PDB-9hth: Crystal structure of the fused, cysteine-less Aes123 PolB1 split ...

ID or keywords:

Entry

Database: PDB / ID: 9hth

Title

Crystal structure of the fused, cysteine-less Aes123 PolB1 split intein (with S1A, N159A mutations)

Components

DNA-directed DNA polymerase

Keywords

SPLICING / intein / protein splicing / cysteine-less / ligation

Function / homology

Function and homology information

intein-mediated protein splicing / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding
Similarity search - Function

Biological species

Aeromonas phage Aes123 (virus)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.378 Å

Authors

Fitzian, K. / Yilmaz, Z. / Humberg, C. / Mootz, H.D. / Kuemmel, D.

Funding support

Germany, 1items

Citation

Journal: Nat Commun / Year: 2025
Title: A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing.
Authors: Humberg, C. / Yilmaz, Z. / Fitzian, K. / Dorner, W. / Kummel, D. / Mootz, H.D.

History

Deposition	Dec 19, 2024	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 7, 2025	Provider: repository / Type: Initial release

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	9hth.cif.gz	153.3 KB	Display	PDBx/mmCIF format
PDB format	pdb9hth.ent.gz		Display	PDB format
PDBx/mmJSON format	9hth.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ht/9hth ftp://data.pdbj.org/pub/pdb/validation_reports/ht/9hth	HTTPS FTP

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Related structure data

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
Related items in Molecule of the Month	#131 - Nov 2010 Inteins similarity (6) #219 - Mar 2018 Vacuolar ATPase similarity (5) #247 - Jul 2020 Myelin-associated Glycoprotein similarity (2) #3 - Mar 2000 DNA Polymerase similarity (2)

Deposited unit

A: DNA-directed DNA polymerase

B: DNA-directed DNA polymerase

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

1

A: DNA-directed DNA polymerase

defined by author
Evidence: gel filtration
19.8 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: DNA-directed DNA polymerase

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

Unit cell

Length a, b, c (Å)	30.129, 63.013, 162.404
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein	DNA-directed DNA polymerase Mass: 19793.750 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Details: IntN,IntC / Source: (gene. exp.) Aeromonas phage Aes123 (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: I6YMS9, DNA-directed DNA polymerase
#2: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 196 / Source method: isolated from a natural source / Formula: H₂O
Has protein modification	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: 1.96 Å³/Da / Density % sol: 37.36 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, sitting drop Details: Morpheus 50 % Precipitant mix 1, 0.1 M buffer system 3 pH 8.5, 0.1 M monosaccharides

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Data collection

Diffraction	Mean temperature: 173 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.97956 Å
Detector	Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 24, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.97956 Å / Relative weight: 1
Reflection	Resolution: 1.378→40.601 Å / Num. obs: 64820 / % possible obs: 96.27 % / Redundancy: 12.8 % / CC_1/2: 0.999 / R_rim(I) all: 0.077 / Net I/σ(I): 16.43
Reflection shell	Resolution: 1.38→1.43 Å / Num. unique obs: 5518 / CC_1/2: 0.782

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Processing

Software

Name	Version	Classification
PHENIX	(1.13_2998: ???)	refinement
XDS		data reduction
XSCALE		data scaling
PHENIX		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.378→40.601 Å / SU ML: 0.15 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 23.15 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.2263	1944	3.09 %
R_work	0.1925	-	-
obs	0.1935	62985	96.67 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 1.378→40.601 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	2747	0	0	196	2943

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	2791
X-RAY DIFFRACTION	f_angle_d	0.939	3752
X-RAY DIFFRACTION	f_dihedral_angle_d	4.721	1050
X-RAY DIFFRACTION	f_chiral_restr	0.091	425
X-RAY DIFFRACTION	f_plane_restr	0.006	474

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
1.3781-1.4124	0.3044	124	0.2753	3733	X-RAY DIFFRACTION	85
1.4124-1.4506	0.2731	128	0.2581	4023	X-RAY DIFFRACTION	90
1.4506-1.4933	0.3043	130	0.232	4047	X-RAY DIFFRACTION	92
1.4933-1.5415	0.2018	134	0.209	4204	X-RAY DIFFRACTION	95
1.5415-1.5966	0.2233	136	0.2056	4342	X-RAY DIFFRACTION	97
1.5966-1.6605	0.224	136	0.2028	4380	X-RAY DIFFRACTION	98
1.6605-1.7361	0.235	140	0.206	4366	X-RAY DIFFRACTION	99
1.7361-1.8276	0.2284	142	0.2093	4424	X-RAY DIFFRACTION	99
1.8276-1.9421	0.2369	144	0.1993	4518	X-RAY DIFFRACTION	100
1.9421-2.0921	0.2083	142	0.1941	4481	X-RAY DIFFRACTION	100
2.0921-2.3026	0.2376	144	0.1949	4539	X-RAY DIFFRACTION	100
2.3026-2.6357	0.2463	144	0.2022	4541	X-RAY DIFFRACTION	100
2.6357-3.3205	0.2535	147	0.2028	4621	X-RAY DIFFRACTION	100
3.3205-40.601	0.1968	153	0.1665	4822	X-RAY DIFFRACTION	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	0.807	-0.6348	-0.3821	0.7237	0.7775	1.0022	-0.0539	-0.0793	-0.0531	0.2161	-0.0228	0.0862	0.0181	0.0214	-0.0002	0.2198	-0.013	0.0406	0.1598	-0.0093	0.1663	-5.642	6.6081	-24.1502
2	0.9235	0.1946	0.5546	0.6218	-0.7148	1.5068	-0.0792	0.2142	-0.0445	0.0113	0.1025	0.1642	-0.3291	-0.3871	0.0457	0.1608	0.0173	0.0181	0.271	-0.0444	0.1987	-13.2762	9.19	-41.339
3	0.5303	0.2602	0.24	0.2414	0.4352	0.6554	-0.04	-0.0187	-0.2132	0.1805	-0.0551	0.1035	0.1638	-0.0833	-0.0027	0.2373	0.0185	0.0593	0.1829	0.0216	0.1923	-4.9854	0.9085	-32.2295
4	0.2885	-0.3215	-0.0375	0.3885	0.1408	0.1597	0.0134	0.1613	-0.3533	0.1455	-0.0918	0.0952	0.2442	0.0637	-0.0096	0.2053	-0.0196	0.0293	0.1896	-0.0493	0.2524	-2.1099	-3.2758	-40.2576
5	1.306	0.8422	-1.0665	0.5974	-0.7016	2.0152	0.0003	0.0825	-0.6453	-0.0959	-0.365	0.3713	0.3717	-0.0629	-0.5237	0.2875	0.0075	-0.0142	0.2557	-0.1245	0.2974	-6.0349	-8.0366	-47.0344
6	0.141	0.1549	-0.0722	0.3372	-0.3293	0.4124	0.2519	0.1331	0.2855	-0.7038	-0.3917	0.1021	-0.6167	-0.0534	-0.0171	0.4076	0.0664	0.0192	0.2547	-0.0332	0.2759	-1.9653	9.7866	-49.8379
7	0.125	-0.093	-0.1719	0.0863	0.0616	0.2822	-0.1409	0.0752	-0.1755	0.0932	0.0031	-0.1375	0.1866	0.1017	-0.0003	0.2168	0.0564	0.0127	0.2601	-0.022	0.2247	2.9909	-3.1209	-45.7485
8	0.5307	0.0034	0.0982	0.6358	-0.0506	0.6295	-0.1646	-0.1738	-0.1009	0.2596	-0.0627	0.1205	0.0245	-0.1689	-0.1624	0.1868	0.0163	0.0443	0.2133	-0.0331	0.195	-5.8183	1.2358	-31.8638
9	0.0811	-0.1873	0.0568	0.5093	-0.0159	0.1463	0.191	-0.6337	-0.0565	0.4893	-0.197	0.1774	-0.1728	-0.405	-0.0035	0.2553	-0.0588	0.0832	0.5221	-0.0405	0.211	-12.0428	1.3812	-32.4721
10	0.4422	0.3384	0.0496	0.3619	0.2859	0.5054	-0.0881	0.0852	0.1881	-0.2592	0.2165	0.1239	-0.0065	-0.1548	0.0002	0.1728	0.0021	-0.0014	0.1815	0.006	0.2287	8.9897	-2.6338	-78.9833
11	0.5522	0.0573	-0.1838	0.4849	0.4858	0.5218	-0.0511	0.1667	0.0101	-0.2897	0.0511	0.0225	-0.0016	0.1212	-0.0022	0.2076	-0.0305	-0.0148	0.13	-0.0011	0.1634	5.5758	-5.893	-88.5586
12	0.5275	0.1908	-0.0959	1.1395	0.2851	1.5862	-0.0249	-0.0824	0.098	-0.0425	0.0636	0.0857	-0.1131	-0.0176	-0.0002	0.1248	0.0111	-0.0158	0.125	-0.0253	0.1469	6.1601	1.4367	-72.0547
13	0.6391	0.2961	0.2812	0.1593	0.2247	0.5738	0.0597	-0.4499	-0.0412	0.0935	-0.0628	-0.339	0.0376	0.2892	-0.0242	0.1968	-0.0066	-0.003	0.2665	-0.0711	0.2358	12.8367	2.0336	-61.7703
14	0.5971	-0.125	0.0739	0.5389	0.5687	0.5558	-0.1094	-0.019	0.0662	-0.2142	0.0878	0.0981	-0.1518	-0.0289	0.001	0.1825	-0.0284	-0.0176	0.1473	-0.0322	0.1707	6.8508	0.0775	-78.2572
15	0.1683	0.2278	-0.0516	0.4297	0.0598	0.1156	0.1075	0.3308	-0.3548	-0.4	-0.038	0.2984	0.0063	-0.0889	-0.0411	0.2219	0.043	-0.1036	0.3005	-0.0843	0.2408	0.5747	0.0714	-77.5278

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 6 through 47 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 48 through 62 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 63 through 85 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 86 through 102 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 103 through 119 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 120 through 130 )
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 131 through 141 )
8	X-RAY DIFFRACTION	8	chain 'A' and (resid 142 through 161 )
9	X-RAY DIFFRACTION	9	chain 'A' and (resid 162 through 166 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 3 through 18 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 19 through 47 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 48 through 113 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 114 through 141 )
14	X-RAY DIFFRACTION	14	chain 'B' and (resid 142 through 161 )
15	X-RAY DIFFRACTION	15	chain 'B' and (resid 162 through 166 )

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