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- PDB-5yqb: Crystal structure of E.coli aminopeptidase N in complex with Puromycin -

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Basic information

Entry
Database: PDB / ID: 5yqb
TitleCrystal structure of E.coli aminopeptidase N in complex with Puromycin
ComponentsAminopeptidase N
KeywordsHYDROLASE / Puromycin / M1 class Aminopeptidase
Function / homology
Function and homology information


alanyl aminopeptidase activity / membrane alanyl aminopeptidase / aminopeptidase activity / metallopeptidase activity / proteolysis / zinc ion binding / identical protein binding / plasma membrane
Similarity search - Function
Aminopeptidase N, middle-beta domain / Peptidase M1, alanyl aminopeptidase, C-terminal domain / Peptidase M1, alanyl aminopeptidase / Peptidase M1, alanyl aminopeptidase, C-terminal / Peptidase M1, alanyl aminopeptidase, Ig-like fold / Peptidase M1, alanyl aminopeptidase, C-terminal domain superfamily / Alanyl aminopeptidase, Ig-like domain superfamily / Domain of unknown function (DUF3458) Ig-like fold / Domain of unknown function (DUF3458_C) ARM repeats / Zincin-like fold ...Aminopeptidase N, middle-beta domain / Peptidase M1, alanyl aminopeptidase, C-terminal domain / Peptidase M1, alanyl aminopeptidase / Peptidase M1, alanyl aminopeptidase, C-terminal / Peptidase M1, alanyl aminopeptidase, Ig-like fold / Peptidase M1, alanyl aminopeptidase, C-terminal domain superfamily / Alanyl aminopeptidase, Ig-like domain superfamily / Domain of unknown function (DUF3458) Ig-like fold / Domain of unknown function (DUF3458_C) ARM repeats / Zincin-like fold / Zincin-like - #30 / Zincin-like / tricorn interacting facor f3 domain / Peptidase M1, alanine aminopeptidase/leukotriene A4 hydrolase / Peptidase M1, membrane alanine aminopeptidase / Aminopeptidase N-like , N-terminal domain / Peptidase family M1 domain / Peptidase M1 N-terminal domain / Aminopeptidase N-like , N-terminal domain superfamliy / Neutral Protease Domain 2 / Neutral Protease; domain 2 / Peptidase M4/M1, CTD superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Alpha Horseshoe / Immunoglobulin-like / Sandwich / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
O-methyl-L-tyrosine / Chem-GMC / Aminopeptidase N
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.56 Å
AuthorsMarapaka, A.K. / Ganji, R.J. / Reddi, R. / Addlagatta, A.
CitationJournal: Int.J.Biol.Macromol. / Year: 2020
Title: Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis
Authors: Reddi, R. / Ganji, R.J. / Marapaka, A.K. / Bala, S.C. / Yerra, N.V. / Haque, N. / Addlagatta, A.
History
DepositionNov 6, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 14, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Refinement description
Category: citation / citation_author ...citation / citation_author / database_2 / refine_hist
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine_hist.d_res_low
Revision 1.2Nov 22, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aminopeptidase N
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,9785
Polymers101,3311
Non-polymers6474
Water15,655869
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area100 Å2
ΔGint-0 kcal/mol
Surface area32990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)120.292, 120.292, 170.123
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number152
Space group name H-MP3121

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Aminopeptidase N / Alpha-aminoacylpeptide hydrolase


Mass: 101331.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: pepN, b0932, JW0915 / Production host: Escherichia coli (E. coli) / References: UniProt: P04825, membrane alanyl aminopeptidase

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Non-polymers , 5 types, 873 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-0A1 / O-methyl-L-tyrosine


Type: L-peptide linking / Mass: 195.215 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H13NO3
#4: Chemical ChemComp-GMC / (2R,3R,4S,5S)-4-AMINO-2-[6-(DIMETHYLAMINO)-9H-PURIN-9-YL]-5-(HYDROXYMETHYL)TETRAHYDRO-3-FURANOL / 6N-DIMETHYL-3'-DEOXY-AMINO-ADENOSINE


Mass: 294.310 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H18N6O3
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 869 / 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: 3.51 Å3/Da / Density % sol: 64.92 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 2.0M Sodium malonate / PH range: 7.2-7.6

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.54 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Dec 10, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.56→50 Å / Num. obs: 198652 / % possible obs: 98.6 % / Redundancy: 4.7 % / Biso Wilson estimate: 22.44 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.035 / Rpim(I) all: 0.016 / Rrim(I) all: 0.039 / Χ2: 1.054 / Net I/av σ(I): 16.63 / Net I/σ(I): 24
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.56-1.624.50.3374.37199860.9060.170.3811.019100
1.62-1.684.50.251199880.9480.1260.2831.074100
1.68-1.764.50.181199580.9730.090.2041.06399.9
1.76-1.854.50.133200490.9850.0650.151.06899.9
1.85-1.974.50.094199420.9920.0460.1061.08799.9
1.97-2.124.70.064200580.9960.0310.0721.08599.9
2.12-2.334.80.047199560.9980.0230.0531.06699.4
2.33-2.6750.039199150.9980.0180.0431.01898.5
2.67-3.365.20.031196300.9990.0140.0341.01896.7
3.36-505.20.023191700.9990.0110.0261.05292.2

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Phasing

PhasingMethod: molecular replacement
Phasing MRR rigid body: 0.414
Highest resolutionLowest resolution
Rotation34.73 Å1.87 Å

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Processing

Software
NameVersionClassification
REFMAC5.8.0135refinement
DENZO2.3.1data collection
SCALEPACK2.2.0data scaling
MOLREP11.4.05; 02.04.2016phasing
PDB_EXTRACT3.22data extraction
Coot0.8.2model building
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2HPO

2hpo


Resolution: 1.56→50 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.967 / SU B: 0.968 / SU ML: 0.035 / Cross valid method: THROUGHOUT / ESU R: 0.058 / ESU R Free: 0.06 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.18189 9844 5 %RANDOM
Rwork0.16136 ---
obs0.16239 187925 98.16 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 23.628 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å2-0 Å2
3----0 Å2
Refinement stepCycle: 1 / Resolution: 1.56→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6940 0 42 869 7851
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0290.0197254
X-RAY DIFFRACTIONr_bond_other_d0.0020.026816
X-RAY DIFFRACTIONr_angle_refined_deg2.61.9569879
X-RAY DIFFRACTIONr_angle_other_deg2.1123.00315660
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.0765903
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.05324.24375
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.239151224
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.4071555
X-RAY DIFFRACTIONr_chiral_restr0.170.21083
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.0218353
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021739
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_it1.8022.083510
X-RAY DIFFRACTIONr_mcbond_other1.8012.0813511
X-RAY DIFFRACTIONr_mcangle_it2.363.1144393
X-RAY DIFFRACTIONr_mcangle_other2.363.1154394
X-RAY DIFFRACTIONr_scbond_it3.8142.4343744
X-RAY DIFFRACTIONr_scbond_other3.8112.4343744
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.4743.5215469
X-RAY DIFFRACTIONr_long_range_B_refined6.21218.4669065
X-RAY DIFFRACTIONr_long_range_B_other6.09617.7178566
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.561→1.601 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.282 744 -
Rwork0.249 14032 -
obs--99.59 %

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