PDB-7fe3: Crystal structure of GH65 alpha-1,2-glucosidase from Flavobacteri...

ID or keywords:

Entry

Database: PDB / ID: 7fe3

Title

Crystal structure of GH65 alpha-1,2-glucosidase from Flavobacterium johnsoniae

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

glycosyltransferase activity /

carbohydrate binding /

hydrolase activity / carbohydrate metabolic process
Similarity search - Function

Biological species

Flavobacterium johnsoniae (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

SAD / Resolution: 1.54 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

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/fe/7fe3 ftp://data.pdbj.org/pub/pdb/validation_reports/fe/7fe3	HTTPS FTP

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Related structure data

Related structure data	7fe4C C: citing same article (ref.)
Similar structure data	7fe4-1 7jfo-d 7jn4-d 3o65-5 5wsk-2 7jsx-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 (1) #74 - Feb 2006 alpha-Amylase similarity (2) #203 - Nov 2016 Aminopeptidase 1 and Autophagy similarity (1) #39 - Mar 2003 lac Repressor 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

231 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
462 kDa, 6 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	122.830, 193.970, 111.735
Angle α, β, γ (deg.)	90.000, 116.601, 90.000
Int Tables number	5
Space group name H-M	C121

Components on special symmetry positions

ID	Model	Components
1	1	C-1278- 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: Chemical	... ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol Mass: 62.068 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C₂H₆O₂
#3: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 1452 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	N

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.59 Å³/Da / Density % sol: 52.57 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 300 mM ammonium citrate, pH 7.0-8.0, 10 mM TCEP, 12% PEG 3350 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: 1 Å
Detector	Type: DECTRIS PILATUS3 S 2M / Detector: PIXEL / Date: Jun 27, 2021
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 1.54→50 Å / Num. obs: 342453 / % possible obs: 99.7 % / Redundancy: 6.8 % / CC_1/2: 1 / R_merge(I) obs: 0.05 / R_pim(I) all: 0.021 / R_rim(I) all: 0.054 / Net I/σ(I): 19.9
Reflection shell	Resolution: 1.54→1.62 Å / R_merge(I) obs: 0.916 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 49838 / CC_1/2: 0.846 / R_pim(I) all: 0.379 / R_rim(I) all: 0.992

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Processing

Software

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

Refinement

Method to determine structure:

SAD / Resolution: 1.54→48.54 Å / Cor.coef. F_o:F_c: 0.974 / Cor.coef. F_o:F_c free: 0.968 / SU B: 3.317 / SU ML: 0.059 / Cross valid method: FREE R-VALUE / ESU R: 0.07 / ESU R Free: 0.07
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.1918	16770	4.919 %
R_work	0.1709	324171	-
all	0.172	-	-
obs	-	340941	99.292 %

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: 29.102 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.698 Å²	0 Å²	0.791 Å²
2-	-	-1.879 Å²	-0 Å²
3-	-	-	1.188 Å²

Refinement step

Cycle: LAST / Resolution: 1.54→48.54 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	15741	0	104	1452	17297

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.009	0.013	16452
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.017	15341
X-RAY DIFFRACTION	r_angle_refined_deg	1.501	1.643	22319
X-RAY DIFFRACTION	r_angle_other_deg	1.416	1.579	35400
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.849	5	2032
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.817	23.819	830
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.173	15	2799
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	13.15	15	63
X-RAY DIFFRACTION	r_chiral_restr	0.08	0.2	2141
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	18853
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	3797
X-RAY DIFFRACTION	r_nbd_refined	0.208	0.2	3184
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.173	0.2	14326
X-RAY DIFFRACTION	r_nbtor_refined	0.171	0.2	7846
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.077	0.2	7497
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.113	0.2	1262
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.003	0.2	1
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.247	0.2	15
X-RAY DIFFRACTION	r_nbd_other	0.149	0.2	90
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.133	0.2	59
X-RAY DIFFRACTION	r_mcbond_it	0.983	2.109	8044
X-RAY DIFFRACTION	r_mcbond_other	0.983	2.109	8043
X-RAY DIFFRACTION	r_mcangle_it	1.477	3.16	10098
X-RAY DIFFRACTION	r_mcangle_other	1.477	3.161	10099
X-RAY DIFFRACTION	r_scbond_it	1.54	2.301	8408
X-RAY DIFFRACTION	r_scbond_other	1.54	2.301	8409
X-RAY DIFFRACTION	r_scangle_it	2.398	3.368	12219
X-RAY DIFFRACTION	r_scangle_other	2.398	3.368	12220
X-RAY DIFFRACTION	r_lrange_it	4.164	25.434	18962
X-RAY DIFFRACTION	r_lrange_other	4.073	24.886	18618

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 (%)
1.54-1.58	0.274	1236	0.272	23944	X-RAY DIFFRACTION	99.2511
1.58-1.623	0.262	1171	0.252	23446	X-RAY DIFFRACTION	99.3663
1.623-1.67	0.268	1148	0.239	22758	X-RAY DIFFRACTION	99.4219
1.67-1.722	0.227	1175	0.217	22064	X-RAY DIFFRACTION	99.5118
1.722-1.778	0.223	1073	0.203	21393	X-RAY DIFFRACTION	99.4511
1.778-1.841	0.22	1103	0.198	20675	X-RAY DIFFRACTION	99.3839
1.841-1.91	0.227	995	0.189	19958	X-RAY DIFFRACTION	99.2657
1.91-1.988	0.215	986	0.182	19169	X-RAY DIFFRACTION	99.2271
1.988-2.076	0.205	930	0.179	18447	X-RAY DIFFRACTION	99.2878
2.076-2.178	0.211	908	0.179	17610	X-RAY DIFFRACTION	99.255
2.178-2.295	0.206	876	0.171	16711	X-RAY DIFFRACTION	99.1431
2.295-2.435	0.192	860	0.166	15780	X-RAY DIFFRACTION	99.1657
2.435-2.603	0.206	797	0.163	14800	X-RAY DIFFRACTION	98.8528
2.603-2.811	0.18	701	0.158	13854	X-RAY DIFFRACTION	98.9665
2.811-3.079	0.189	616	0.158	12806	X-RAY DIFFRACTION	99.2825
3.079-3.442	0.175	588	0.164	11560	X-RAY DIFFRACTION	99.4027
3.442-3.974	0.169	560	0.154	10182	X-RAY DIFFRACTION	99.3985
3.974-4.865	0.154	463	0.141	8582	X-RAY DIFFRACTION	98.9498
4.865-6.872	0.165	359	0.153	6729	X-RAY DIFFRACTION	99.9295
6.872-48.54	0.16	225	0.154	3703	X-RAY DIFFRACTION	99.4934

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	0.1268	-0.0275	-0.0626	0.4401	0.2626	0.1953	-0.0079	0.0186	-0.032	-0.0206	-0.0325	0.1181	-0.0241	-0.0396	0.0404	0.0395	-0.006	0.026	0.0458	-0.0041	0.1196	-35.11	-11.858	0.71
2	0.3501	0.1363	0.061	0.3966	0.1287	0.1058	0.0175	-0.071	0.0735	0.0779	-0.0578	0.1277	-0.0039	-0.0036	0.0403	0.0919	0.0174	0.0735	0.0523	-0.0066	0.088	-30.553	12.264	24.898
3	0.0852	0.0166	0.0092	0.4094	-0.159	0.2282	0.0197	-0.0269	0.0165	0.0166	-0.0501	-0.0566	-0.0288	0.0331	0.0304	0.0716	-0.0132	0.0328	0.045	0.0012	0.0755	9.37	14.572	16.172
4	0.2353	0.5629	-0.2417	1.8547	-0.7931	0.4506	0.1196	-0.1331	-0.0952	0.312	-0.3131	-0.2345	-0.0883	0.1524	0.1936	0.1353	-0.0481	-0.0112	0.1022	0.0714	0.1109	12.915	-8.776	44.007
5	0.3996	0.0539	-0.0233	0.1349	-0.0402	0.0398	0.0184	-0.0042	-0.0609	0.0471	-0.0151	-0.0302	0.014	-0.0013	-0.0033	0.0933	0.011	0.0257	0.0171	0.0185	0.0908	6.523	-42.586	19.21
6	0.5628	-0.0641	0.1772	0.2128	-0.0773	0.1623	-0.0029	-0.1242	-0.0841	0.0432	0.0217	0.052	-0.0001	-0.0809	-0.0187	0.0936	-0.0212	0.0695	0.06	0.0124	0.0807	-27.291	-42.291	26.519

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	ALL	A	23 - 360
2	X-RAY DIFFRACTION	2	ALL	A	361 - 681
3	X-RAY DIFFRACTION	3	ALL	B	23 - 284
4	X-RAY DIFFRACTION	4	ALL	B	285 - 681
5	X-RAY DIFFRACTION	5	ALL	C	23 - 353
6	X-RAY DIFFRACTION	6	ALL	C	354 - 681

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