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- PDB-5kkg: Crystal structure of E72A mutant of ancestral protein ancMT of AD... -

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Basic information

Entry
Database: PDB / ID: 5kkg
TitleCrystal structure of E72A mutant of ancestral protein ancMT of ADP-dependent sugar kinases family
ComponentsancMT E72A
KeywordsDE NOVO PROTEIN / ancestral protein reconstruction / ADP-dependent sugar kinases family / glucokinase / phosphofructokinase / bifuncional enzymes / enzyme evolution
Function / homologyAdenosine kinase, small domain - #20 / Adenosine kinase, small domain / Ribokinase / UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta / ADENOSINE MONOPHOSPHATE / IODIDE ION
Function and homology information
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.608 Å
AuthorsCastro-Fernandez, V. / Herrera-Morande, A. / Zamora, R. / Merino, F. / Pereira, H.M. / Brandao-Neto, J. / Garratt, R. / Guixe, V.
Funding support Chile, 1items
OrganizationGrant numberCountry
FONDECYT1150460 Chile
CitationJournal: J. Biol. Chem. / Year: 2017
Title: Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.
Authors: Castro-Fernandez, V. / Herrera-Morande, A. / Zamora, R. / Merino, F. / Gonzalez-Ordenes, F. / Padilla-Salinas, F. / Pereira, H.M. / Brandao-Neto, J. / Garratt, R.C. / Guixe, V.
History
DepositionJun 21, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 4, 2018Group: Data collection / Database references / Category: citation / citation_author
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.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 27, 2023Group: 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: ancMT E72A
B: ancMT E72A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,65718
Polymers108,5102
Non-polymers2,14616
Water1,820101
1
A: ancMT E72A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,61810
Polymers54,2551
Non-polymers1,3629
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: ancMT E72A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,0398
Polymers54,2551
Non-polymers7847
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)132.529, 132.529, 117.438
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein ancMT E72A


Mass: 54255.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET-28a / Details (production host): TEV clivage site / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE / Adenosine monophosphate


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Comment: AMP*YM
#3: Chemical
ChemComp-IOD / IODIDE ION / Iodide


Mass: 126.904 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: I
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 101 / 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: 2.48 Å3/Da / Density % sol: 50.36 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.8
Details: 18 % (w/v) PEG 3350, 0.2 M NaI Protein 8 mg/mL, 20 mM F6P, 30 mM MgCl2, 25 mM HEPES pH 7.8, 20 mM AMP

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.92 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Apr 26, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
ReflectionResolution: 2.608→59.27 Å / Num. obs: 31763 / % possible obs: 97.7 % / Redundancy: 8.5 % / Biso Wilson estimate: 56.06 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.122 / Net I/σ(I): 11.8
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsDiffraction-ID% possible all
2.61-2.727.60.937197.4
9.03-59.278.20.065199.8

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Processing

Software
NameVersionClassification
Aimless0.5.15data scaling
PHENIX1.10.1_2155refinement
PDB_EXTRACT3.2data extraction
XDSOct 15, 2015data reduction
PHASER2.6.0phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5K27

5k27


Resolution: 2.608→59.269 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.4
RfactorNum. reflection% reflection
Rfree0.261 1574 4.97 %
Rwork0.2231 --
obs0.2249 31663 97.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 142.35 Å2 / Biso mean: 64.8791 Å2 / Biso min: 23.24 Å2
Refinement stepCycle: final / Resolution: 2.608→59.269 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6887 0 53 101 7041
Biso mean--68.82 56.19 -
Num. residues----902
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0027050
X-RAY DIFFRACTIONf_angle_d0.5449555
X-RAY DIFFRACTIONf_chiral_restr0.0431135
X-RAY DIFFRACTIONf_plane_restr0.0031205
X-RAY DIFFRACTIONf_dihedral_angle_d13.6964304
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.6075-2.69170.33651400.30632631277196
2.6917-2.78790.34881510.311527372888100
2.7879-2.89950.35431520.296827542906100
2.8995-3.03150.33081660.294227552921100
3.0315-3.19130.30721540.273527512905100
3.1913-3.39120.30651450.263527782923100
3.3912-3.6530.25451400.24427892929100
3.653-4.02060.27021050.23552187229277
4.0206-4.60220.21011330.175428272960100
4.6022-5.79750.2391370.188828703007100
5.7975-59.28410.21321510.185330103161100
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)
10.44910.57940.08360.6865-0.1150.0035-0.0033-0.1522-0.07430.0154-0.0718-0.0506-0.0096-0.0187-00.35390.03280.02060.33190.01180.3313-8.286129.369319.159
20.9633-0.25660.01030.8271-0.06851.6911-0.02560.08020.0202-0.04480.02890.00220.0391-0.0085-00.28880.0147-0.00990.2062-0.0140.2682-2.773445.41952.1891
30.5153-0.0980.4740.41280.26560.1589-0.1351-0.11330.1292-0.0852-0.12960.1896-0.217-0.1706-00.4841-0.0479-0.0910.5061-0.04890.4682-34.78811.0599-12.642
4-0.00180.02850.34670.52180.06890.4627-0.2841-0.2127-0.07220.00910.18870.3594-0.33020.0459-0.06530.3826-0.0354-0.01180.3677-0.02170.3752-41.491.4719-12.4006
51.6343-0.0538-0.3190.78930.43790.3160.0202-0.4556-0.04530.10550.0832-0.0831-0.0550.082800.3564-0.0218-0.04740.61190.0320.4351-21.0608-6.2478-18.023
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 0 through 202 )A0 - 202
2X-RAY DIFFRACTION2chain 'A' and (resid 203 through 451 )A203 - 451
3X-RAY DIFFRACTION3chain 'B' and (resid -2 through 116 )B-2 - 116
4X-RAY DIFFRACTION4chain 'B' and (resid 117 through 215 )B117 - 215
5X-RAY DIFFRACTION5chain 'B' and (resid 216 through 450 )B216 - 450

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