYS	LYS	LYS	LYS	3	A	A	378	378
58	29	LYS	LYS	LYS	LYS	3	B	B	378	378
59	30	GLY	GLY	PHE	PHE	1	A	A	379 - 388	379 - 388
60	30	GLY	GLY	PHE	PHE	1	B	B	379 - 388	379 - 388
61	31	GLN	GLN	GLN	GLN	3	A	A	389	389
62	31	GLN	GLN	GLN	GLN	3	B	B	389	389
63	32	TYR	TYR	THR	THR	1	A	A	390 - 395	390 - 395
64	32	TYR	TYR	THR	THR	1	B	B	390 - 395	390 - 395
65	33	PRO	PRO	PRO	PRO	5	A	A	396	396
66	33	PRO	PRO	PRO	PRO	5	B	B	396	396

#1: Protein

Glycosyl Hydrolase Family 53

Mass: 43720.398 Da / Num. of mol.: 2 / Mutation: E263A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Bacillus licheniformis (bacteria) / Production host:

Bacillus subtilis (bacteria)
References: GenBank: 52005769, UniProt: Q65CX5*PLUS, arabinogalactan endo-beta-1,4-galactanase

#2: Polysaccharide

beta-D-galactopyranose-(1-4)-beta-D-galactopyranose-(1-4)-beta-D-galactopyranose / 4beta-beta-galactotriose

Type: oligosaccharide, Oligosaccharide / Class: Metabolism / Mass: 504.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: 4beta-beta-galactotriose

Descriptor	Type	Program
DGalpb1-4DGalpb1-4DGalpb1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/1,3,2/[a2112h-1b_1-5]/1-1-1/a4-b1_b4-c1	WURCS	PDB2Glycan 1.1.0
[][b-D-Galp]{[(4+1)][b-D-Galp]{[(4+1)][b-D-Galp]{}}}	LINUCS	PDB-CARE

#3: Chemical

ChemComp-CA / CALCIUM ION

Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

#4: Water

ChemComp-HOH / water

Mass: 18.015 Da / Num. of mol.: 294 / Source method: isolated from a natural source / Formula: H₂O

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.33 Å³/Da / Density % sol: 47.2 %
Crystal grow	Temperature: 296 K / Method: vapor diffusion, hanging drop Details: 24-35% (w/v) PEG 1500 Soak: few seconds, 0.3-0.5 M methyl- (1 4)-galactotetraoside, 15% PEG 1500 and 15% PEG 400., VAPOR DIFFUSION, HANGING DROP, temperature 296K

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Data collection

Diffraction	Mean temperature: 100 K
Diffraction source	Source: SYNCHROTRON / Site: MAX II / Beamline: I711
Detector	Type: MAR CCD 165 mm / Detector: CCD / Date: Mar 2, 2004
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Relative weight: 1
Reflection	Resolution: 2.3→30 Å / Num. obs: 31812 / % possible obs: 88 % / R_merge(I) obs: 0.064
Reflection shell	Resolution: 2.3→2.38 Å / R_merge(I) obs: 0.346 / % possible all: 81.9

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Processing

Software

Name	Version	Classification
REFMAC	5.2.0005	refinement
DENZO		data reduction
SCALEPACK		data scaling

Refinement

Method to determine structure:

FOURIER SYNTHESIS / Resolution: 2.3→30 Å / Cor.coef. F_o:F_c: 0.954 / Cor.coef. F_o:F_c free: 0.922 / SU B: 7.186 / SU ML: 0.176 / Cross valid method: THROUGHOUT / ESU R: 0.507 / ESU R Free: 0.259 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.23156	1606	5.1 %	RANDOM
R_work	0.17985	-	-	-
obs	0.18253	30191	87.25 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 32.164 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.25 Å²	0 Å²	1.56 Å²
2-	-	2.71 Å²	0 Å²
3-	-	-	-1.88 Å²

Refinement step

Cycle: LAST / Resolution: 2.3→30 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5997	0	70	294	6361

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.012	0.022	6230
X-RAY DIFFRACTION	r_bond_other_d
X-RAY DIFFRACTION	r_angle_refined_deg	1.293	1.937	8478
X-RAY DIFFRACTION	r_angle_other_deg
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	5.858	5	771
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	37.685	24.846	293
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	15.947	15	945
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	25.974	15	20
X-RAY DIFFRACTION	r_chiral_restr	0.089	0.2	907
X-RAY DIFFRACTION	r_gen_planes_refined	0.005	0.02	4817
X-RAY DIFFRACTION	r_gen_planes_other
X-RAY DIFFRACTION	r_nbd_refined	0.203	0.2	3022
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined	0.304	0.2	4252
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.155	0.2	439
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined	0.131	0.2	10
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined	0.238	0.2	57
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined	0.138	0.2	13
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	0.604	1.5	3898
X-RAY DIFFRACTION	r_mcbond_other
X-RAY DIFFRACTION	r_mcangle_it	1.022	2	6080
X-RAY DIFFRACTION	r_scbond_it	1.533	3	2731
X-RAY DIFFRACTION	r_scangle_it	2.449	4.5	2398
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

Number	Type	Rms dev position (Å)	Weight position
2786	tight positional	0.08	0.05
21	medium positional	0.79	0.5
187	loose positional	1.18	5
2786	tight thermal	0.13	0.5
21	medium thermal	0.39	2
187	loose thermal	2.58	10

LS refinement shell

Resolution: 2.3→2.345 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.326	91	-
R_work	0.235	1884	-
obs	-	-	75.1 %

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