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- PDB-5tpq: E. coli alkaline phosphatase D101A, D153A, R166S, E322A, K328A mutant -

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Basic information

Entry
Database: PDB / ID: 5tpq
TitleE. coli alkaline phosphatase D101A, D153A, R166S, E322A, K328A mutant
ComponentsAlkaline phosphatase
KeywordsHYDROLASE / Alkaline Phosphatase / generalist enzyme / bimetallo motif / catalytic promiscuity / evolution / D101A/D153A/R166S/E322A/K328A
Function / homology
Function and homology information


oxidoreductase activity, acting on phosphorus or arsenic in donors / alkaline phosphatase / alkaline phosphatase activity / hydrogenase (acceptor) activity / phosphoprotein phosphatase activity / dephosphorylation / protein dephosphorylation / outer membrane-bounded periplasmic space / periplasmic space / magnesium ion binding / zinc ion binding
Similarity search - Function
Alkaline phosphatase, active site / Alkaline phosphatase active site. / Alkaline phosphatase / Alkaline phosphatase / Alkaline phosphatase homologues / Alkaline-phosphatase-like, core domain superfamily
Similarity search - Domain/homology
PHOSPHATE ION / Alkaline phosphatase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.45 Å
AuthorsSunden, F. / AlSadhan, I. / Lyubimov, A.Y. / Doukov, T. / Swan, J. / Herschlag, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM64798 United States
CitationJournal: J. Biol. Chem. / Year: 2017
Title: Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.
Authors: Sunden, F. / AlSadhan, I. / Lyubimov, A. / Doukov, T. / Swan, J. / Herschlag, D.
History
DepositionOct 20, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 1, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jan 3, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.3Apr 18, 2018Group: Data collection / Category: diffrn_detector / Item: _diffrn_detector.detector
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr1_symmetry / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alkaline phosphatase
B: Alkaline phosphatase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,76911
Polymers93,1212
Non-polymers6489
Water7,404411
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8570 Å2
ΔGint-238 kcal/mol
Surface area27670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)160.720, 160.720, 138.360
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number182
Space group name H-MP6322

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Components

#1: Protein Alkaline phosphatase / APase


Mass: 46560.742 Da / Num. of mol.: 2 / Fragment: UNP residues 30-471 / Mutation: D101A, D153A, R166S, E322A, K328A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: phoA, b0383, JW0374 / Production host: Escherichia coli (E. coli) / Strain (production host): SM547lambdaDE3 / References: UniProt: P00634, alkaline phosphatase
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 411 / 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: 2.79 Å3/Da / Density % sol: 55.9 % / Description: Small cube
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 5 mg/mL protein in 10 mM MOPS pH 7.0, 50 mM NaCl, 100 mM ZnCl2. Protein solution mixed with equal volume of precipitant solution: 23% PEG 3350, 0.2 M NH3F, 0.2 M HEPES pH=8.0
Temp details: Room temperature

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Oxford Cryo
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 13, 2016
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.45→38.6 Å / Num. obs: 39204 / % possible obs: 99.5 % / Redundancy: 35.5 % / Biso Wilson estimate: 50.3 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.594 / Net I/σ(I): 7.8
Reflection shellResolution: 2.45→2.54 Å / Redundancy: 30.4 % / Rmerge(I) obs: 5.6 / Mean I/σ(I) obs: 1.2 / CC1/2: 0.318 / % possible all: 95.6

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Processing

Software
NameVersionClassification
BUSTER2.10.2refinement
XDSdata reduction
Aimless0.5.25data scaling
PHASER2.6.1phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3TG0

3tg0


Resolution: 2.45→38.6 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.927 / Rfactor Rfree error: 0.03 / SU R Cruickshank DPI: 0.322 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.375 / SU Rfree Blow DPI: 0.232 / SU Rfree Cruickshank DPI: 0.227
RfactorNum. reflection% reflectionSelection details
Rfree0.219 1955 4.99 %RANDOM
Rwork0.165 ---
obs0.167 39204 100 %-
Displacement parametersBiso mean: 55.92 Å2
Baniso -1Baniso -2Baniso -3
1--5.1165 Å20 Å20 Å2
2---5.1165 Å20 Å2
3---10.233 Å2
Refine analyzeLuzzati coordinate error obs: 0.28 Å
Refinement stepCycle: 1 / Resolution: 2.45→38.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6520 0 17 493 7030
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.016663HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.139054HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d3053SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes181HARMONIC2
X-RAY DIFFRACTIONt_gen_planes984HARMONIC5
X-RAY DIFFRACTIONt_it6663HARMONIC20
X-RAY DIFFRACTIONt_nbd6SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion3.2
X-RAY DIFFRACTIONt_other_torsion2.98
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion912SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact8250SEMIHARMONIC4
LS refinement shellResolution: 2.45→2.51 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.27 153 5.35 %
Rwork0.228 2706 -
all0.23 2859 -
obs--99.86 %
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)
10.56440.27940.06910.8538-0.13621.78310.16140.036-0.146-0.1926-0.0714-0.03730.38640.0803-0.090.18570.0673-0.0572-0.13580.0445-0.2268-62.23340.0506-24.3533
20.46290.36720.12221.2485-0.31171.43090.1607-0.14270.07730.1861-0.1415-0.071-0.28840.2271-0.01920.0927-0.09880.0475-0.01670.0297-0.2489-53.845531.1764-10.023
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|8 - A|505 }
2X-RAY DIFFRACTION2{ B|9 - B|504 }

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