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- PDB-4mat: E.COLI METHIONINE AMINOPEPTIDASE HIS79ALA MUTANT -

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Basic information

Entry
Database: PDB / ID: 4mat
TitleE.COLI METHIONINE AMINOPEPTIDASE HIS79ALA MUTANT
ComponentsPROTEIN (METHIONINE AMINOPEPTIDASE)
KeywordsHYDROLASE / HYDROLASE(ALPHA-AMINOACYLPEPTIDE) / SITE-DIRECTED MUTANT
Function / homology
Function and homology information


: / methionyl aminopeptidase / initiator methionyl aminopeptidase activity / metalloaminopeptidase activity / ferrous iron binding / proteolysis / metal ion binding / cytosol
Similarity search - Function
Methionine aminopeptidase subfamily 1 signature. / Peptidase M24A, methionine aminopeptidase, subfamily 1 / Peptidase M24, methionine aminopeptidase / Creatine Amidinohydrolase / Creatinase/methionine aminopeptidase superfamily / Peptidase M24 / Metallopeptidase family M24 / Creatinase/aminopeptidase-like / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Methionine aminopeptidase / Methionine aminopeptidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsLowther, W.T. / Orville, A.M. / Madden, D.T. / Lim, S. / Rich, D.H. / Matthews, B.W.
Citation
Journal: Biochemistry / Year: 1999
Title: Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis.
Authors: Lowther, W.T. / Orville, A.M. / Madden, D.T. / Lim, S. / Rich, D.H. / Matthews, B.W.
#1: Journal: Proc.Natl.Acad.Sci.USA / Year: 1998
Title: The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase.
Authors: Lowther, W.T. / McMillen, D.A. / Orville, A.M. / Matthews, B.W.
#2: Journal: Biochemistry / Year: 1993
Title: Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme.
Authors: Roderick, S.L. / Matthews, B.W.
History
DepositionMar 29, 1999Deposition site: BNL / Processing site: RCSB
Revision 1.0Jun 18, 1999Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2007Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Mar 14, 2018Group: Database references / Category: struct_ref_seq_dif / Item: _struct_ref_seq_dif.details
Revision 1.4Nov 20, 2019Group: Database references / Category: citation / citation_author / struct_ref_seq_dif
Item: _citation.page_last / _citation.pdbx_database_id_DOI ..._citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _struct_ref_seq_dif.details
Revision 1.5Sep 20, 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_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

Downloads & links

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Assembly

Deposited unit
A: PROTEIN (METHIONINE AMINOPEPTIDASE)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0472
Polymers31,0241
Non-polymers231
Water2,792155
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)38.648, 68.298, 55.449
Angle α, β, γ (deg.)90.00, 105.98, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein PROTEIN (METHIONINE AMINOPEPTIDASE)


Mass: 31023.641 Da / Num. of mol.: 1 / Mutation: R175Q, H79A
Source method: isolated from a genetically manipulated source
Details: SITE-DIRECTED MUTANT / Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: PET28B / Cell line (production host): BL21(DE3) / Production host: Escherichia coli (E. coli)
References: UniProt: P07906, UniProt: P0AE18*PLUS, methionyl aminopeptidase
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 155 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsPOLY-HISTIDINE TAGGED VARIANT THIS PROTEIN WAS NOT TREATED WITH THROMBIN AND THEREFORE CONTAINED ...POLY-HISTIDINE TAGGED VARIANT THIS PROTEIN WAS NOT TREATED WITH THROMBIN AND THEREFORE CONTAINED ADDITIONAL RESIDUES (LEU VAL PRO ARG GLY SER LEU GLU HIS HIS HIS HIS HIS HIS)

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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: 2.38 Å3/Da / Density % sol: 47.9 %
Crystal growpH: 7
Details: Crystals of the HIS79ALA mutant were obtained by mixing the apoenzyme retaining the C-terminal HISs-tag (6.7 mg/ml, 20 mM DTT, 25 mM HEPES pH 6.8, 25 mM K2SO4, 100 mM NaCl) with an equal ...Details: Crystals of the HIS79ALA mutant were obtained by mixing the apoenzyme retaining the C-terminal HISs-tag (6.7 mg/ml, 20 mM DTT, 25 mM HEPES pH 6.8, 25 mM K2SO4, 100 mM NaCl) with an equal volume of well solution (22-27 % PEG 3400, 0.1 M HEPES pH 7.0, 200 mM NaCl). Diffraction quality crystals were obtained after macroseeding into 20 uL hanging drops.
Crystal grow
*PLUS
Method: vapor diffusion, hanging drop / Details: macroseeding
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
16.7 mg/mlprotein1drop
220 mMdithiothreitol1drop
322-27 %PEG34001reservoir
40.1 MHEPES1reservoir
5200 mM1reservoirNaCl

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Data collection

DiffractionMean temperature: 103 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Oct 10, 1997
RadiationMonochromator: NI FILTER / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2→28.8 Å / Num. obs: 16062 / % possible obs: 85.2 % / Redundancy: 3 % / Biso Wilson estimate: 16.2 Å2 / Rsym value: 4.7 / Net I/σ(I): 22.4
Reflection shellResolution: 2→2.07 Å / Mean I/σ(I) obs: 4.4 / Rsym value: 21 / % possible all: 70.8
Reflection
*PLUS
Num. measured all: 48052 / Rmerge(I) obs: 0.047
Reflection shell
*PLUS
% possible obs: 70.8 % / Rmerge(I) obs: 0.21

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Processing

Software
NameVersionClassification
AMoREphasing
TNT5F PRERELEASErefinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2MAT

2mat


Resolution: 2→28.8 Å / Isotropic thermal model: TNT BCORREL V1.0 / σ(F): 0 / Stereochemistry target values: TNT PROTGEO
RfactorNum. reflection% reflection
all0.184 16062 -
obs-16062 100 %
Solvent computationSolvent model: TNT / Bsol: 207.2 Å2 / ksol: 0.915 e/Å3
Refinement stepCycle: LAST / Resolution: 2→28.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2052 0 1 155 2208
Refine LS restraints
Refine-IDTypeDev idealNumberWeight
X-RAY DIFFRACTIONt_bond_d0.01220870.8
X-RAY DIFFRACTIONt_angle_deg2.3828201.3
X-RAY DIFFRACTIONt_dihedral_angle_d17.112640
X-RAY DIFFRACTIONt_incorr_chiral_ct0
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes0.008522
X-RAY DIFFRACTIONt_gen_planes0.0123045
X-RAY DIFFRACTIONt_it4.920871
X-RAY DIFFRACTIONt_nbd0.0166410
Software
*PLUS
Name: TNT / Version: 5F / Classification: refinement
Refine LS restraints
*PLUS
Refine-IDTypeDev idealWeight
X-RAY DIFFRACTIONt_dihedral_angle_d
X-RAY DIFFRACTIONt_dihedral_angle_deg17.10
X-RAY DIFFRACTIONt_plane_restr0.0125

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