PDB-7k0a: Puromycin N-acetyltransferase in complex with acetylated puromyci...

ID or keywords:

Entry

Database: PDB / ID: 7k0a

Title

Puromycin N-acetyltransferase in complex with acetylated puromycin and CoA

Components

Puromycin N-acetyltransferase

Keywords

TRANSFERASE / Enzyme acetyletransferase complex selection marker / ANTIBIOTIC

Function / homology

Function and homology information

Transferases; Acyltransferases / acyltransferase activity, transferring groups other than amino-acyl groups / response to antibiotic
Similarity search - Function

Biological species

Streptomyces alboniger (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2 Å

Authors

Peat, T.S. / Caputo, A.T. / Newman, J. / Adams, T.E.

Citation

Journal: Sci Rep / Year: 2021
Title: Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.
Authors: Caputo, A.T. / Eder, O.M. / Bereznakova, H. / Pothuis, H. / Ardevol, A. / Newman, J. / Nuttall, S. / Peat, T.S. / Adams, T.E.

History

Deposition	Sep 3, 2020	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Mar 17, 2021	Provider: repository / Type: Initial release
Revision 1.1	Oct 18, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_radiation_wavelength / pdbx_initial_refinement_model Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_radiation_wavelength.wavelength

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	7k0a.cif.gz	106.7 KB	Display	PDBx/mmCIF format
PDB format	pdb7k0a.ent.gz	77 KB	Display	PDB format
PDBx/mmJSON format	7k0a.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	7k0a_validation.pdf.gz	1.6 MB	Display	wwPDB validaton report
Full document	7k0a_full_validation.pdf.gz	1.6 MB	Display
Data in XML	7k0a_validation.xml.gz	21.5 KB	Display
Data in CIF	7k0a_validation.cif.gz	29.8 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/k0/7k0a ftp://data.pdbj.org/pub/pdb/validation_reports/k0/7k0a	HTTPS FTP

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Related structure data

Related structure data	7k09SC S: Starting model for refinement C: citing same article (ref.)
Similar structure data	3gcd-2 7k09-1 6mro-d 5cs2-d 3u0d-3 1uhi-2 3gcd-1 7kpp-2 3i0a-1 4ory-6 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
Related items in Molecule of the Month	#146 - Feb 2012 Aminoglycoside Antibiotics similarity (5) #97 - Jan 2008 Circadian Clock Proteins similarity (4) #93 - Sep 2007 Citrate Synthase similarity (1) #154 - Oct 2012 Citric Acid Cycle similarity (1) #216 - Dec 2017 Biodegradable Plastic similarity (1) #39 - Mar 2003 lac Repressor similarity (1)

Deposited unit

A: Puromycin N-acetyltransferase

B: Puromycin N-acetyltransferase

hetero molecules

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

1

A: Puromycin N-acetyltransferase

hetero molecules

defined by author&software
Evidence: gel filtration, Elutes on Superdex 75 column at expect volume for a monomeric globular protein
24.5 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Puromycin N-acetyltransferase

hetero molecules

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

Unit cell

Length a, b, c (Å)	36.596, 141.509, 98.985
Angle α, β, γ (deg.)	90.000, 100.341, 90.000
Int Tables number	5
Space group name H-M	I121

Components on special symmetry positions

ID	Model	Components
1	1	A-441- HOH
2	1	A-527- HOH

Noncrystallographic symmetry (NCS)

NCS domain: (Details: Chains A B)

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Protein , 1 types, 2 molecules AB

#1: Protein	Puromycin N-acetyltransferase Mass: 22581.490 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptomyces alboniger (bacteria) / Gene: pac / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P13249, Transferases; Acyltransferases

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Non-polymers , 6 types, 288 molecules

#2: Chemical	ChemComp-VQ1 / N-Acetylpuromycin / 3'-[(N-acetyl-O-methyl-L-tyrosyl)amino]-3'-deoxy-N,N-dimethyladenosine Mass: 513.546 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₂₄H₃₁N₇O₆ / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical	ChemComp-COA / COENZYME A Mass: 767.534 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₂₁H₃₆N₇O₁₆P₃S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical	ChemComp-PEG / DI(HYDROXYETHYL)ETHER Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₄H₁₀O₃
#5: Chemical	ChemComp-SO4 / SULFATE ION Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO₄
#6: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C₂H₆O₂
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 273 / Source method: isolated from a natural source / Formula: H₂O

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Details

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: 2.79 Å³/Da / Density % sol: 55.94 %
Crystal grow	Temperature: 281 K / Method: vapor diffusion, sitting drop / pH: 8.26 Details: 9.9% (w/v) 1,6-hexanediol, 1.59 M ammonium sulfate, 7.4 % (v/v) polyethylene glycol 400, 0.1 M HEPES pH 8.26, 0.5 n-hexyl-b-D-glucopyranoside

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Data collection

Diffraction

ID	Mean temperature (K)	Crystal-ID	Serial crystal experiment
1	100	1	N
2	100	1	N
3	100	1	N

Diffraction source

Source	Site	Beamline	ID	Wavelength (Å)
SYNCHROTRON	Australian Synchrotron	MX2	1	0.9537
SYNCHROTRON	Australian Synchrotron	MX2	2	0.9537
SYNCHROTRON	Australian Synchrotron	MX2	3	0.9537

Detector

Type	ID	Detector	Date
ADSC QUANTUM 315r	1	CCD	Mar 11, 2016
ADSC QUANTUM 315r	2	CCD	Mar 11, 2016
ADSC QUANTUM 315r	3	CCD	Apr 8, 2016

Radiation

ID	Protocol	Monochromatic (M) / Laue (L)	Scattering type	Wavelength-ID
1	SINGLE WAVELENGTH	M	x-ray	1
2	SINGLE WAVELENGTH	M	x-ray	2
3	SINGLE WAVELENGTH	M	x-ray	3

Radiation wavelength

ID	Wavelength (Å)	Relative weight
1	0.9537	1
2		1
3		1

Reflection

Resolution: 2→70.75 Å / Num. obs: 33396 / % possible obs: 99.9 % / Redundancy: 17.2 % / CC_1/2: 0.989 / R_merge(I) obs: 0.376 / R_pim(I) all: 0.136 / R_rim(I) all: 0.4 / Χ²: 1.02 / Net I/σ(I): 7.7

Reflection shell

Resolution: 2→2.05 Å / Redundancy: 15.2 % / Mean I/σ(I) obs: 2.3 / Num. unique obs: 2460 / CC_1/2: 0.857 / R_pim(I) all: 0.608 / % possible all: 99.4

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0258	refinement
DIALS		data reduction
Aimless		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 7K09

7k09

Resolution: 2→42.488 Å / Cor.coef. F_o:F_c: 0.92 / Cor.coef. F_o:F_c free: 0.895 / SU B: 4.723 / SU ML: 0.126 / Cross valid method: FREE R-VALUE / ESU R: 0.187 / ESU R Free: 0.162
Details: Hydrogens have been added in their riding positions

	R_factor	Num. reflection	% reflection
R_free	0.2405	1619	4.852 %
R_work	0.2071	31752	-
all	0.209	-	-
obs	-	33371	99.97 %

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT

Displacement parameters

B_iso mean: 16.23 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-1.713 Å²	-0 Å²	0.798 Å²
2-	-	-2.055 Å²	-0 Å²
3-	-	-	3.26 Å²

Refinement step

Cycle: LAST / Resolution: 2→42.488 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	2968	0	226	273	3467

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.01	0.013	3298
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.017	3040
X-RAY DIFFRACTION	r_angle_refined_deg	1.621	1.7	4521
X-RAY DIFFRACTION	r_angle_other_deg	1.331	1.597	7008
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.514	5	396
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	28.425	19.518	166
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	13.779	15	460
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	15.643	15	33
X-RAY DIFFRACTION	r_chiral_restr	0.082	0.2	425
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	3645
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	698
X-RAY DIFFRACTION	r_nbd_refined	0.213	0.2	665
X-RAY DIFFRACTION	r_symmetry_nbd_other	0.183	0.2	2839
X-RAY DIFFRACTION	r_nbtor_refined	0.162	0.2	1595
X-RAY DIFFRACTION	r_symmetry_nbtor_other	0.083	0.2	1496
X-RAY DIFFRACTION	r_xyhbond_nbd_refined	0.192	0.2	229
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_other	0.13	0.2	4
X-RAY DIFFRACTION	r_symmetry_nbd_refined	0.163	0.2	17
X-RAY DIFFRACTION	r_nbd_other	0.196	0.2	68
X-RAY DIFFRACTION	r_symmetry_xyhbond_nbd_refined	0.182	0.2	23
X-RAY DIFFRACTION	r_mcbond_it	1.268	1.451	1581
X-RAY DIFFRACTION	r_mcbond_other	1.263	1.451	1580
X-RAY DIFFRACTION	r_mcangle_it	2.023	2.171	1978
X-RAY DIFFRACTION	r_mcangle_other	2.023	2.171	1979
X-RAY DIFFRACTION	r_scbond_it	2.201	1.932	1716
X-RAY DIFFRACTION	r_scbond_other	2.092	1.881	1693
X-RAY DIFFRACTION	r_scangle_it	3.355	2.784	2542
X-RAY DIFFRACTION	r_scangle_other	3.234	2.708	2507
X-RAY DIFFRACTION	r_lrange_it	6.204	29.454	14277
X-RAY DIFFRACTION	r_lrange_other	6.109	29.281	14105
X-RAY DIFFRACTION	r_ncsr_local_group_1	0.069	0.05	6254

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 (%)
2-2.052	0.275	111	0.258	2356	X-RAY DIFFRACTION	99.9595
2.052-2.108	0.301	97	0.258	2303	X-RAY DIFFRACTION	99.9584
2.108-2.169	0.231	114	0.219	2216	X-RAY DIFFRACTION	100
2.169-2.236	0.282	118	0.257	2129	X-RAY DIFFRACTION	99.9111
2.236-2.309	0.318	121	0.289	2101	X-RAY DIFFRACTION	99.9101
2.309-2.39	0.242	96	0.182	2012	X-RAY DIFFRACTION	100
2.39-2.48	0.222	119	0.179	1955	X-RAY DIFFRACTION	100
2.48-2.581	0.244	114	0.181	1839	X-RAY DIFFRACTION	100
2.581-2.696	0.193	80	0.177	1816	X-RAY DIFFRACTION	100
2.696-2.828	0.24	81	0.171	1739	X-RAY DIFFRACTION	100
2.828-2.98	0.226	70	0.179	1636	X-RAY DIFFRACTION	99.9414
2.98-3.161	0.223	61	0.191	1556	X-RAY DIFFRACTION	100
3.161-3.379	0.206	76	0.183	1476	X-RAY DIFFRACTION	100
3.379-3.649	0.207	77	0.198	1359	X-RAY DIFFRACTION	100
3.649-3.996	0.268	63	0.2	1246	X-RAY DIFFRACTION	100
3.996-4.467	0.204	68	0.182	1132	X-RAY DIFFRACTION	100
4.467-5.155	0.206	58	0.189	1000	X-RAY DIFFRACTION	100
5.155-6.307	0.291	42	0.231	843	X-RAY DIFFRACTION	100
6.307-8.892	0.243	29	0.233	672	X-RAY DIFFRACTION	100
8.892-42.488	0.267	24	0.246	366	X-RAY DIFFRACTION	99.4898

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