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- PDB-5ne9: Crystal structure of H60A H307A mutant of Thermotoga maritima TmP... -

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Basic information

Entry
Database: PDB / ID: 5ne9
TitleCrystal structure of H60A H307A mutant of Thermotoga maritima TmPEP1050 aminopeptidase
ComponentsAMINOPEPTIDASE
KeywordsLYASE / leucylaminopeptidase / M42 family / tetrahedral structure
Function / homology
Function and homology information


aminopeptidase activity / proteolysis / metal ion binding
Similarity search - Function
Peptidase M42, domain 2 / Peptidase M42, domain 2 / : / M42 glutamyl aminopeptidase / Peptidase M42 / Zn peptidases / Elongation Factor Tu (Ef-tu); domain 3 / Aminopeptidase / Beta Barrel / 3-Layer(aba) Sandwich ...Peptidase M42, domain 2 / Peptidase M42, domain 2 / : / M42 glutamyl aminopeptidase / Peptidase M42 / Zn peptidases / Elongation Factor Tu (Ef-tu); domain 3 / Aminopeptidase / Beta Barrel / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Biological speciesThermotoga maritima MSB8 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.374 Å
AuthorsDutoit, R.
Funding support Belgium, 1items
OrganizationGrant numberCountry
BAG-Belgium20151139 Belgium
Citation
Journal: J Vis Exp / Year: 2020
Title: X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050.
Authors: Dutoit, R. / Brandt, N. / Van Elder, D. / Droogmans, L.
#1: Journal: PLoS ONE / Year: 2012
Title: Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.
Authors: Dutoit, R. / Brandt, N. / Legrain, C. / Bauvois, C.
History
DepositionMar 10, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 16, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2019Group: Data collection / Category: reflns_shell / Item: _reflns_shell.percent_possible_all
Revision 1.2Feb 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Jan 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

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Assembly

Deposited unit
A: AMINOPEPTIDASE
B: AMINOPEPTIDASE


Theoretical massNumber of molelcules
Total (without water)71,9522
Polymers71,9522
Non-polymers00
Water1,72996
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Size exclusion chromatography was performed with a Superdex75 column (GE Healthcare). The apparent molecular weight is 67.6 kDa
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1040 Å2
ΔGint-6 kcal/mol
Surface area27390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)43.240, 137.790, 61.110
Angle α, β, γ (deg.)90.000, 110.690, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein AMINOPEPTIDASE / Endoglucanase M


Mass: 35976.160 Da / Num. of mol.: 2 / Mutation: H60A H307A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima MSB8 (bacteria) / Gene: TM_1050, Tmari_1054 / Plasmid: pBAD-TOPO / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: Q9X0E0, leucyl aminopeptidase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 96 / 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: 2.37 Å3/Da / Density % sol: 48.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.2
Details: TmPep1050 H60A H307A (320 uM in 50 mM MOPS 0.5 M ammonium sulfate pH 7.2) was crystallised in 0.1M sodium citrate 20% PEG3350 pH 5.2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.9801 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Nov 25, 2016
RadiationMonochromator: Si (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9801 Å / Relative weight: 1
Reflection twinOperator: h,-k,-h-l / Fraction: 0.35
ReflectionResolution: 2.37→43.995 Å / Num. obs: 26900 / % possible obs: 99.4 % / Observed criterion σ(I): -3 / Redundancy: 6.815 % / CC1/2: 0.992 / Rmerge(I) obs: 0.135 / Rrim(I) all: 0.146 / Χ2: 0.944 / Net I/σ(I): 8.75
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
2.37-2.446.3870.5452.4119230.8140.59396
2.44-2.56.9230.4952.9319410.8570.53599.9
2.5-2.586.6070.4263.4818720.8810.46299.8
2.58-2.656.6180.3434.418680.9340.37399.9
2.65-2.746.9070.3115.2317500.9510.33699.8
2.74-2.846.6450.2476.2517410.9570.26999.7
2.84-2.957.3520.237.4116370.9680.24799.8
2.95-3.077.3270.1988.9116280.9730.214100
3.07-3.27.2880.17310.1515140.9840.18799.7
3.2-3.367.1170.15911.314570.980.17299.8
3.36-3.546.9330.15612.0213870.9880.16899.8
3.54-3.756.7770.14912.8313280.9810.16199.6
3.75-4.016.5810.13413.412330.9760.14699.4
4.01-4.346.5060.12313.9611660.9830.13499.1
4.34-4.756.030.11813.8710580.9780.13100
4.75-5.316.3040.11314.229520.9830.12399.7
5.31-6.136.5860.11514.48690.9850.12599.1
6.13-7.517.3720.115.556980.9880.10899.6
7.51-10.627.2610.09815.875680.9940.105100
10.62-43.9956.9290.08315.553100.9850.09197.5

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation5.94 Å43.99 Å
Translation5.94 Å43.99 Å

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Processing

Software
NameVersionClassification
XSCALE20160617data scaling
PHASER2.6.0phasing
PHENIX1.10.1-2155refinement
PDB_EXTRACT3.22data extraction
XDS20160617data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4P6Y

4p6y


Resolution: 2.374→43.995 Å / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 39.17
RfactorNum. reflection% reflection
Rfree0.2343 1350 5.02 %
Rwork0.2056 --
obs0.2086 26900 99.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 185.25 Å2 / Biso mean: 57.2936 Å2 / Biso min: 16.41 Å2
Refinement stepCycle: final / Resolution: 2.374→43.995 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4545 0 0 96 4641
Biso mean---51.98 -
Num. residues----616
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0054623
X-RAY DIFFRACTIONf_angle_d0.7386264
X-RAY DIFFRACTIONf_chiral_restr0.049742
X-RAY DIFFRACTIONf_plane_restr0.004815
X-RAY DIFFRACTIONf_dihedral_angle_d13.9682797
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.3763-2.46120.3521340.31452530266494
2.4612-2.55970.30611340.29472559269395
2.5597-2.67620.29811340.29012538267295
2.6762-2.81720.31651330.27962525265895
2.8172-2.99370.2551340.26522556269095
2.9937-3.22470.27421360.2482587272395
3.2247-3.54910.26511340.2312537267195
3.5491-4.06220.23441340.19972551268595
4.0622-5.11640.18281350.1472567270295
5.1164-40.45710.1951370.16662586272395

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