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- PDB-7k09: Puromycin N-acetyltransferase in complex with acetyl-CoA -

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Basic information

Entry
Database: PDB / ID: 7k09
TitlePuromycin N-acetyltransferase in complex with acetyl-CoA
ComponentsPuromycin N-acetyltransferase
KeywordsTRANSFERASE / Enzyme acetyletransferase complex selection marker / ANTIBIOTIC
Function / homologyTransferases; Acyltransferases / Acetyltransferase (GNAT) family / acetyltransferase activity / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / Acyl-CoA N-acyltransferase / response to antibiotic / ACETYL COENZYME *A / Puromycin N-acetyltransferase
Function and homology information
Biological speciesStreptomyces alboniger (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.313 Å
AuthorsCaputo, A.T. / Newman, J. / Adams, T.E. / Peat, T.S.
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 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
C: Puromycin N-acetyltransferase
D: Puromycin N-acetyltransferase
E: Puromycin N-acetyltransferase
F: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,34612
Polymers135,4896
Non-polymers4,8576
Water43224
1
A: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
5
E: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
6
F: Puromycin N-acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,3912
Polymers22,5811
Non-polymers8101
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)149.074, 73.63, 106.242
Angle α, β, γ (deg.)90, 90, 90
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein
Puromycin N-acetyltransferase


Mass: 22581.490 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces alboniger (bacteria) / Gene: pac / Plasmid: pET43 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P13249, Transferases; Acyltransferases
#2: Chemical
ChemComp-ACO / ACETYL COENZYME *A / Acetyl-CoA


Mass: 809.571 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C23H38N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O
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: 2.15 Å3/Da / Density % sol: 42.84 % / Description: small 20 um chunks
Crystal growTemperature: 281 K / Method: vapor diffusion, sitting drop / pH: 9
Details: 2.5 M ammonium sulfate, 10% v/v DL-malate-MES-Tris pH 9.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95372 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 19, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95372 Å / Relative weight: 1
ReflectionResolution: 2.361→106.242 Å / Num. obs: 27792 / % possible obs: 93.1 % / Redundancy: 16.9 % / CC1/2: 0.994 / Rmerge(I) obs: 0.502 / Rpim(I) all: 0.125 / Rrim(I) all: 0.517 / Net I/σ(I): 7.7
Reflection shellResolution: 2.361→2.589 Å / Redundancy: 15.8 % / Mean I/σ(I) obs: 1.5 / Num. unique obs: 1192 / CC1/2: 0.504 / Rpim(I) all: 0.795 / % possible all: 67

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSdata reduction
Aimlessdata scaling
MR-Rosettaphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2QEC

2qec


Resolution: 2.313→106.24 Å / Cor.coef. Fo:Fc: 0.882 / Cor.coef. Fo:Fc free: 0.867 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.4
RfactorNum. reflection% reflectionSelection details
Rfree0.2523 1346 -RANDOM
Rwork0.2363 ---
obs0.2371 27849 53.7 %-
Displacement parametersBiso mean: 45.44 Å2
Baniso -1Baniso -2Baniso -3
1-0.6727 Å20 Å20 Å2
2---2.5916 Å20 Å2
3---1.919 Å2
Refine analyzeLuzzati coordinate error obs: 0.46 Å
Refinement stepCycle: LAST / Resolution: 2.313→106.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8829 0 306 24 9159
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0118355HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.0633262HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d5401SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes2956HARMONIC5
X-RAY DIFFRACTIONt_it9369HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion1219SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact12558SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.2
X-RAY DIFFRACTIONt_other_torsion16.81
LS refinement shellResolution: 2.361→2.5 Å
RfactorNum. reflection% reflection
Rfree0.328 29 -
Rwork0.2346 --
obs--5.24 %
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDL11 (°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)
10.6569-0.3143-0.12050-0.5910.36250.0010.00460.00590.0046-0.00490.00460.00590.00460.004-0.00810.0022-0.01720.01080.019-0.010515.525218.940834.4616
20.22920.2049-0.039800.04880.2782-0.0005-0.00840.0066-0.0084-0.000500.006600.001-0.0072-0.02160.00210.0041-0.019-0.000314.543217.251970.4481
30.2635-0.07850.06240.1495-0.40730.0652-0.00070.00670.01280.00670.00650.0010.01280.001-0.0058-0.0020.0006-0.0091-0.01480.00810.018513.265217.4322-0.1655
40.3973-0.16650.20420.05260.13910.5485-0.00110.00140.00330.00140.00070.00330.00330.00330.0004-0.01460.0191-0.01260.00830.0016-0.01150.474213.766838.6051
50.47940.25290.04740-0.16290.45590.00060.00430.00320.00430.00060.0080.00320.008-0.0013-0.0065-0.02260.00040.0109-0.0044-0.01624.0995-22.045531.5296
60.122-0.19830.01470.3927-0.16520-0.00050.0054-0.0130.00540.0007-0.0057-0.013-0.0057-0.00020.00990.0141-0.0221-0.01460.00720.009849.127812.62643.4332
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|* }
2X-RAY DIFFRACTION2{ B|* }
3X-RAY DIFFRACTION3{ C|* }
4X-RAY DIFFRACTION4{ D|* }
5X-RAY DIFFRACTION5{ E|* }
6X-RAY DIFFRACTION6{ F|* }

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