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- PDB-3tyk: Crystal structure of aminoglycoside phosphotransferase APH(4)-Ia -

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Basic information

Entry
Database: PDB / ID: 3tyk
TitleCrystal structure of aminoglycoside phosphotransferase APH(4)-Ia
ComponentsHygromycin-B 4-O-kinase
KeywordsTRANSFERASE/ANTIBIOTIC / Midwest Center for Structural Genomics / MCSG / PSI-2 / AMINOGLYCOSIDE PHOSPHOTRANSFERASE / EUKARYOTIC PROTEIN KINASE-LIKE FOLD / PHOSPHOTRANSFERASE/KINASE / ANTIBIOTIC RESISTANCE / AMINOGLYCOSIDE BINDING / PHOSPHORYLATION / TRANSFERASE-ANTIBIOTIC COMPLEX / cytoplasmic / Protein Structure Initiative
Function / homology
Function and homology information


hygromycin B 4-O-kinase / kinase activity / phosphorylation / response to antibiotic / ATP binding
Similarity search - Function
Phosphorylase Kinase; domain 1 - #150 / Aminoglycoside 3'-phosphotransferase; Chain: A, domain 2 / Aminoglycoside phosphotransferase (APH), C-terminal lobe / Aminoglycoside phosphotransferase / Phosphotransferase enzyme family / Phosphorylase Kinase; domain 1 / Protein kinase-like domain superfamily / Alpha-Beta Complex / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
HYGROMYCIN B VARIANT / Hygromycin-B 4-O-kinase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.95 Å
AuthorsStogios, P.J. / Shabalin, I.G. / Shakya, T. / Evdokmova, E. / Fan, Y. / Chruszcz, M. / Minor, W. / Wright, G.D. / Savchenko, A. / Anderson, W.F. / Midwest Center for Structural Genomics (MCSG)
CitationJournal: J.Biol.Chem. / Year: 2011
Title: Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
Authors: Stogios, P.J. / Shakya, T. / Evdokimova, E. / Savchenko, A. / Wright, G.D.
History
DepositionSep 26, 2011Deposition site: RCSB / Processing site: RCSB
SupersessionOct 12, 2011ID: 3OVC
Revision 1.0Oct 12, 2011Provider: repository / Type: Initial release
Revision 1.1Apr 25, 2012Group: Structure summary
Revision 1.2May 2, 2012Group: Other
Revision 1.3Nov 28, 2012Group: Structure summary
Revision 1.4Apr 13, 2022Group: Database references / Derived calculations / Structure summary
Category: audit_author / chem_comp ...audit_author / chem_comp / database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _audit_author.identifier_ORCID / _chem_comp.pdbx_synonyms ..._audit_author.identifier_ORCID / _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Hygromycin-B 4-O-kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,5033
Polymers40,9401
Non-polymers5632
Water5,116284
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)70.640, 70.640, 125.880
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221
Detailsbiological assembly is the single chain in the asymmetric unit

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Components

#1: Protein Hygromycin-B 4-O-kinase / APH(4) / Hygromycin B phosphotransferase / Hygromycin-B kinase


Mass: 40940.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: Plasmid pKC222 / Gene: APH(4), hph / Plasmid: P15TV-L / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00557, hygromycin B 4-O-kinase
#2: Chemical ChemComp-HY0 / HYGROMYCIN B VARIANT / (2R,3'R,3aS,4S,4'S,5'R,6R,6'R,7S,7aS)-4-[(1R,2S,3R,5S,6R)-3-azanyl-2,6-dihydroxy-5-(methylamino)cyclohexyl]oxy-6'-[(1S) -1-azanyl-2-hydroxy-ethyl]-6-(hydroxymethyl)spiro[4,6,7,7a-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-2,2'-oxane]-3',4',5', 7-tetrol


Mass: 527.520 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H37N3O13
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 284 / Source method: isolated from a natural source / Formula: H2O

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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: 2.36 Å3/Da / Density % sol: 47.82 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.2 M DI-AMMONIUM HYDROGEN CITRATE, 16% PEG3350, 2MM HYGROMYCIN B, pH 7, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 0.9791532 Å
DetectorType: ADSC QUANTUM 210r / Detector: CCD / Date: Oct 29, 2009 / Details: MIRRORS
RadiationMonochromator: SI 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791532 Å / Relative weight: 1
ReflectionResolution: 1.95→50 Å / Num. obs: 27293 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 2 / Redundancy: 10.3 % / Rsym value: 0.068 / Net I/σ(I): 52.18
Reflection shellResolution: 1.95→1.98 Å / Redundancy: 7.6 % / Mean I/σ(I) obs: 3.46 / Rsym value: 0.697 / % possible all: 100

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Processing

Software
NameVersionClassification
HKL-3000data collection
SOLVEphasing
REFMAC5.6.0117refinement
HKL-2000data reduction
HKL-2000data scaling
AutoSolphasing
RefinementMethod to determine structure: SAD / Resolution: 1.95→34.01 Å / Cor.coef. Fo:Fc: 0.968 / Cor.coef. Fo:Fc free: 0.955 / SU B: 5.55 / SU ML: 0.086 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.137 / ESU R Free: 0.134 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2027 1310 5 %RANDOM
Rwork0.15744 ---
obs0.1597 24723 95.78 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 38.095 Å2
Baniso -1Baniso -2Baniso -3
1-0.81 Å20.41 Å20 Å2
2--0.81 Å20 Å2
3----1.22 Å2
Refinement stepCycle: LAST / Resolution: 1.95→34.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2314 0 37 284 2635
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.022430
X-RAY DIFFRACTIONr_bond_other_d0.0010.021598
X-RAY DIFFRACTIONr_angle_refined_deg1.7181.9633317
X-RAY DIFFRACTIONr_angle_other_deg1.05433872
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1495299
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.07223.984123
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.95115340
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.5771517
X-RAY DIFFRACTIONr_chiral_restr0.1020.2365
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.0212749
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02532
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.95→2.001 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.248 88 -
Rwork0.183 1515 -
obs--86.7 %
Refinement TLS params.

Method: refined / 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)
13.0106-1.2746-0.38991.7693-0.22813.0757-0.1974-0.3694-0.14610.12830.06730.00980.1360.26080.13010.0572-0.020.01540.15140.07970.147633.599-43.85432.604
21.9387-0.431-0.34210.8503-0.07081.15670.02460.12120.0968-0.0326-0.0282-0.0408-0.14590.00670.00350.0263-0.0121-0.01780.11380.02010.073724.46-24.22920.35
34.3302-1.32640.39812.6769-0.3393.472-0.0681-0.03510.4499-0.02980.0544-0.2851-0.4140.09550.01380.0807-0.0324-0.01340.02320.01710.103228.905-13.57222.531
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 95
2X-RAY DIFFRACTION2A96 - 255
3X-RAY DIFFRACTION3A256 - 302

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