PDB-7rag: Structure of the CwlD amidase from Clostridioides difficile in co...

ID or keywords:

Entry

Database: PDB / ID: 7rag

Title

Structure of the CwlD amidase from Clostridioides difficile in complex with the GerS lipoprotein

Components

Germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin
Lipoprotein

Keywords

HYDROLASE / N-acetylmuramic acid (NAM) hydrolase Cortex lytic enzyme Amidase_3 family LolA family

Function / homology

Function and homology information

N-acetylmuramoyl-L-alanine amidase /

N-acetylmuramoyl-L-alanine amidase activity / peptidoglycan catabolic process / membrane => GO:0016020
Similarity search - Function

Biological species

Clostridioides difficile (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MIR / Resolution: 2.4 Å

Authors

Eckenroth, B.E. / Doublie, S.

Funding support

United States, 2items

Organization	Grant number	Country
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)	GM140361	United States
National Institutes of Health/National Cancer Institute (NIH/NCI)	CA233185	United States

Citation

Journal: Plos Genet. / Year: 2021
Title: A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation.
Authors: Alves Feliciano, C. / Eckenroth, B.E. / Diaz, O.R. / Doublie, S. / Shen, A.

History

Deposition	Jul 1, 2021	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Sep 8, 2021	Provider: repository / Type: Initial release
Revision 1.1	Oct 6, 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7rag.cif.gz	161.5 KB	Display	PDBx/mmCIF format
PDB format	pdb7rag.ent.gz	132.7 KB	Display	PDB format
PDBx/mmJSON format	7rag.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/ra/7rag ftp://data.pdbj.org/pub/pdb/validation_reports/ra/7rag	HTTPS FTP

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Related structure data

Similar structure data	2ec5-1 2ec5-2 4zo0-d 5v68-2 5v68-1 2ebh-d 2ebf-d 3dad-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

Deposited unit

A: Lipoprotein

B: Germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin

hetero molecules

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

1

A: Lipoprotein

B: Germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin

hetero molecules

A: Lipoprotein

B: Germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin

hetero molecules

defined by author&software
Evidence: gel filtration, Dynamic light scattering and SEC support dimer assembly
91.8 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	105.185, 105.185, 154.771
Angle α, β, γ (deg.)	90.000, 90.000, 120.000
Int Tables number	179
Space group name H-M	P6₅22

#1: Protein	Lipoprotein / / Lipoprotein localisation LolA/LolB/LppX / Outer membrane lipoprotein-sorting protein / Putative lipoprotein Mass: 20823.184 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridioides difficile (bacteria) Gene: BN1096_760049, BN1097_760051, cdgr_15010, E5F32_07620, E5F39_08860, E5F43_07640, GSQ22_27115, IB136_2570, SAMEA1402406_02874, SAMEA3374989_03440, SAMEA3375041_03139, SAMEA708418_02871 Production host: Escherichia coli (E. coli) / References: UniProt: A0A031WJD5
#2: Protein	Germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin Mass: 24951.879 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: cwlD, CD630_01060 / Production host: Escherichia coli (E. coli) References: UniProt: Q18CJ4, N-acetylmuramoyl-L-alanine amidase
#3: Chemical	ChemComp-ZN / ZINC ION Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#5: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 14 / 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: 2.72 Å³/Da / Density % sol: 54.77 %
Crystal grow	Temperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5 Details: 12-18 % PEG 3350, 5 % glycerol, 0.25 M ammonium nitrate, 50 mM BisTris pH 7.5. 1:1 ratio of reservoir to 30 mg/ml complex

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

APS

/ Beamline: 23-ID-D / Wavelength: 0.9794 Å

Detector

Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 16, 2020 / Details: mirrors

Radiation

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

Radiation wavelength

Wavelength: 0.9794 Å / Relative weight: 1

Reflection

Resolution: 2.4→46 Å / Num. obs: 37392 / % possible obs: 99.9 % / Redundancy: 10.2 % / CC_1/2: 0.999 / R_merge(I) obs: 0.109 / R_pim(I) all: 0.036 / R_rim(I) all: 0.115 / Net I/σ(I): 10.7

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. unique obs	CC_1/2	R_pim(I) all	R_rim(I) all	% possible all
5.17-45.5		0.048	24.9	3748	0.999	0.017	0.051	99.6
4.1-5.17	10.1	0.058	24.2	3753	0.998	0.019	0.061	100
3.59-4.1	10.5	0.083	19.7	3728	0.997	0.027	0.087	100
3.26-3.59	10.1	0.133	14.2	3758	0.994	0.044	0.14	100
3.02-3.26	10.4	0.243	9.1	3713	0.991	0.079	0.255	100
2.85-3.02	10.3	0.422	5.9	3745	0.958	0.137	0.444	100
2.7-2.85	10.3	0.697	3.7	3749	0.9	0.227	0.734	100
2.59-2.7	10.3	1.123	2.4	3748	0.775	0.367	1.181	100
2.49-2.59	10.6	1.576	1.7	3763	0.682	0.506	1.656	100
2.4-2.49	9.7	2.205	1.1	3687	0.406	0.745	2.329	98.9

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Phasing

Phasing	Method: MIR

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Processing

Software

Name	Version	Classification
PROTEUM PLUS	V3	data reduction
PROTEUM PLUS	V3	data scaling
SHARP	2.8.10	phasing
PHENIX	1.18.2_3874	refinement
PDB_EXTRACT	3.27	data extraction

Refinement

Method to determine structure:

MIR / Resolution: 2.4→45.55 Å / SU ML: 0.39 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 30.14 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.249	3742	10.02 %
R_work	0.203	33598	-
obs	0.2076	37340	99.72 %

Solvent computation

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

Displacement parameters

B_iso max: 168.21 Å² / B_iso mean: 69.1844 Å² / B_iso min: 36.29 Å²

Refinement step

Cycle: final / Resolution: 2.4→45.55 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	2890	0	5	14	2909
B_iso mean	-	-	60.38	60.11	-
Num. residues	-	-	-	-	367

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / R_factor R_free error: 0 / Total num. of bins used: 27

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Num. reflection all	% reflection obs (%)
2.4-2.43	0.3871	120	0.3826	1207	1327	96
2.43-2.46	0.4148	154	0.3654	1243	1397	100
2.46-2.5	0.3576	148	0.3509	1235	1383	100
2.5-2.53	0.3828	140	0.3461	1229	1369	100
2.53-2.57	0.3989	138	0.3105	1257	1395	100
2.57-2.61	0.3309	138	0.3146	1222	1360	100
2.61-2.65	0.3604	144	0.2969	1247	1391	100
2.65-2.7	0.3535	149	0.2871	1252	1401	100
2.7-2.75	0.3135	155	0.2847	1231	1386	100
2.75-2.8	0.2839	144	0.2675	1209	1353	100
2.8-2.86	0.3435	143	0.2432	1235	1378	100
2.86-2.92	0.3233	150	0.2574	1278	1428	100
2.92-2.99	0.316	139	0.2376	1239	1378	100
2.99-3.06	0.3191	116	0.2452	1262	1378	100
3.06-3.14	0.3167	136	0.2394	1245	1381	100
3.15-3.24	0.2165	128	0.2255	1250	1378	100
3.24-3.34	0.2689	141	0.2168	1241	1382	100
3.34-3.46	0.2804	157	0.2045	1232	1389	100
3.46-3.6	0.3164	150	0.1964	1238	1388	100
3.6-3.76	0.2499	103	0.2056	1286	1389	100
3.76-3.96	0.2091	123	0.1756	1262	1385	100
3.96-4.21	0.2081	112	0.1612	1287	1399	100
4.21-4.54	0.1895	132	0.1457	1244	1376	100
4.54-4.99	0.2123	134	0.1521	1254	1388	100
4.99-5.71	0.2023	176	0.188	1211	1387	100
5.72-7.19	0.2183	143	0.1947	1250	1393	100
7.2-45.55	0.199	129	0.1756	1252	1381	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	-0.2245	-0.3838	-0.5983	1.5458	-0.8006	-0.3096	0.0601	0.0666	-0.2832	-0.3272	-0.0952	0.385	0.3138	-0.1989	0.0023	0.6323	0.0621	0.0446	0.8998	-0.0211	0.6767	28.5868	41.4828	65.7657
2	0.9215	1.1292	-0.2549	5.413	-0.1991	0.9669	-0.0827	-0.0893	-0.2509	0.5097	0.1781	0.3425	0.235	-0.0643	-0.0001	0.7374	0.1788	0.0376	0.6388	-0.0619	0.5634	21.177	53.7241	34.4524
3	1.3904	-0.1513	1.4061	1.6269	0.7913	1.9292	0.095	0.3255	0.6381	-0.8223	0.2966	-0.1475	-0.4557	0.3965	-0.0002	0.8587	0.0032	0.1815	0.6552	-0.0678	0.6585	31.1547	81.0789	19.4406
4	1.8277	-0.3523	0.7347	0.9568	0.766	1.2032	-0.1039	-0.1157	-0.008	-1.2805	-0.0958	-1.1286	0.7371	0.4876	-0.0004	0.5753	0.1311	0.0997	0.6005	-0.1126	0.5716	31.362	71.8534	23.8547
5	1.8959	0.0495	-0.5786	1.1969	0.907	0.8821	-0.4404	1.2459	0.1196	-1.7094	0.3281	-0.2072	1.0631	-0.4347	-0.0005	0.8963	-0.0462	-0.0626	0.9057	0.0651	0.5027	22.0817	77.6726	13.8086
6	1.7956	-0.1598	-0.5333	2.0399	1.1616	0.8828	0.0103	0.393	0.2711	-0.0541	0.2716	0.4193	-0.3423	-0.2726	-0	0.6198	0.1512	0.0069	0.7166	0.0433	0.6726	19.3198	82.0185	21.6102
7	0.0876	0.1248	-0.0896	0.1043	-0.101	0.0667	0.2909	-0.6113	1.288	0.1405	0.0284	-1.5215	-0.2737	0.3824	-0.0022	0.7771	-0.0901	0.1066	0.7259	-0.2284	1.2543	41.2689	91.2878	26.9276
8	0.8794	1.2622	0.4745	2.0168	0.4538	1.0395	-0.0487	-0.7337	0.5411	0.9969	0.1775	-0.2184	0.0763	0.1424	-0.0004	0.6666	0.1115	-0.066	0.6198	-0.1994	0.6641	29.1409	83.1827	38.4617
9	0.0473	-0.0132	-0.063	-0.0107	0.0029	0.1207	-0.0866	-0.9957	-0.1949	0.825	0.3601	-1.0098	-0.7685	0.1504	-0.0027	0.7116	0.012	-0.0863	0.8057	-0.3895	1.0395	40.2154	85.6351	38.6096
10	0.5588	-0.6117	0.2277	0.7671	0.1602	0.4777	-0.3798	-0.5922	1.3849	0.6985	0.3817	0.4532	-1.0237	-0.6252	-0.0017	0.8004	0.1987	0.0224	0.569	-0.0476	0.8868	20.2508	88.6112	30.1042
11	0.0957	-0.0957	0.0542	0.2654	0.1894	0.5655	0.1692	-0.1765	0.5391	-0.5592	0.077	-0.9604	-0.3759	0.3964	-0.0001	0.6254	-0.012	0.2766	0.7077	-0.1585	1.083	38.7107	83.0724	26.1822
12	1.3332	0.2793	1.1495	2.9465	-0.5585	1.2582	0.0054	-0.082	0.3938	0.6062	0.1689	-0.8458	-0.0911	0.0154	0.0004	0.5457	0.1655	-0.04	0.5461	-0.1399	0.5993	33.2265	72.3706	34.6577

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 22 through 78 )	A	22 - 78
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 79 through 195 )	A	79 - 195
3	X-RAY DIFFRACTION	3	chain 'B' and (resid 40 through 64 )	B	40 - 64
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 65 through 83 )	B	65 - 83
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 84 through 105 )	B	84 - 105
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 106 through 133 )	B	106 - 133
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 134 through 145 )	B	134 - 145
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 146 through 171 )	B	146 - 171
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 172 through 179 )	B	172 - 179
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 180 through 193 )	B	180 - 193
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 194 through 214 )	B	194 - 214
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 215 through 236 )	B	215 - 236

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