PDB-7r29: Crystal structure of TaCel5A E133Q Y200F variant with covalently ...

ID or keywords:

Entry

Database: PDB / ID: 7r29

Title

Crystal structure of TaCel5A E133Q Y200F variant with covalently linked cellotriose

Components

EGI

Keywords

HYDROLASE /

glycoside hydrolase /

cellulase / GH5_5 family / GH-A clan / (a/b)8 barrel / Thermoascus aurantiacus

Function / homology

Glycoside hydrolase, family 5 / Cellulase (glycosyl hydrolase family 5) /

cellulase /

cellulase activity / cellulose catabolic process / Glycoside hydrolase superfamily / alpha-cellotriose /

cellulase

Function and homology information

Biological species

Thermoascus aurantiacus ATCC 26904 (fungus)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT /

molecular replacement / Resolution: 1.28 Å

Authors

Dutoit, R.

Funding support

Belgium, 1items

Citation

Journal: To Be Published
Title: Crystal structure of TaCel5A E133Q Y200F variant with covalently linked cellotriose
Authors: Dutoit, R.

History

Deposition	Feb 4, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Feb 15, 2023	Provider: repository / Type: Initial release
Revision 1.1	Feb 7, 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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Download

PDBx/mmCIF format	7r29.cif.gz	275.1 KB	Display	PDBx/mmCIF format
PDB format	pdb7r29.ent.gz	220.6 KB	Display	PDB format
PDBx/mmJSON format	7r29.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/r2/7r29 ftp://data.pdbj.org/pub/pdb/validation_reports/r2/7r29	HTTPS FTP

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Related structure data

Related structure data	1gzjS S: Starting model for refinement
Similar structure data	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: EGI

B: EGI

hetero molecules

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

1

A: EGI

hetero molecules

defined by author
Evidence: gel filtration, Ta_Cel5A E133Q Y200F elutes as a monomer using a Superdex 75 column (V=120ml)
34.8 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: EGI

hetero molecules

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

Unit cell

Length a, b, c (Å)	76.200, 85.290, 89.400
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein

EGI / Endoglucanase

Mass: 33712.344 Da / Num. of mol.: 2 / Mutation: E133Q, Y200F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Thermoascus aurantiacus ATCC 26904 (fungus)
Gene: eg1 / Plasmid: pET30b / Production host:

Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8TG26,

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
[][D-1-deoxy-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}}	LINUCS	PDB-CARE

#3: Chemical

ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL /

Glycerol

Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C₃H₈O₃

#4: Chemical

ChemComp-SO4 / SULFATE ION /

Sulfate

Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO₄

#5: Water

ChemComp-HOH / water /

Water

Mass: 18.015 Da / Num. of mol.: 901 / 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: 2.15 Å³/Da / Density % sol: 42.91 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6 Details: Reservoir solution: 0.1 M Tris pH6.0, 1.3 M ammonium sulfate Drop: 2 ul of protein solution mixed with 2 ul of reservoir solution and 0.2 ul microseeds Protein solution: 465 uM enzyme, 5 mM ...Details: Reservoir solution: 0.1 M Tris pH6.0, 1.3 M ammonium sulfate Drop: 2 ul of protein solution mixed with 2 ul of reservoir solution and 0.2 ul microseeds Protein solution: 465 uM enzyme, 5 mM 2-chloro-4-nitrophenyl-cellotriose

Crystal

Density Matthews: 2.15 Å³/Da / Density % sol: 42.91 %

Crystal grow

Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6
Details: Reservoir solution: 0.1 M Tris pH6.0, 1.3 M ammonium sulfate Drop: 2 ul of protein solution mixed with 2 ul of reservoir solution and 0.2 ul microseeds Protein solution: 465 uM enzyme, 5 mM ...

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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: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Oct 2, 2021

Radiation

Monochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.9801 Å / Relative weight: 1

Reflection

Resolution: 1.28→44.7 Å / Num. obs: 148849 / % possible obs: 99 % / Redundancy: 13.023 % / B_iso Wilson estimate: 14.67 Å² / CC_1/2: 0.999 / R_merge(I) obs: 0.086 / R_rim(I) all: 0.09 / Χ²: 0.869 / Net I/σ(I): 16.14 / Num. measured all: 1938511 / Scaling rejects: 1133

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
1.28-1.31	11.465	1.915	1.45	110934	11043	9676	0.768	2.001	87.6
1.31-1.35	13.568	1.245	2.09	145010	10690	10688	0.883	1.293	100
1.35-1.39	13.493	0.999	2.64	141182	10464	10463	0.917	1.039	100
1.39-1.43	13.354	0.845	3.18	135373	10138	10137	0.941	0.879	100
1.43-1.48	12.987	0.736	3.9	127633	9830	9828	0.941	0.766	100
1.48-1.53	12.575	0.558	5.03	120316	9573	9568	0.966	0.582	99.9
1.53-1.59	13.607	0.394	7.18	125269	9206	9206	0.981	0.409	100
1.59-1.65	13.411	0.293	9.22	118771	8856	8856	0.989	0.305	100
1.65-1.72	12.665	0.227	11.41	107740	8507	8507	0.992	0.236	100
1.72-1.81	13.788	0.166	15.84	112372	8151	8150	0.996	0.172	100
1.81-1.91	13.713	0.147	19.33	106618	7795	7775	0.996	0.153	99.7
1.91-2.02	12.709	0.121	23.88	92495	7317	7278	0.997	0.126	99.5
2.02-2.16	13.27	0.088	29.83	91947	6929	6929	0.998	0.091	100
2.16-2.34	12.082	0.079	31.86	77819	6471	6441	0.998	0.083	99.5
2.34-2.56	12.285	0.065	35.85	73366	5973	5972	0.998	0.068	100
2.56-2.86	13.633	0.056	42.33	73672	5404	5404	0.999	0.059	100
2.86-3.3	12.682	0.051	44.58	60875	4800	4800	0.999	0.053	100
3.3-4.05	12.229	0.046	47.57	50250	4109	4109	0.999	0.048	100
4.05-5.72	13.771	0.038	52.48	44176	3208	3208	0.999	0.039	100
5.72-44.7	12.24	0.035	48.4	22693	1863	1854	0.999	0.036	99.5

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Phasing

Phasing

Method:

molecular replacement

Phasing MR

	Highest resolution	Lowest resolution
Rotation	3.59 Å	47.96 Å
Translation	3.59 Å	47.96 Å

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Processing

Software

Name	Version	Classification
XDS	20210323	data reduction
XSCALE	20210323	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: 1GZJ

1gzj

Resolution: 1.28→44.7 Å / SU ML: 0.14 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 17.95 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1686	7439	5 %
R_work	0.1507	141356	-
obs	0.1515	148795	98.99 %

Solvent computation

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

Displacement parameters

B_iso max: 71.51 Å² / B_iso mean: 18.2651 Å² / B_iso min: 9.42 Å²

Refinement step

Cycle: final / Resolution: 1.28→44.7 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4754	0	150	901	5805
B_iso mean	-	-	22.85	30.98	-
Num. residues	-	-	-	-	610

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 (%)
1.28-1.29	0.3551	181	0.3518	3449	3630	73
1.29-1.31	0.318	246	0.3159	4676	4922	100
1.31-1.32	0.278	245	0.2574	4668	4913	100
1.32-1.34	0.2802	250	0.2461	4744	4994	100
1.34-1.36	0.2645	249	0.2425	4720	4969	100
1.36-1.38	0.2556	247	0.2414	4714	4961	100
1.38-1.4	0.2429	247	0.2318	4717	4964	100
1.4-1.42	0.2823	247	0.2298	4694	4941	100
1.42-1.44	0.2373	248	0.222	4713	4961	100
1.44-1.46	0.2233	249	0.1971	4728	4977	100
1.46-1.49	0.2441	250	0.208	4730	4980	100
1.49-1.52	0.1946	247	0.1738	4703	4950	100
1.52-1.55	0.1776	251	0.1484	4768	5019	100
1.55-1.58	0.1838	247	0.1409	4682	4929	100
1.58-1.61	0.1623	251	0.138	4763	5014	100
1.61-1.65	0.1599	249	0.1427	4731	4980	100
1.65-1.69	0.1674	249	0.1466	4734	4983	100
1.69-1.74	0.1634	250	0.1511	4755	5005	100
1.74-1.79	0.1814	249	0.1553	4733	4982	100
1.79-1.84	0.1691	250	0.1489	4744	4994	100
1.84-1.91	0.1653	248	0.1493	4723	4971	99
1.91-1.99	0.1629	250	0.1534	4743	4993	99
1.99-2.08	0.1582	252	0.1371	4779	5031	100
2.08-2.19	0.1571	251	0.1397	4770	5021	100
2.19-2.32	0.1657	249	0.146	4732	4981	99
2.32-2.5	0.157	254	0.1411	4827	5081	100
2.5-2.76	0.157	253	0.1405	4818	5071	100
2.76-3.15	0.1498	255	0.1416	4837	5092	100
3.15-3.97	0.1558	257	0.1287	4889	5146	100
3.97-44.7	0.1341	268	0.1272	5072	5340	100

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	0.8925	-0.5399	-0.7081	1.5074	0.1747	1.9667	-0.0308	0.0015	-0.1663	0.0405	-0.0021	0.0133	0.1427	0.011	0.0432	0.1221	-0.0082	-0.0329	0.1137	0.009	0.1354	4.0575	16.4783	10.2973
2	1.1783	-0.0533	-0.3357	1.083	-0.0797	1.0609	0.0159	0.1224	-0.2489	-0.1243	-0.0586	0.0585	0.1889	-0.0127	0.0442	0.1553	-0.0024	-0.0237	0.1352	-0.0221	0.1736	5.3421	11.9551	2.8336
3	1.3723	-0.2062	-0.0471	1.1114	0.0482	1.3976	0.0632	0.157	-0.08	-0.1798	-0.0489	0.0313	0.0816	0.0359	-0.0099	0.1416	0.0226	-0.0146	0.1555	-0.0104	0.1293	9.5101	21.3051	-3.6409
4	0.598	-0.1392	0.1226	0.4231	-0.1494	0.5486	-0.0102	0.0291	0.0173	0.0062	0.023	-0.0318	0.0027	0.0131	-0.0121	0.0991	-0.0011	-0.0049	0.1146	-0.0003	0.1243	9.026	28.8857	11.8284
5	0.4046	-0.3226	0.1733	1.8982	-0.6651	0.6569	-0.0339	-0.1377	-0.0316	0.2622	0.0096	-0.0079	-0.0614	-0.0256	0.0177	0.1551	0.0117	0.0033	0.161	0.006	0.1316	2.0333	27.4496	28.9752
6	0.6113	0.156	0.0025	0.5578	0.1348	0.7068	0.0321	-0.0551	-0.0897	0.0243	-0.0052	-0.0235	0.0302	-0.1174	-0.0289	0.1183	-0.0047	-0.0001	0.1303	0.0197	0.1398	-0.4893	17.7604	22.1626
7	1.0736	0.2098	-0.5116	0.8843	-0.1236	1.7875	-0.0294	0.047	-0.1884	-0.1365	0.024	0.0797	0.2391	-0.1069	0.0203	0.1881	-0.0154	-0.0319	0.1261	0.0105	0.2253	1.7545	5.4733	14.8153
8	0.9113	0.2768	-0.6426	1.4216	-0.3062	1.9772	-0.0664	0.0634	-0.0417	-0.0379	0.0092	-0.0237	0.1481	0.0262	0.0509	0.1087	0.0042	-0.0089	0.1437	-0.009	0.1121	38.1711	19.4067	34.2717
9	1.4767	-0.0172	-0.1929	0.897	0.0196	1.1038	0.0078	-0.0462	-0.1466	0.1272	0.0017	-0.0164	0.2064	0.0427	-0.0013	0.1563	0.0135	-0.011	0.1395	-0.004	0.1213	38.4026	14.5477	41.5176
10	0.3349	0.0432	-0.0294	0.4869	0.006	0.6993	0.0106	-0.051	0.0025	0.0319	-0.0037	0.0105	0.0589	-0.021	-0.0061	0.1106	-0.0029	0.0016	0.1449	0.0021	0.117	29.8541	24.4926	44.3115
11	0.3624	-0.0343	-0.0631	0.5443	0.0922	0.6638	-0.0129	0.0089	0.0341	-0.0339	0.0186	0.0187	-0.0523	0.024	-0.0058	0.1044	0.0006	-0.0053	0.1202	-0.0017	0.12	31.7371	33.9884	33.9783
12	0.2535	-0.256	0.0189	0.7945	0.05	0.5291	0.0234	0.06	-0.04	-0.1598	-0.0249	-0.0074	0.0459	0.0281	-0.0028	0.1624	0.0064	0.0127	0.1598	-0.0014	0.1246	35.6328	23.5015	18.21
13	0.8528	0.5296	0.1253	2.6502	0.3397	0.7176	0.009	0.0498	-0.0404	-0.2175	-0.0412	-0.1445	0.1113	0.1182	0.0299	0.1888	0.0258	0.0298	0.1891	0.0042	0.141	43.6617	19.3167	16.0443
14	0.4264	-0.0752	0.0886	0.9262	0.1026	0.6216	0.0278	0.0075	-0.0611	0.0464	-0.0057	-0.0919	0.1693	0.1056	-0.016	0.1715	0.0295	-0.0053	0.165	-0.0085	0.146	42.0225	13.4525	28.4718

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 20 )	A	1 - 20
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 21 through 52 )	A	21 - 52
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 53 through 84 )	A	53 - 84
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 85 through 216 )	A	85 - 216
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 217 through 232 )	A	217 - 232
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 233 through 277 )	A	233 - 277
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 278 through 305 )	A	278 - 305
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 1 through 20 )	B	1 - 20
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 21 through 52 )	B	21 - 52
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 53 through 105 )	B	53 - 105
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 106 through 200 )	B	106 - 200
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 201 through 246 )	B	201 - 246
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 247 through 263 )	B	247 - 263
14	X-RAY DIFFRACTION	14	chain 'B' and (resid 264 through 305 )	B	264 - 305

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