PDB-7p6h: Crystal structure of the endoglucanase RBcel1 E135Q in complex wi...

ID or keywords:

Entry

Database: PDB / ID: 7p6h

Title

Crystal structure of the endoglucanase RBcel1 E135Q in complex with cellotriose

Components

Endoglucanase

Keywords

HYDROLASE / Transglycosylase / GH5_5 family / complex

Function / homology

glucan catabolic process / cellulase / Glycoside hydrolase, family 5 / Cellulase (glycosyl hydrolase family 5) / cellulase activity / Glycoside hydrolase superfamily / alpha-cellotriose / Endoglucanase

Function and homology information

Biological species

uncultured bacterium (environmental samples)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT /

molecular replacement / Resolution: 1.73 Å

Authors

Collet, L. / Dutoit, R.

Citation

Journal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-beta-glucanase RBcel1.
Authors: Collet, L. / Vander Wauven, C. / Oudjama, Y. / Galleni, M. / Dutoit, R.

History

Deposition	Jul 16, 2021	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Mar 2, 2022	Provider: repository / Type: Initial release
Revision 1.1	Mar 16, 2022	Group: Database references / Category: citation / citation_author Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2	Jan 31, 2024	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.3	Oct 16, 2024	Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	7p6h.cif.gz	302.7 KB	Display	PDBx/mmCIF format
PDB format	pdb7p6h.ent.gz	246.3 KB	Display	PDB format
PDBx/mmJSON format	7p6h.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	7p6h_validation.pdf.gz	1.1 MB	Display	wwPDB validaton report
Full document	7p6h_full_validation.pdf.gz	1.2 MB	Display
Data in XML	7p6h_validation.xml.gz	32.5 KB	Display
Data in CIF	7p6h_validation.cif.gz	49.6 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/p6/7p6h ftp://data.pdbj.org/pub/pdb/validation_reports/p6/7p6h	HTTPS FTP

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Related structure data

Related structure data	7p6gC 7p6iC 7p6jC 4ee9S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	7p6h-2 4g3h-2 7a0t-1 2xzj-2 3avf-d 3avn-d 1vcu-1 3gja-2 3avl-d 5d8m-d 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: Endoglucanase

B: Endoglucanase

hetero molecules

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

1

A: Endoglucanase

hetero molecules

defined by author
Evidence: gel filtration, RBcel1 E135Q variant is monomeric based on sized exclusion chromatography using a Superdex75 column.
37 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Endoglucanase

hetero molecules

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

Unit cell

Length a, b, c (Å)	45.740, 99.790, 149.030
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein

Endoglucanase

Mass: 36381.066 Da / Num. of mol.: 2 / Mutation: E135Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

uncultured bacterium (environmental samples)
Plasmid: pET22b / Production host:

Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: C1JI15, cellulase

#2: Polysaccharide

beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-alpha-D-glucopyranose

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

Descriptor	Type	Program
DGlcpb1-4DGlcpb1-4DGlcpa1-ROH	Glycam Condensed Sequence	GMML 1.0
WURCS=2.0/2,3,2/[a2122h-1a_1-5][a2122h-1b_1-5]/1-2-2/a4-b1_b4-c1	WURCS	PDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}}	LINUCS	PDB-CARE

#3: Chemical

ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL

Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₂H₆O₂

#4: Water

ChemComp-HOH / water

Mass: 18.015 Da / Num. of mol.: 607 / Source method: isolated from a natural source / Formula: H₂O

Has ligand of interest

Y

Has protein modification

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: 2.34 Å³/Da / Density % sol: 47.44 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.4 Details: Reservoir: 0.1M Tris, 17.5% PEG600, pH7.4. Protein: 14 mg/ml in 20 mM sodium phosphate buffer, pH6.5. Drop: 2 ul reservoir + 2 ul protein + 0.4 ul 2 mM cellotriose + 0.2 ul microseeds.

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

SOLEIL

/ Beamline: PROXIMA 2 / Wavelength: 0.9801 Å

Detector

Type: ADSC QUANTUM 315r / Detector: CCD / Date: Jun 11, 2015

Radiation

Monochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.9801 Å / Relative weight: 1

Reflection

Resolution: 1.73→41.46 Å / Num. obs: 71941 / % possible obs: 99.3 % / Redundancy: 7.12 % / B_iso Wilson estimate: 25.07 Å² / CC_1/2: 0.999 / R_merge(I) obs: 0.081 / R_rim(I) all: 0.088 / Χ²: 1.063 / Net I/σ(I): 14.59 / Num. measured all: 512215 / Scaling rejects: 10

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. measured obs	Num. possible	Num. unique obs	CC_1/2	R_rim(I) all	% possible all
1.73-1.77	6.421	1.485	1.41	31733	5313	4942	0.663	1.611	93
1.77-1.82	7.297	1.194	2.13	37587	5156	5151	0.793	1.284	99.9
1.82-1.87	7.284	0.879	3.04	36327	4989	4987	0.866	0.946	100
1.87-1.93	7.261	0.647	4.2	35506	4890	4890	0.91	0.697	100
1.93-2	7.276	0.499	5.38	34697	4769	4769	0.941	0.537	100
2-2.07	7.292	0.349	7.48	33076	4540	4536	0.968	0.375	99.9
2.07-2.14	7.266	0.252	9.59	31969	4406	4400	0.979	0.272	99.9
2.14-2.23	7.261	0.197	11.33	30912	4267	4257	0.987	0.212	99.8
2.23-2.33	7.246	0.149	13.91	29803	4120	4113	0.991	0.16	99.8
2.33-2.44	7.24	0.118	16.38	28214	3903	3897	0.993	0.128	99.8
2.44-2.58	7.239	0.102	17.84	26986	3742	3728	0.995	0.11	99.6
2.58-2.73	7.208	0.083	21.06	25400	3536	3524	0.996	0.089	99.7
2.73-2.92	7.12	0.07	23.88	23697	3335	3328	0.996	0.076	99.8
2.92-3.16	7.031	0.057	27.39	21959	3131	3123	0.997	0.062	99.7
3.16-3.46	6.951	0.05	30.99	20068	2893	2887	0.998	0.055	99.8
3.46-3.86	6.716	0.046	32.8	17503	2618	2606	0.998	0.05	99.5
3.86-4.46	7.044	0.039	35.62	16399	2330	2328	0.998	0.043	99.9
4.46-5.46	7.019	0.037	36.7	13996	1998	1994	0.999	0.04	99.8
5.46-7.73	6.877	0.037	35.82	10872	1584	1581	0.998	0.04	99.8
7.73-41.46	6.123	0.03	35.19	5511	930	900	0.998	0.034	96.8

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Phasing

Phasing

Method:

molecular replacement

Phasing MR

	Highest resolution	Lowest resolution
Rotation	3.78 Å	47.31 Å
Translation	3.78 Å	47.31 Å

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Processing

Software

Name	Version	Classification
XDS	20190315	data reduction
XSCALE	20190315	data scaling
PHASER	2.8.3	phasing
PHENIX	1.19.2	refinement
PDB_EXTRACT	3.27	data extraction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 4EE9

4ee9

Resolution: 1.73→41.46 Å / SU ML: 0.19 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 19.53 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.194	3595	5 %
R_work	0.1666	68314	-
obs	0.168	71909	99.27 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL

Displacement parameters

B_iso max: 88.14 Å² / B_iso mean: 30.5354 Å² / B_iso min: 11.9 Å²

Refinement step

Cycle: final / Resolution: 1.73→41.46 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5108	0	122	607	5837
B_iso mean	-	-	32.73	39.34	-
Num. residues	-	-	-	-	638

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / R_factor R_free error: 0 / Total num. of bins used: 26

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Num. reflection all	% reflection obs (%)
1.73-1.75	0.3757	121	0.3701	2300	2421	87
1.75-1.77	0.3718	135	0.3179	2574	2709	100
1.77-1.8	0.3146	137	0.2754	2595	2732	100
1.8-1.83	0.2606	140	0.2402	2651	2791	100
1.83-1.86	0.2651	134	0.2269	2573	2707	100
1.86-1.89	0.2396	138	0.2127	2617	2755	100
1.89-1.92	0.2384	138	0.2116	2616	2754	100
1.92-1.95	0.2645	136	0.2125	2592	2728	100
1.95-1.99	0.2859	141	0.2061	2663	2804	100
1.99-2.03	0.2011	135	0.182	2584	2719	100
2.03-2.08	0.2273	140	0.1684	2651	2791	100
2.08-2.12	0.1653	136	0.1669	2575	2711	100
2.12-2.18	0.1949	139	0.1657	2650	2789	100
2.18-2.24	0.201	137	0.1646	2598	2735	100
2.24-2.3	0.1723	139	0.1639	2651	2790	100
2.3-2.38	0.2008	138	0.1579	2619	2757	100
2.38-2.46	0.202	138	0.1638	2620	2758	100
2.46-2.56	0.2088	140	0.1669	2663	2803	100
2.56-2.68	0.2235	138	0.1653	2620	2758	100
2.68-2.82	0.2213	139	0.17	2643	2782	100
2.82-2.99	0.1792	139	0.1687	2646	2785	100
2.99-3.22	0.1994	141	0.1658	2671	2812	100
3.22-3.55	0.1622	141	0.1487	2681	2822	100
3.55-4.06	0.1632	141	0.1425	2683	2824	100
4.06-5.12	0.1535	145	0.1278	2741	2886	100
5.12-41.46	0.1752	149	0.1669	2837	2986	99

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	1.8193	-0.0008	-0.0181	0.7224	-1.1612	1.5757	0.0276	-0.2391	-0.1681	0.1455	-0.0218	-0.2999	-0.0058	0.928	-0.1041	0.1036	-0.0081	-0.0123	0.3834	0.0482	0.2342	11.5679	5.2354	33.9614
2	2.0078	0.0974	0.0602	1.2729	-0.8837	2.6261	0.0599	-0.0907	0.052	0.0785	-0.0658	-0.0526	-0.5573	0.5584	-0.0378	0.1692	-0.0741	-0.0041	0.2151	0.0052	0.1451	6.1709	14.9032	30.645
3	1.428	0.3306	0.3468	1.9295	-0.4091	3.3811	0.0782	-0.0871	0.1472	0.0898	-0.0071	-0.0284	-0.6005	0.3141	-0.0282	0.2002	-0.0463	0.0329	0.1256	-0.0025	0.1537	2.0015	16.3862	26.0486
4	2.2846	0.6654	-0.1682	2.1011	-0.0147	3.3511	0.0237	0.1559	0.1281	-0.0764	0.0607	0.0477	-0.3134	-0.0661	-0.1204	0.1214	-0.0021	0.0022	0.1472	0.0253	0.1455	-3.3849	8.9112	20.2929
5	2.3098	0.6504	0.4795	2.8321	0.1115	1.3592	-0.0319	0.2722	0.1977	-0.3032	0.1643	0.3406	-0.2883	-0.5884	-0.1263	0.166	0.0455	-0.0123	0.2492	0.05	0.1988	-9.7176	9.5239	16.4302
6	1.073	-0.1604	0.0335	2.1318	-0.1803	0.0673	-0.0819	-0.1661	-0.2818	0.4404	0.1798	0.46	1.0136	-0.7749	-0.0742	0.5415	-0.1713	0.0221	0.3453	0.0681	0.3155	-11.3556	-11.0073	35.7798
7	1.6131	-0.11	-0.2522	1.6909	-0.0675	1.7374	0.002	-0.0054	-0.3711	0.0661	0.0379	0.068	0.8497	-0.0892	-0.0777	0.3048	-0.0268	-0.0143	0.1248	0.0115	0.216	-6.5599	-7.2532	22.3291
8	1.8228	0.3793	0.4276	1.9083	0.3954	0.6433	0.0163	-0.4319	-0.3169	0.3165	-0.152	-0.282	0.5303	0.5346	-0.0948	0.2369	0.0839	-0.0214	0.314	0.1274	0.2906	6.4627	-4.8169	35.6877
9	1.8771	-0.3106	-0.2079	3.2884	-1.3422	0.6497	-0.1354	0.4715	-0.5306	-0.5442	0.0337	-0.3168	1.0598	0.3954	-0.1533	0.5032	0.2272	0.0652	0.2589	-0.0182	0.3893	4.2971	-8.6309	17.6894
10	3.1038	1.2039	-0.1305	3.2575	-0.1464	3.1344	0.0204	0.3064	0.1497	-0.1609	0.061	0.333	-0.2626	-0.3228	-0.0716	0.2188	0.0553	0.0254	0.2216	0.0105	0.1964	-10.7379	8.8707	57.2765
11	1.5724	0.0888	-0.1926	1.7397	-0.5776	2.6285	-0.0588	0.1563	-0.1989	-0.0621	-0.0151	0.0289	0.1965	-0.1214	0.0507	0.1507	-0.0112	0.0423	0.132	-0.0533	0.1934	-5.4725	-4.9877	61.5729
12	2.4479	0.1349	-0.0677	1.6758	-0.9251	2.6143	-0.0609	-0.5195	-0.087	0.4827	0.042	0.1047	-0.3731	0.0651	-0.0089	0.3347	0.0009	-0.004	0.3181	0.0217	0.21	-0.4905	3.7612	78.7574
13	2.0724	-0.0117	0.196	2.2404	-1.08	3.0956	-0.0395	-0.0862	0.2899	0.4012	-0.0816	-0.1956	-0.7653	0.238	0.0056	0.3471	-0.0502	-0.0044	0.166	-0.0433	0.1819	1.3069	13.9168	71.2432

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 1 through 40 )	A	1 - 40
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 41 through 64 )	A	41 - 64
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 65 through 158 )	A	65 - 158
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 159 through 176 )	A	159 - 176
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 177 through 199 )	A	177 - 199
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 200 through 219 )	A	200 - 219
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 220 through 269 )	A	220 - 269
8	X-RAY DIFFRACTION	8	chain 'A' and (resid 270 through 297 )	A	270 - 297
9	X-RAY DIFFRACTION	9	chain 'A' and (resid 298 through 320 )	A	298 - 320
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 1 through 40 )	B	1 - 40
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 41 through 176 )	B	41 - 176
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 177 through 219 )	B	177 - 219
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 220 through 318 )	B	220 - 318

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