PDB-7fe4: Crystal structure of GH65 alpha-1,2-glucosidase from Flavobacteri...

ID or keywords:

Entry

Database: PDB / ID: 7fe4

Title

Crystal structure of GH65 alpha-1,2-glucosidase from Flavobacterium johnsoniae in complex with glucose

Components

Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

Keywords

HYDROLASE /

Glycoside hydrolase / GH65 / (alpha/alpha)6-barrel /

kojibiose /

dextran

Function / homology

Function and homology information

carbohydrate binding /

hydrolase activity / carbohydrate metabolic process
Similarity search - Function

Biological species

Flavobacterium johnsoniae (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.4 Å

Authors

Nakamura, S. / Miyazaki, T.

Funding support

Japan, 1items

Citation

Journal: J.Biol.Chem. / Year: 2021
Title: Structure of a bacterial alpha-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases.
Authors: Nakamura, S. / Nihira, T. / Kurata, R. / Nakai, H. / Funane, K. / Park, E.Y. / Miyazaki, T.

History

Deposition	Jul 19, 2021	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Nov 10, 2021	Provider: repository / Type: Initial release
Revision 1.1	Dec 8, 2021	Group: Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2	Nov 29, 2023	Group: Data collection / Derived calculations / Refinement description Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7fe4.cif.gz	759.7 KB	Display	PDBx/mmCIF format
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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/fe/7fe4 ftp://data.pdbj.org/pub/pdb/validation_reports/fe/7fe4	HTTPS FTP

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Related structure data

Related structure data	7fe3SC S: Starting model for refinement C: citing same article (ref.)
Similar structure data	7fe3-1 6u7a-d 7jfo-d 3wky-1 7jsx-d 7l2p-d 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
Related items in Molecule of the Month	#198 - Jun 2016 Beta-galactosidase similarity (2) #74 - Feb 2006 alpha-Amylase similarity (2) #203 - Nov 2016 Aminopeptidase 1 and Autophagy similarity (1) #39 - Mar 2003 lac Repressor similarity (1) #75 - Mar 2006 Tissue Factor similarity (1) #77 - May 2006 Glucose Oxidase similarity (1)

Deposited unit

A: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

B: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

C: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

hetero molecules

232 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

B: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

C: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

hetero molecules

A: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

B: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

C: Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65

hetero molecules

defined by author&software
Evidence: gel filtration
463 kDa, 6 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	123.505, 194.201, 112.006
Angle α, β, γ (deg.)	90.000, 116.598, 90.000
Int Tables number	5
Space group name H-M	C121

Components on special symmetry positions

ID	Model	Components
1	1	C-1604- HOH

#1: Protein

Candidate alpha glycoside phosphorylase Glycoside hydrolase family 65 / alpha-1 / 2-glucosidase

Mass: 76489.219 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Flavobacterium johnsoniae (strain ATCC 17061 / DSM 2064 / JCM 8514 / NBRC 14942 / NCIMB 11054 / UW101) (bacteria)
Strain: ATCC 17061 / DSM 2064 / JCM 8514 / NBRC 14942 / NCIMB 11054 / UW101
Gene: Fjoh_4428 / Plasmid: pET28a / Production host:

Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A5FBJ5

#2: Sugar

ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose /

Glucose

Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C₆H₁₂O₆ / Feature type: SUBJECT OF INVESTIGATION

#3: Water

ChemComp-HOH / water /

Water

Mass: 18.015 Da / Num. of mol.: 1668 / Source method: isolated from a natural source / Formula: H₂O

Has ligand of interest

Y

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.62 Å³/Da / Density % sol: 53 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 300 mM ammonium citrate, pH 7.0-8.0, 10 mM TCEP, 12% PEG3350 PH range: 7.0-8.0

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NW12A / Wavelength: 0.9795 Å
Detector	Type: DECTRIS PILATUS3 S 2M / Detector: PIXEL / Date: Nov 14, 2020
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.9795 Å / Relative weight: 1
Reflection	Resolution: 1.4→50 Å / Num. obs: 451842 / % possible obs: 98 % / Redundancy: 6.8 % / CC_1/2: 0.999 / R_merge(I) obs: 0.06 / R_pim(I) all: 0.025 / R_rim(I) all: 0.065 / Net I/σ(I): 15.6
Reflection shell	Resolution: 1.4→1.48 Å / Redundancy: 6.5 % / R_merge(I) obs: 0.755 / Mean I/σ(I) obs: 2.4 / Num. unique obs: 64988 / CC_1/2: 0.81 / R_pim(I) all: 0.318 / R_rim(I) all: 0.82 / % possible all: 96.6

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0267	refinement
XDS		data reduction
SCALA		data scaling
MOLREP		phasing
Coot		model building

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 7FE3

7fe3

Resolution: 1.4→44.704 Å / Cor.coef. F_o:F_c: 0.977 / Cor.coef. F_o:F_c free: 0.972 / SU B: 1.993 / SU ML: 0.039 / Cross valid method: FREE R-VALUE / ESU R: 0.05 / ESU R Free: 0.05
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.1702	22421	4.965 %
R_work	0.1566	429162	-
all	0.157	-	-
obs	-	451583	97.986 %

Solvent computation

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

Displacement parameters

B_iso mean: 25.135 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.073 Å²	-0 Å²	0.105 Å²
2-	-	0.162 Å²	0 Å²
3-	-	-	-0.129 Å²

Refinement step

Cycle: LAST / Resolution: 1.4→44.704 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	15741	0	144	1668	17553

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.009	0.013	16411
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.017	15206
X-RAY DIFFRACTION	r_angle_refined_deg	1.526	1.652	22314
X-RAY DIFFRACTION	r_angle_other_deg	1.454	1.59	35127
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.866	5	2010
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.912	23.883	819
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.065	15	2771
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	13.57	15	60
X-RAY DIFFRACTION	r_chiral_restr	0.078	0.2	2190
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.02	18672
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	3756
X-RAY DIFFRACTION	r_nbd_refined	0.209	0.2	3155
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.175	0.2	14218
X-RAY DIFFRACTION	r_nbtor_refined	0.172	0.2	7880
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.079	0.2	7273
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.112	0.2	1295
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.321	0.2	18
X-RAY DIFFRACTION	r_nbd_other	0.202	0.2	105
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.128	0.2	44
X-RAY DIFFRACTION	r_mcbond_it	0.612	1.171	7995
X-RAY DIFFRACTION	r_mcbond_other	0.612	1.171	7994
X-RAY DIFFRACTION	r_mcangle_it	0.979	1.756	10020
X-RAY DIFFRACTION	r_mcangle_other	0.979	1.756	10021
X-RAY DIFFRACTION	r_scbond_it	1.131	1.318	8414
X-RAY DIFFRACTION	r_scbond_other	1.131	1.318	8414
X-RAY DIFFRACTION	r_scangle_it	1.745	1.925	12293
X-RAY DIFFRACTION	r_scangle_other	1.745	1.925	12294
X-RAY DIFFRACTION	r_lrange_it	4.721	15.102	18940
X-RAY DIFFRACTION	r_lrange_other	4.6	14.379	18537

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
1.4-1.436	0.268	1688	0.255	31156	X-RAY DIFFRACTION	96.4837
1.436-1.476	0.244	1538	0.236	30570	X-RAY DIFFRACTION	96.6759
1.476-1.518	0.237	1572	0.218	29641	X-RAY DIFFRACTION	96.8927
1.518-1.565	0.21	1497	0.203	28977	X-RAY DIFFRACTION	97.1531
1.565-1.617	0.21	1481	0.195	28092	X-RAY DIFFRACTION	97.3789
1.617-1.673	0.205	1418	0.187	27328	X-RAY DIFFRACTION	97.6393
1.673-1.736	0.188	1393	0.176	26312	X-RAY DIFFRACTION	97.8457
1.736-1.807	0.195	1261	0.176	25508	X-RAY DIFFRACTION	98.0657
1.807-1.888	0.19	1239	0.166	24520	X-RAY DIFFRACTION	98.2118
1.888-1.98	0.174	1206	0.159	23411	X-RAY DIFFRACTION	98.4404
1.98-2.087	0.168	1208	0.153	22308	X-RAY DIFFRACTION	98.6616
2.087-2.213	0.162	1088	0.148	21186	X-RAY DIFFRACTION	98.8681
2.213-2.366	0.153	1049	0.14	19862	X-RAY DIFFRACTION	98.9261
2.366-2.555	0.154	986	0.141	18600	X-RAY DIFFRACTION	99.0593
2.555-2.799	0.149	856	0.142	17122	X-RAY DIFFRACTION	99.1452
2.799-3.129	0.162	826	0.146	15428	X-RAY DIFFRACTION	99.0433
3.129-3.613	0.16	731	0.147	13571	X-RAY DIFFRACTION	98.6481
3.613-4.423	0.147	617	0.13	11615	X-RAY DIFFRACTION	99.5119
4.423-6.249	0.147	498	0.133	8976	X-RAY DIFFRACTION	99.9262
6.249-44.704	0.161	269	0.153	4979	X-RAY DIFFRACTION	99.2999

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°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)
1	0.4776	0.0363	-0.197	0.4886	0.2703	0.9505	-0.0115	0.2339	-0.0167	-0.1375	-0.0097	0.1223	-0.0947	-0.396	0.0211	0.0698	0.0171	-0.0399	0.2377	0.008	0.1311	-33.893	-4.72	-3.232
2	0.5219	-0.0016	-0.3074	0.6687	0.2353	1.2048	-0.081	0.2033	-0.0669	-0.0693	-0.0225	0.1355	0.1155	-0.4483	0.1035	0.0512	-0.083	-0.0172	0.2151	-0.0367	0.1122	-34.202	-19.646	-4.057
3	0.4754	0.0987	-0.1531	0.5292	0.175	0.871	0.0791	0.0003	0.0675	0.068	-0.0526	0.116	-0.1567	-0.2692	-0.0265	0.0774	0.0675	0.0376	0.1059	0.0142	0.1059	-32.234	9.694	23.051
4	0.4228	0.029	-0.4024	0.4324	-0.2504	0.9619	0.1049	-0.171	0.133	-0.0046	-0.0616	-0.0774	-0.2161	0.3959	-0.0433	0.0944	-0.0876	0.0001	0.1745	-0.0391	0.1313	17.21	8.91	20.378
5	0.4447	0.0528	-0.3197	0.4449	-0.0765	0.9825	0.1318	-0.0979	0.1183	0.0142	-0.0187	-0.015	-0.2911	0.1675	-0.1131	0.1268	-0.0584	0.0334	0.0367	-0.0336	0.1195	6.108	16.993	14.459
6	0.553	0.0902	-0.579	0.74	-0.1765	1.3058	-0.0082	-0.3777	-0.0169	0.179	-0.0423	-0.03	0.0173	0.5607	0.0505	0.0669	0.0211	-0.0307	0.3174	0.0142	0.0341	12.99	-8.938	44.069
7	0.7458	-0.1746	-0.0987	0.35	0.0763	0.4205	-0.1543	0.0439	-0.186	0.0584	0.001	-0.0416	0.3466	0.104	0.1533	0.311	0.077	0.1189	0.0324	0.0282	0.1737	2.312	-46.542	13.418
8	0.5266	-0.0604	-0.1468	0.4877	0.005	0.3921	-0.1293	-0.0811	-0.1158	0.0958	0.0092	-0.0512	0.2564	0.1684	0.1201	0.2115	0.1244	0.0558	0.0815	0.0579	0.1206	14.674	-39.735	18.421
9	0.7209	-0.0801	-0.1876	0.2545	-0.0247	0.7021	-0.199	-0.0749	-0.1527	0.0907	0.019	0.046	0.3318	-0.1177	0.18	0.2692	-0.0593	0.123	0.0448	-0.0098	0.1208	-24.298	-43.003	26.706

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	A	23 - 101
2	X-RAY DIFFRACTION	2	ALL	A	102 - 284
3	X-RAY DIFFRACTION	3	ALL	A	285 - 681
4	X-RAY DIFFRACTION	4	ALL	B	23 - 101
5	X-RAY DIFFRACTION	5	ALL	B	102 - 284
6	X-RAY DIFFRACTION	6	ALL	B	285 - 681
7	X-RAY DIFFRACTION	7	ALL	C	23 - 101
8	X-RAY DIFFRACTION	8	ALL	C	102 - 284
9	X-RAY DIFFRACTION	9	ALL	C	285 - 681

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