PDB-7p6g: Crystal structure of the endoglucanase RBcel1 E135Q

ID or keywords:

Entry

Database: PDB / ID: 7p6g

Title

Crystal structure of the endoglucanase RBcel1 E135Q

Components

Endoglucanase

Keywords

HYDROLASE / Transglycosylase / GH5_5 family

Function / homology

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

Function and homology information

Biological species

uncultured bacterium (environmental samples)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT /

molecular replacement / Resolution: 1.49 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

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7p6g.cif.gz	295.6 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	7p6g_validation.pdf.gz	453.1 KB	Display	wwPDB validaton report
Full document	7p6g_full_validation.pdf.gz	456.6 KB	Display
Data in XML	7p6g_validation.xml.gz	33.7 KB	Display
Data in CIF	7p6g_validation.cif.gz	53 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/p6/7p6g ftp://data.pdbj.org/pub/pdb/validation_reports/p6/7p6g	HTTPS FTP

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Related structure data

Related structure data	7p6hC 7p6iC 7p6jC 4ee9S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	6zz3-4 5ljf-1 7p6g-2 4ee9-d 4m24-d 6zz3-1 7p6j-3 7p6j-1 6zz3-3 2abb-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

73.1 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 is monomeric according to size exclusion chromatography using a Superdex75 column.
36.5 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Endoglucanase

hetero molecules

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

Unit cell

Length a, b, c (Å)	46.380, 99.520, 149.930
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: Chemical	ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER Mass: 122.143 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₄H₁₂NO₃ / Comment: pH buffer*YM
#3: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#4: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 877 / 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: 2.4 Å³/Da / Density % sol: 48.7 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 Details: Reservoir: 0.1M Tris, 17.5% PEG600, pH8. Protein: 14 mg/ml in 20 mM sodium phosphate buffer pH 6.5. Drop: 2 ul reservoir + 2 ul protein

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

ESRF

/ Beamline: BM30A / Wavelength: 0.9798 Å

Detector

Type: ADSC QUANTUM 315r / Detector: CCD / Date: Dec 15, 2014

Radiation

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

Radiation wavelength

Wavelength: 0.9798 Å / Relative weight: 1

Reflection

Resolution: 1.49→15.57 Å / Num. obs: 112931 / % possible obs: 99.4 % / Redundancy: 7.09 % / B_iso Wilson estimate: 20.94 Å² / CC_1/2: 1 / R_merge(I) obs: 0.043 / R_rim(I) all: 0.046 / Χ²: 0.912 / Net I/σ(I): 22.27 / Num. measured all: 800713 / Scaling rejects: 86

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
1.49-1.53	6.444	1.109	1.69	49725	8289	7716	0.623	1.202	93.1
1.53-1.57	7.255	0.868	2.34	58787	8107	8103	0.792	0.935	100
1.57-1.62	7.271	0.666	3.07	57274	7878	7877	0.862	0.717	100
1.62-1.67	7.285	0.482	4.27	55823	7666	7663	0.926	0.519	100
1.67-1.72	7.28	0.367	5.54	54334	7465	7463	0.954	0.395	100
1.72-1.78	7.296	0.265	7.61	52577	7207	7206	0.976	0.286	100
1.78-1.85	7.299	0.195	10.17	50445	6912	6911	0.988	0.21	100
1.85-1.93	7.108	0.143	14.19	47692	6715	6710	0.992	0.155	99.9
1.93-2.01	7.24	0.104	18.24	46440	6425	6414	0.996	0.112	99.8
2.01-2.11	7.094	0.075	24.36	43911	6197	6190	0.997	0.082	99.9
2.11-2.22	7.237	0.06	29.28	42194	5832	5830	0.998	0.065	100
2.22-2.36	7.031	0.051	34.56	38771	5525	5514	0.998	0.055	99.8
2.36-2.52	7.166	0.042	39.98	37521	5238	5236	0.999	0.045	100
2.52-2.72	7.039	0.037	43.56	34717	4937	4932	0.999	0.04	99.9
2.72-2.98	7.039	0.031	50.24	31533	4490	4480	0.999	0.034	99.8
2.98-3.34	6.781	0.029	55.57	27952	4129	4122	0.999	0.031	99.8
3.34-3.85	6.679	0.025	60.27	24357	3651	3647	0.999	0.027	99.9
3.85-4.72	6.951	0.021	64.24	21541	3106	3099	1	0.023	99.8
4.72-6.67	6.92	0.019	64.3	17009	2459	2458	1	0.021	100
6.67-15.57	5.963	0.02	58.57	8110	1438	1360	0.999	0.022	94.6

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Phasing

Phasing

Method:

molecular replacement

Phasing MR

	Highest resolution	Lowest resolution
Rotation	3.78 Å	47.23 Å
Translation	3.78 Å	47.23 Å

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Processing

Software

Name	Version	Classification
XDS	20200417	data reduction
XSCALE	20200417	data scaling
PHASER	2.7.15	phasing
PHENIX	1.19.2	refinement
PDB_EXTRACT	3.27	data extraction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 4EE9

4ee9

Resolution: 1.49→15.57 Å / SU ML: 0.15 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 19.38 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1933	5642	5 %
R_work	0.1741	107185	-
obs	0.1751	112827	99.37 %

Solvent computation

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

Displacement parameters

B_iso max: 146.6 Å² / B_iso mean: 30.5152 Å² / B_iso min: 12.13 Å²

Refinement step

Cycle: final / Resolution: 1.49→15.57 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5108	0	20	877	6005
B_iso mean	-	-	37.83	39.86	-
Num. residues	-	-	-	-	638

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / R_factor R_free error: 0 / Total num. of bins used: 30

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Num. reflection all	% reflection obs (%)
1.49-1.51	0.3323	159	0.2947	3013	3172	85
1.51-1.53	0.2912	187	0.2658	3558	3745	100
1.53-1.55	0.2686	188	0.2502	3566	3754	100
1.55-1.57	0.2572	183	0.2266	3487	3670	100
1.57-1.59	0.2355	189	0.2209	3580	3769	100
1.59-1.61	0.2768	187	0.2151	3565	3752	100
1.61-1.63	0.2484	186	0.2051	3536	3722	100
1.63-1.65	0.2082	188	0.198	3557	3745	100
1.65-1.68	0.2067	187	0.1962	3558	3745	100
1.68-1.71	0.2295	188	0.187	3569	3757	100
1.71-1.74	0.2409	187	0.192	3552	3739	100
1.74-1.77	0.2217	188	0.1895	3570	3758	100
1.77-1.8	0.2074	187	0.1896	3570	3757	100
1.8-1.84	0.214	189	0.1929	3581	3770	100
1.84-1.88	0.2304	186	0.1947	3541	3727	100
1.88-1.92	0.2114	189	0.1899	3587	3776	100
1.92-1.97	0.2274	187	0.1918	3551	3738	100
1.97-2.02	0.2145	191	0.1945	3628	3819	100
2.02-2.08	0.2186	187	0.184	3556	3743	100
2.08-2.15	0.1984	188	0.1769	3579	3767	100
2.15-2.23	0.2154	190	0.1762	3606	3796	100
2.23-2.32	0.1915	190	0.18	3596	3786	100
2.32-2.42	0.1786	188	0.1709	3589	3777	100
2.42-2.55	0.2108	190	0.1787	3611	3801	100
2.55-2.71	0.1848	191	0.1739	3618	3809	100
2.71-2.92	0.2066	190	0.1807	3622	3812	100
2.92-3.21	0.1809	192	0.1679	3644	3836	100
3.21-3.66	0.1738	193	0.1652	3669	3862	100
3.67-4.6	0.1478	196	0.1426	3713	3909	100
4.6-15.57	0.1735	201	0.1531	3813	4014	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	0.6864	0.1438	0.3384	0.3414	-0.3367	0.9426	0.0588	-0.0115	-0.1194	0.0773	-0.0356	-0.027	-0.0428	0.262	0.056	0.123	-0.0017	-0.034	0.2266	0.0026	0.1985	-37.0342	107.6486	32.5416
2	0.4308	0.1349	0.5977	0.7244	0.0803	1.3578	-0.0002	0.0174	0.0599	0.0017	-0.0164	0.0156	-0.1647	0.0912	0	0.1273	-0.0212	0.0014	0.1652	0.0136	0.1753	-45.4581	114.665	25.4968
3	0.1366	0.071	0.1223	0.134	0.093	0.1091	0.0362	0.1432	-0.001	-0.1409	0.0261	0.1902	-0.0268	-0.1406	0.0034	0.14	0.0007	-0.0351	0.228	0.0309	0.2112	-56.7619	108.9811	16.8029
4	0.0404	-0.1237	-0.0199	0.271	0.0965	0.0842	0.091	-0.0433	-0.0884	0.0303	0.0858	0.1149	0.2511	-0.3908	0.0057	0.2623	-0.0781	-0.0773	0.2262	0.0328	0.2522	-58.425	92.1647	28.1357
5	0.542	0.2231	-0.3716	0.1682	-0.1662	0.344	0.1627	-0.044	-0.2196	-0.0213	-0.1164	-0.0853	0.4483	0.2255	0.0275	0.2768	0.0121	-0.093	0.1867	0.0256	0.2637	-45.9685	92.2108	30.0654
6	0.2358	0.1471	-0.0133	0.0834	-0.018	0.048	0.0612	0.2415	-0.299	-0.166	0.0438	-0.3816	0.4103	0.07	0.0037	0.3436	0.0868	-0.0608	0.3062	-0.0752	0.3693	-43.6187	91.253	17.42
7	0.0551	-0.0258	-0.1106	0.257	-0.0099	0.6325	0.1377	0.0476	0.1103	0.1128	0.012	0.1868	-0.5082	-0.1483	-0.0078	0.2686	0.042	0.0836	0.2242	0.0524	0.2441	-56.7246	108.4746	58.1224
8	0.8377	-0.0489	-0.0968	1.0147	-0.3208	1.3027	0.0507	0.0343	-0.0812	-0.0628	-0.0418	0.1156	0.097	0.0437	0	0.2085	-0.0026	0.0468	0.1625	-0.0254	0.1979	-51.1281	94.4591	63.3215
9	0.3448	0.1043	-0.067	0.1216	-0.1663	0.4404	0.0767	-0.304	-0.0845	0.4204	-0.0907	-0.0592	-0.3125	0.3313	-0.0031	0.4482	-0.0149	0.1047	0.2562	-0.0275	0.1469	-46.9801	105.6246	77.6261
10	0.4143	0.1719	-0.1847	0.9245	-0.662	1.8669	0.0719	-0.1924	0.0871	0.7182	-0.2988	-0.1828	-1.4685	0.6995	-0.4221	0.6434	-0.0997	0.0955	0.1026	-0.1209	0.1405	-44.7932	113.2793	71.5893

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 64 )	A	1 - 64
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 65 through 176 )	A	65 - 176
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 177 through 199 )	A	177 - 199
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 200 through 237 )	A	200 - 237
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 238 through 297 )	A	238 - 297
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 298 through 320 )	A	298 - 320
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 1 through 40 )	B	1 - 40
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 41 through 184 )	B	41 - 184
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 185 through 221 )	B	185 - 221
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 222 through 318 )	B	222 - 318

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