PDB-8j53: Crystal structure of Bacteroides salyersiae GH31 alpha-galactosidase

Basic information

• Entry: Database: PDB / ID: 8j53
• Title: Crystal structure of Bacteroides salyersiae GH31 alpha-galactosidase
• Components: GH31 alpha-galactosidase
• Keywords: HYDROLASE / Glycoside hydrolase / (beta/alpha)8 barrel / Globo sphingolipid / Carbohydrate

Function / homology

Function:

hydrolase activity, hydrolyzing O-glycosyl compounds / carbohydrate metabolic process

Domain/homology:

: / : / Glycosyl hydrolase family 31 C-terminal domain / Glycoside hydrolase family 31 / Glycosyl hydrolases family 31 TIM-barrel domain / Glycosyl hydrolase, all-beta / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta

Component:

Glycoside hydrolase

• Biological species: Bacteroides salyersiae JCM 12988 (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.5 Å
• Authors: Ikegaya, M. / Miyazaki, T.

Funding support

Japan, 1items

Organization	Grant number	Country
Japan Society for the Promotion of Science (JSPS)	19K15748	Japan

• Citation: Journal: Febs J. / Year: 2023; Title: Structure-function analysis of bacterial GH31 alpha-galactosidases specific for alpha-(1→4)-galactobiose.; Authors: Ikegaya, M. / Park, E.Y. / Miyazaki, T.

History

Deposition	Apr 21, 2023	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Jul 26, 2023	Provider: repository / Type: Initial release
Revision 1.1	Nov 1, 2023	Group: Data collection / Database references Category: chem_comp_atom / chem_comp_bond / citation / citation_author Item: _citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

Assembly

Deposited unit

A: GH31 alpha-galactosidase

B: GH31 alpha-galactosidase

C: GH31 alpha-galactosidase

D: GH31 alpha-galactosidase

E: GH31 alpha-galactosidase

Summary:

• 306 kDa, 5 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	306,004	5
Polymers	306,004	5
Non-polymers	0	0
Water	0	0

1

A: GH31 alpha-galactosidase

B: GH31 alpha-galactosidase

Summary:

• defined by author
• Evidence: gel filtration
• 122 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	122,401	2
Polymers	122,401	2
Non-polymers	0	0
Water	0

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

2

C: GH31 alpha-galactosidase

C: GH31 alpha-galactosidase

Summary:

• defined by author
• 122 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	122,401	2
Polymers	122,401	2
Non-polymers	0	0
Water	0

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1
crystal symmetry operation	2_555	-x,y,-z	1

3

D: GH31 alpha-galactosidase

E: GH31 alpha-galactosidase

Summary:

• defined by author
• 122 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	122,401	2
Polymers	122,401	2
Non-polymers	0	0
Water	0

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Unit cell

Length a, b, c (Å)	200.521, 157.535, 131.408
Angle α, β, γ (deg.)	90.000, 116.823, 90.000
Int Tables number	5
Space group name H-M	C121

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	A
3	2	A
4	2	A
5	3	A
6	3	A
7	4	A
8	4	A
9	5	A
10	5	A
11	6	A
12	6	A
13	7	A
14	7	A
15	8	A
16	8	A
17	9	A
18	9	A
19	10	A
20	10	A

NCS domain segments:

Beg auth comp-ID: VAL / Beg label comp-ID: VAL / End auth comp-ID: LYS / End label comp-ID: LYS / Auth asym-ID: A / Label asym-ID: A / Auth seq-ID: 26 - 532 / Label seq-ID: 26 - 532

Dom-ID	Component-ID	Ens-ID
1	1	1
2	1	1
3	2	2
4	2	2
5	3	3
6	3	3
7	4	4
8	4	4
9	5	5
10	5	5
11	6	6
12	6	6
13	7	7
14	7	7
15	8	8
16	8	8
17	9	9
18	9	9
19	10	10
20	10	10

NCS ensembles :

ID	Details
1	Local NCS retraints between domains: 1 2
2	Local NCS retraints between domains: 3 4
3	Local NCS retraints between domains: 5 6
4	Local NCS retraints between domains: 7 8
5	Local NCS retraints between domains: 9 10
6	Local NCS retraints between domains: 11 12
7	Local NCS retraints between domains: 13 14
8	Local NCS retraints between domains: 15 16
9	Local NCS retraints between domains: 17 18
10	Local NCS retraints between domains: 19 20

Components

#1: Protein

A B C D E
GH31 alpha-galactosidase

Details:

Mass: 61200.734 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteroides salyersiae JCM 12988 (bacteria)
Strain: JCM 12988 / Gene: F3F73_10765 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A7J4XIY8

Experimental details

Experiment

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

Sample preparation

• Crystal: Density Matthews: 3.03 ų/Da / Density % sol: 59.36 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion, hanging drop; Details: 16% PEG 3350, 40 mM citric acid, 60 mM bis-tris propane

Data collection

• Diffraction: Mean temperature: 100 K / Serial crystal experiment: N
• Diffraction source: Source: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
• Detector: Type: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: May 15, 2022
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1 Å / Relative weight: 1
• Reflection: Resolution: 3.5→48.635 Å / Num. obs: 45955 / % possible obs: 99.9 % / Redundancy: 6.8 % / CC_1/2: 0.996 / R_merge(I) obs: 0.168 / R_pim(I) all: 0.106 / R_rim(I) all: 0.199 / Net I/σ(I): 9.3
• Reflection shell: Resolution: 3.5→3.62 Å / R_merge(I) obs: 1.05 / Mean I/σ(I) obs: 2 / Num. unique obs: 4479 / CC_1/2: 0.797 / R_pim(I) all: 0.651 / R_rim(I) all: 1.238

Processing

Software

Name	Version	Classification
REFMAC	5.8.0405	refinement
XDS		data reduction
Aimless		data scaling
MOLREP		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.5→48.635 Å / Cor.coef. F_o:F_c: 0.906 / Cor.coef. F_o:F_c free: 0.853 / SU B: 51.701 / SU ML: 0.737 / Cross valid method: FREE R-VALUE / ESU R Free: 0.764
Details: Hydrogens have been added in their riding positions

	Rfactor	Num. reflection	% reflection
Rfree	0.3128	2223	4.838 %
Rwork	0.2569	43730	-
all	0.26	-	-
obs	-	45953	99.922 %

• 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: 102.778 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	5.234 Å2	0 Å2	-4.531 Å2
2-	-	7.794 Å2	-0 Å2
3-	-	-	-5.588 Å2

Refinement step

Cycle: LAST / Resolution: 3.5→48.635 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	20611	0	0	0	20611

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.008	0.012	21210
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.016	19628
X-RAY DIFFRACTION	r_ext_dist_refined_b	0.316	0.1	211221
X-RAY DIFFRACTION	r_angle_refined_deg	1.498	1.651	28706
X-RAY DIFFRACTION	r_angle_other_deg	0.481	1.573	45339
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.349	5	2532
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	15.932	5	100
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	16.858	10	3629
X-RAY DIFFRACTION	r_dihedral_angle_6_deg	15.217	10	1034
X-RAY DIFFRACTION	r_chiral_restr	0.066	0.2	2896
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	24660
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	5052
X-RAY DIFFRACTION	r_nbd_refined	0.198	0.2	4824
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.214	0.2	20535
X-RAY DIFFRACTION	r_nbtor_refined	0.187	0.2	10245
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.085	0.2	11004
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.157	0.2	423
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.087	0.2	9
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.285	0.2	51
X-RAY DIFFRACTION	r_nbd_other	0.265	0.2	107
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.077	0.2	6
X-RAY DIFFRACTION	r_mcbond_it	12.742	10.077	10149
X-RAY DIFFRACTION	r_mcbond_other	12.741	10.077	10149
X-RAY DIFFRACTION	r_mcangle_it	19.997	18.047	12674
X-RAY DIFFRACTION	r_mcangle_other	19.996	18.047	12675
X-RAY DIFFRACTION	r_scbond_it	11.05	10.474	11061
X-RAY DIFFRACTION	r_scbond_other	11.05	10.474	11062
X-RAY DIFFRACTION	r_scangle_it	17.767	19.094	16032
X-RAY DIFFRACTION	r_scangle_other	17.766	19.094	16033
X-RAY DIFFRACTION	r_lrange_it	28.841	210.348	247013
X-RAY DIFFRACTION	r_lrange_other	28.841	210.347	247014
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.109	0.05	17308
X-RAY DIFFRACTION	r_ncsr_local_group_2	0.114	0.05	17363
X-RAY DIFFRACTION	r_ncsr_local_group_3	0.106	0.05	17409
X-RAY DIFFRACTION	r_ncsr_local_group_4	0.107	0.05	17283
X-RAY DIFFRACTION	r_ncsr_local_group_5	0.097	0.05	17484
X-RAY DIFFRACTION	r_ncsr_local_group_6	0.088	0.05	17463
X-RAY DIFFRACTION	r_ncsr_local_group_7	0.089	0.05	17426
X-RAY DIFFRACTION	r_ncsr_local_group_8	0.098	0.05	17565
X-RAY DIFFRACTION	r_ncsr_local_group_9	0.101	0.05	17382
X-RAY DIFFRACTION	r_ncsr_local_group_10	0.095	0.05	17488

Refine LS restraints NCS

Ens-ID	Dom-ID	Auth asym-ID	Refine-ID	Type	Rms dev position (Å)	Weight position
1	1	A	X-RAY DIFFRACTION	Local ncs	0.10883	0.05009
1	2	A	X-RAY DIFFRACTION	Local ncs	0.10883	0.05009
2	3	A	X-RAY DIFFRACTION	Local ncs	0.11445	0.05009
2	4	A	X-RAY DIFFRACTION	Local ncs	0.11445	0.05009
3	5	A	X-RAY DIFFRACTION	Local ncs	0.10587	0.05009
3	6	A	X-RAY DIFFRACTION	Local ncs	0.10587	0.05009
4	7	A	X-RAY DIFFRACTION	Local ncs	0.10746	0.05009
4	8	A	X-RAY DIFFRACTION	Local ncs	0.10746	0.05009
5	9	A	X-RAY DIFFRACTION	Local ncs	0.09722	0.05009
5	10	A	X-RAY DIFFRACTION	Local ncs	0.09722	0.05009
6	11	A	X-RAY DIFFRACTION	Local ncs	0.08834	0.05009
6	12	A	X-RAY DIFFRACTION	Local ncs	0.08834	0.05009
7	13	A	X-RAY DIFFRACTION	Local ncs	0.08883	0.0501
7	14	A	X-RAY DIFFRACTION	Local ncs	0.08883	0.0501
8	15	A	X-RAY DIFFRACTION	Local ncs	0.09791	0.05009
8	16	A	X-RAY DIFFRACTION	Local ncs	0.09791	0.05009
9	17	A	X-RAY DIFFRACTION	Local ncs	0.10134	0.05009
9	18	A	X-RAY DIFFRACTION	Local ncs	0.10134	0.05009
10	19	A	X-RAY DIFFRACTION	Local ncs	0.09458	0.05009
10	20	A	X-RAY DIFFRACTION	Local ncs	0.09458	0.05009

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
3.5-3.59	0.386	146	0.362	3209	0.363	3356	0.891	0.911	99.9702	0.351
3.59-3.688	0.389	168	0.332	3098	0.335	3266	0.894	0.928	100	0.323
3.688-3.795	0.354	146	0.31	3070	0.312	3216	0.899	0.935	100	0.295
3.795-3.911	0.38	173	0.323	2897	0.326	3070	0.898	0.925	100	0.305
3.911-4.038	0.35	141	0.309	2880	0.311	3021	0.909	0.934	100	0.289
4.038-4.179	0.358	144	0.287	2786	0.291	2932	0.911	0.943	99.9318	0.26
4.179-4.335	0.307	147	0.265	2668	0.267	2817	0.938	0.953	99.929	0.234
4.335-4.511	0.305	126	0.249	2587	0.251	2716	0.942	0.959	99.8895	0.219
4.511-4.709	0.338	128	0.254	2468	0.258	2598	0.928	0.956	99.923	0.221
4.709-4.937	0.327	130	0.251	2369	0.255	2499	0.926	0.958	100	0.221
4.937-5.201	0.314	105	0.228	2239	0.232	2347	0.932	0.966	99.8722	0.203
5.201-5.513	0.287	93	0.214	2176	0.217	2269	0.949	0.97	100	0.19
5.513-5.888	0.293	108	0.212	2006	0.216	2115	0.95	0.973	99.9527	0.189
5.888-6.352	0.287	94	0.22	1885	0.224	1979	0.941	0.969	100	0.199
6.352-6.946	0.349	88	0.217	1720	0.223	1808	0.928	0.97	100	0.201
6.946-7.746	0.256	68	0.211	1587	0.213	1655	0.959	0.971	100	0.2
7.746-8.907	0.254	68	0.212	1392	0.214	1460	0.961	0.971	100	0.209
8.907-10.819	0.25	62	0.218	1188	0.219	1250	0.962	0.971	100	0.221
10.819-14.935	0.251	52	0.243	934	0.243	987	0.956	0.961	99.8987	0.249
14.935-48.635	0.302	36	0.324	571	0.323	608	0.925	0.93	99.8355	0.338