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

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Basic information

Entry
Database: PDB / ID: 7k0a
TitlePuromycin N-acetyltransferase in complex with acetylated puromycin and CoA
ComponentsPuromycin N-acetyltransferase
KeywordsTRANSFERASE / Enzyme acetyletransferase complex selection marker / ANTIBIOTIC
Function / homology
Function and homology information


Transferases; Acyltransferases / acetyltransferase activity / response to antibiotic
Similarity search - Function
Acetyltransferase (GNAT) family / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / Acyl-CoA N-acyltransferase
Similarity search - Domain/homology
COENZYME A / DI(HYDROXYETHYL)ETHER / N-Acetylpuromycin / Puromycin N-acetyltransferase
Similarity search - Component
Biological speciesStreptomyces alboniger (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsPeat, T.S. / Caputo, A.T. / Newman, J. / Adams, T.E.
CitationJournal: 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
DepositionSep 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 18, 2023Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Puromycin N-acetyltransferase
B: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,70017
Polymers45,1632
Non-polymers3,53715
Water4,918273
1
A: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,4599
Polymers22,5811
Non-polymers1,8788
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,2418
Polymers22,5811
Non-polymers1,6597
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
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 number5
Space group name H-MI121
Components on special symmetry positions
IDModelComponents
11A-441-

HOH

21A-527-

HOH

Noncrystallographic symmetry (NCS)NCS domain: (Details: Chains A B)

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Components

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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 / 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 / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H10O3
#5: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#7: Water ChemComp-HOH / water / 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 interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.79 Å3/Da / Density % sol: 55.94 %
Crystal growTemperature: 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
IDMean temperature (K)Crystal-IDSerial crystal experiment
11001N
21001N
31001N
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONAustralian Synchrotron MX210.9537
SYNCHROTRONAustralian Synchrotron MX220.9537
SYNCHROTRONAustralian Synchrotron MX230.9537
Detector
TypeIDDetectorDate
ADSC QUANTUM 315r1CCDMar 11, 2016
ADSC QUANTUM 315r2CCDMar 11, 2016
ADSC QUANTUM 315r3CCDApr 8, 2016
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2SINGLE WAVELENGTHMx-ray2
3SINGLE WAVELENGTHMx-ray3
Radiation wavelength
IDWavelength (Å)Relative weight
10.95371
21
31
ReflectionResolution: 2→70.75 Å / Num. obs: 33396 / % possible obs: 99.9 % / Redundancy: 17.2 % / CC1/2: 0.989 / Rmerge(I) obs: 0.376 / Rpim(I) all: 0.136 / Rrim(I) all: 0.4 / Χ2: 1.02 / Net I/σ(I): 7.7
Reflection shellResolution: 2→2.05 Å / Redundancy: 15.2 % / Mean I/σ(I) obs: 2.3 / Num. unique obs: 2460 / CC1/2: 0.857 / Rpim(I) all: 0.608 / % possible all: 99.4

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7K09

7k09


Resolution: 2→42.488 Å / Cor.coef. Fo:Fc: 0.92 / Cor.coef. Fo:Fc 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
RfactorNum. reflection% reflection
Rfree0.2405 1619 4.852 %
Rwork0.2071 31752 -
all0.209 --
obs-33371 99.97 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 16.23 Å2
Baniso -1Baniso -2Baniso -3
1--1.713 Å2-0 Å20.798 Å2
2---2.055 Å2-0 Å2
3---3.26 Å2
Refinement stepCycle: LAST / Resolution: 2→42.488 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2968 0 226 273 3467
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0133298
X-RAY DIFFRACTIONr_bond_other_d0.0020.0173040
X-RAY DIFFRACTIONr_angle_refined_deg1.6211.74521
X-RAY DIFFRACTIONr_angle_other_deg1.3311.5977008
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.5145396
X-RAY DIFFRACTIONr_dihedral_angle_2_deg28.42519.518166
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.77915460
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.6431533
X-RAY DIFFRACTIONr_chiral_restr0.0820.2425
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.023645
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02698
X-RAY DIFFRACTIONr_nbd_refined0.2130.2665
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1830.22839
X-RAY DIFFRACTIONr_nbtor_refined0.1620.21595
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0830.21496
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1920.2229
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.130.24
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1630.217
X-RAY DIFFRACTIONr_nbd_other0.1960.268
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1820.223
X-RAY DIFFRACTIONr_mcbond_it1.2681.4511581
X-RAY DIFFRACTIONr_mcbond_other1.2631.4511580
X-RAY DIFFRACTIONr_mcangle_it2.0232.1711978
X-RAY DIFFRACTIONr_mcangle_other2.0232.1711979
X-RAY DIFFRACTIONr_scbond_it2.2011.9321716
X-RAY DIFFRACTIONr_scbond_other2.0921.8811693
X-RAY DIFFRACTIONr_scangle_it3.3552.7842542
X-RAY DIFFRACTIONr_scangle_other3.2342.7082507
X-RAY DIFFRACTIONr_lrange_it6.20429.45414277
X-RAY DIFFRACTIONr_lrange_other6.10929.28114105
X-RAY DIFFRACTIONr_ncsr_local_group_10.0690.056254
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.0520.2751110.2582356X-RAY DIFFRACTION99.9595
2.052-2.1080.301970.2582303X-RAY DIFFRACTION99.9584
2.108-2.1690.2311140.2192216X-RAY DIFFRACTION100
2.169-2.2360.2821180.2572129X-RAY DIFFRACTION99.9111
2.236-2.3090.3181210.2892101X-RAY DIFFRACTION99.9101
2.309-2.390.242960.1822012X-RAY DIFFRACTION100
2.39-2.480.2221190.1791955X-RAY DIFFRACTION100
2.48-2.5810.2441140.1811839X-RAY DIFFRACTION100
2.581-2.6960.193800.1771816X-RAY DIFFRACTION100
2.696-2.8280.24810.1711739X-RAY DIFFRACTION100
2.828-2.980.226700.1791636X-RAY DIFFRACTION99.9414
2.98-3.1610.223610.1911556X-RAY DIFFRACTION100
3.161-3.3790.206760.1831476X-RAY DIFFRACTION100
3.379-3.6490.207770.1981359X-RAY DIFFRACTION100
3.649-3.9960.268630.21246X-RAY DIFFRACTION100
3.996-4.4670.204680.1821132X-RAY DIFFRACTION100
4.467-5.1550.206580.1891000X-RAY DIFFRACTION100
5.155-6.3070.291420.231843X-RAY DIFFRACTION100
6.307-8.8920.243290.233672X-RAY DIFFRACTION100
8.892-42.4880.267240.246366X-RAY DIFFRACTION99.4898

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