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- PDB-5mq5: A protease-resistant N24S Escherichia coli Asparaginase mutant wi... -

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Basic information

Entry
Database: PDB / ID: 5mq5
TitleA protease-resistant N24S Escherichia coli Asparaginase mutant with outstanding stability and enhanced anti-leukaemic activity
ComponentsL-asparaginase 2
KeywordsHYDROLASE / L-asparaginase / amidohydrolase / chemotherapeutic drug
Function / homology
Function and homology information


asparagine catabolic process / asparaginase / asparaginase activity / outer membrane-bounded periplasmic space / protein homotetramerization / periplasmic space / protein-containing complex / identical protein binding / cytosol
Similarity search - Function
L-asparaginase, N-terminal domain / Rossmann fold - #40 / L-asparaginase, type II / Asparaginase/glutaminase, active site 1 / Asparaginase / glutaminase active site signature 1. / L-asparaginase, C-terminal / Asparaginase/glutaminase, active site 2 / Asparaginase/glutaminase, C-terminal / Glutaminase/Asparaginase C-terminal domain / Asparaginase / glutaminase active site signature 2. ...L-asparaginase, N-terminal domain / Rossmann fold - #40 / L-asparaginase, type II / Asparaginase/glutaminase, active site 1 / Asparaginase / glutaminase active site signature 1. / L-asparaginase, C-terminal / Asparaginase/glutaminase, active site 2 / Asparaginase/glutaminase, C-terminal / Glutaminase/Asparaginase C-terminal domain / Asparaginase / glutaminase active site signature 2. / Asparaginase / Asparaginase/glutaminase-like / L-asparaginase, N-terminal / Asparaginase/glutaminase-like superfamily / L-asparaginase, N-terminal domain superfamily / Asparaginase, N-terminal / Asparaginase / glutaminase domain profile. / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
ASPARTIC ACID / L-asparaginase 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsMaggi, M. / Mittelman, S.D. / Parmentier, J.H. / Whitmire, J.M. / Merrell, D.S. / Scotti, C.
CitationJournal: Sci Rep / Year: 2017
Title: A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro.
Authors: Maggi, M. / Mittelman, S.D. / Parmentier, J.H. / Colombo, G. / Meli, M. / Whitmire, J.M. / Merrell, D.S. / Whitelegge, J. / Scotti, C.
History
DepositionDec 20, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 15, 2017Provider: repository / Type: Initial release
Revision 2.0Oct 16, 2019Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Other / Refinement description / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / cell / computing / diffrn / entity / pdbx_distant_solvent_atoms / pdbx_entry_details / pdbx_nonpoly_scheme / pdbx_refine_tls / pdbx_refine_tls_group / pdbx_struct_special_symmetry / pdbx_validate_close_contact / pdbx_validate_symm_contact / refine / refine_hist / refine_ls_restr / refine_ls_shell / reflns / reflns_shell / software / struct_site / struct_site_gen / symmetry
Item: _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] ..._atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][3] / _cell.volume / _diffrn.pdbx_serial_crystal_experiment / _entity.pdbx_number_of_molecules / _entity.src_method / _pdbx_distant_solvent_atoms.auth_seq_id / _pdbx_struct_special_symmetry.auth_seq_id / _refine.B_iso_mean / _refine.ls_R_factor_R_free / _refine.ls_R_factor_R_work / _refine.ls_R_factor_obs / _refine.ls_d_res_low / _refine.ls_number_reflns_R_free / _refine.ls_percent_reflns_R_free / _refine.ls_percent_reflns_obs / _refine.overall_SU_ML / _refine.pdbx_ls_cross_valid_method / _refine.pdbx_ls_sigma_F / _refine.pdbx_overall_phase_error / _refine.pdbx_solvent_shrinkage_radii / _refine.pdbx_solvent_vdw_probe_radii / _refine_hist.d_res_low / _refine_hist.number_atoms_solvent / _refine_hist.number_atoms_total / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _refine_ls_restr.type / _reflns.d_resolution_high / _reflns.d_resolution_low / _reflns.number_obs / _reflns.observed_criterion_sigma_F / _reflns.pdbx_Rmerge_I_obs / _reflns.pdbx_Rpim_I_all / _reflns.pdbx_Rrim_I_all / _reflns.pdbx_netI_over_av_sigmaI / _reflns.pdbx_netI_over_sigmaI / _reflns.pdbx_redundancy / _reflns.percent_possible_obs / _reflns_shell.Rmerge_I_obs / _reflns_shell.d_res_low / _reflns_shell.meanI_over_sigI_obs / _reflns_shell.number_unique_obs / _reflns_shell.pdbx_CC_half / _reflns_shell.pdbx_Rpim_I_all / _reflns_shell.pdbx_Rrim_I_all / _reflns_shell.pdbx_redundancy / _reflns_shell.percent_possible_all / _software.version / _symmetry.space_group_name_Hall
Description: Atoms with unrealistic or zero occupancies / Provider: author / Type: Coordinate replacement
Revision 2.1Jan 17, 2024Group: 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: L-asparaginase 2
C: L-asparaginase 2
B: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,2478
Polymers141,7154
Non-polymers5324
Water22,0681225
1
A: L-asparaginase 2
C: L-asparaginase 2
hetero molecules

A: L-asparaginase 2
C: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,2478
Polymers141,7154
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_757-x+2,y,-z+21
Buried area15740 Å2
ΔGint-50 kcal/mol
Surface area40130 Å2
MethodPISA
2
B: L-asparaginase 2
D: L-asparaginase 2
hetero molecules

B: L-asparaginase 2
D: L-asparaginase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,2478
Polymers141,7154
Non-polymers5324
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_858-x+3,y,-z+31
Buried area15520 Å2
ΔGint-53 kcal/mol
Surface area40470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)152.003, 62.394, 142.271
Angle α, β, γ (deg.)90.000, 117.920, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Components on special symmetry positions
IDModelComponents
11A-699-

HOH

21C-1204-

HOH

31B-667-

HOH

41D-675-

HOH

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Components

#1: Protein
L-asparaginase 2 / L-asparaginase II / L-ASNase II / L-asparagine amidohydrolase II


Mass: 35428.660 Da / Num. of mol.: 4 / Mutation: N24S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ansB, b2957, JW2924 / Plasmid: pET101 Directional TOPO / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): DELTAansA-/DELTAansB- / References: UniProt: P00805, asparaginase
#2: Chemical
ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H7NO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1225 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.16 Å3/Da / Density % sol: 43.12 % / Description: Long, prismatic crystals.
Crystal growTemperature: 294.15 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 100 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 5% w/v PEG 8000, 4% v/v ethylene glycol. Soaking in 0.1 mM L-asparatate.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.976251 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 26, 2016
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976251 Å / Relative weight: 1
ReflectionResolution: 1.427→48.16 Å / Num. obs: 195240 / % possible obs: 90 % / Redundancy: 2.6 % / CC1/2: 0.998 / Rmerge(I) obs: 0.061 / Rpim(I) all: 0.044 / Rrim(I) all: 0.076 / Net I/σ(I): 8.6
Reflection shellResolution: 1.43→1.48 Å / Redundancy: 1.9 % / Rmerge(I) obs: 1.284 / Num. unique obs: 11874 / CC1/2: 0.233 / Rpim(I) all: 1.284 / Rrim(I) all: 1.816 / % possible all: 55.9

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Processing

Software
NameVersionClassification
PHENIXrefinement
iMOSFLM7.2.1data reduction
SCALACCP4i v7data scaling
PHASERCCP4i v7phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3ECA

3eca


Resolution: 1.6→12.97 Å / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflection
Rfree0.1979 --
Rwork0.1473 --
obs-150249 96.9 %
Displacement parametersBiso mean: 27.29 Å2
Refinement stepCycle: LAST / Resolution: 1.6→12.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9574 0 36 1225 10835
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00759752
X-RAY DIFFRACTIONf_angle_d0.794213282
X-RAY DIFFRACTIONf_chiral_restr0.05331596
X-RAY DIFFRACTIONf_plane_restr0.00611738
X-RAY DIFFRACTIONf_dihedral_angle_d11.51345878

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