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- PDB-7p75: Re-engineered 2-deoxy-D-ribose-5-phosphate aldolase catalysing as... -

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Basic information

Entry
Database: PDB / ID: 7p75
TitleRe-engineered 2-deoxy-D-ribose-5-phosphate aldolase catalysing asymmetric Michael addition reactions in substrate-free state
ComponentsDeoxyribose-phosphate aldolase
KeywordsLYASE / Protein engineering / directed evolution / DERA / aldolase / carboligase / Michael addition / carbon-carbon lyase
Function / homology
Function and homology information


deoxyribose phosphate catabolic process / deoxyribose-phosphate aldolase / deoxyribose-phosphate aldolase activity / deoxyribonucleotide catabolic process / carbohydrate catabolic process / cytoplasm
Similarity search - Function
Deoxyribose-phosphate aldolase type II / Deoxyribose-phosphate aldolase / DeoC/LacD family aldolase / DeoC/FbaB/LacD aldolase / DeoC/LacD family aldolase / Aldolase-type TIM barrel
Similarity search - Domain/homology
Deoxyribose-phosphate aldolase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.23 Å
AuthorsThunnissen, A.M.W.H. / Rozeboom, H.J. / Kunzendorf, A. / Poelarends, G.J.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)724.016.002 Netherlands
CitationJournal: Acs Catalysis / Year: 2021
Title: Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution.
Authors: Kunzendorf, A. / Xu, G. / van der Velde, J.J.H. / Rozeboom, H.J. / Thunnissen, A.W.H. / Poelarends, G.J.
History
DepositionJul 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 31, 2024Group: Data collection / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Deoxyribose-phosphate aldolase
BBB: Deoxyribose-phosphate aldolase


Theoretical massNumber of molelcules
Total (without water)57,3672
Polymers57,3672
Non-polymers00
Water9,242513
1
AAA: Deoxyribose-phosphate aldolase


Theoretical massNumber of molelcules
Total (without water)28,6841
Polymers28,6841
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
BBB: Deoxyribose-phosphate aldolase


Theoretical massNumber of molelcules
Total (without water)28,6841
Polymers28,6841
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)47.634, 67.497, 73.657
Angle α, β, γ (deg.)90.000, 101.045, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11AAA
21BBB

NCS domain segments:

Ens-ID: 1 / Beg auth comp-ID: THR / Beg label comp-ID: THR / End auth comp-ID: HIS / End label comp-ID: HIS / Auth seq-ID: 2 - 250 / Label seq-ID: 2 - 250

Dom-IDComponent-IDAuth asym-IDLabel asym-ID
11AAAA
22BBBB

NCS ensembles : (Details: Local NCS retraints between domains: 1 2)

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Components

#1: Protein Deoxyribose-phosphate aldolase / / DERA / 2-deoxy-D-ribose 5-phosphate aldolase / Phosphodeoxyriboaldolase / Deoxyriboaldolase


Mass: 28683.738 Da / Num. of mol.: 2
Mutation: T18S D22G D24Y C47S F52S T142S K172L T197S P202V A203T V206A S239G
Source method: isolated from a genetically manipulated source
Details: Engineered Escherichia coli DERA variant with 12 mutations
Source: (gene. exp.) Escherichia coli (strain ATCC 8739 / DSM 1576 / NBRC 3972 / NCIMB 8545 / WDCM 00012 / Crooks) (bacteria)
Strain: ATCC 8739 / DSM 1576 / NBRC 3972 / NCIMB 8545 / WDCM 00012 / Crooks
Gene: deoC, EcolC_3675 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B1IS38, deoxyribose-phosphate aldolase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 513 / 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: 2.03 Å3/Da / Density % sol: 39.28 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Protein was concentrated to 16 mg/ml in 10 mM potassium phosphate. Crystals grew from hanging-drop vapour diffusion experiments using 13% PEG 3350, 10% isopropanol and 0.1 M HEPES pH 7.5 as reservoir solution.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-1 / Wavelength: 0.9655 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 6, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9655 Å / Relative weight: 1
ReflectionResolution: 1.229→47 Å / Num. obs: 128757 / % possible obs: 96.9 % / Redundancy: 2.3 % / CC1/2: 0.99 / Rmerge(I) obs: 0.057 / Rpim(I) all: 0.057 / Rrim(I) all: 0.081 / Net I/σ(I): 6.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
6.73-472.40.0268160.9690.0260.03796.1
1.23-1.252.20.62958620.5390.6170.882

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Processing

Software
NameVersionClassification
XDS20210205data reduction
Aimless0.7.7data scaling
PHASER2.8.3phasing
Coot0.9.5model building
REFMAC5.8.0267refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1P1X

1p1x


Resolution: 1.23→47 Å / Cor.coef. Fo:Fc: 0.978 / Cor.coef. Fo:Fc free: 0.971 / SU B: 1.954 / SU ML: 0.036 / Cross valid method: FREE R-VALUE / ESU R: 0.042 / ESU R Free: 0.041
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1714 6430 4.996 %
Rwork0.1477 122274 -
all0.149 --
obs-128704 96.803 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL PLUS MASK
Displacement parametersBiso mean: 17.444 Å2
Baniso -1Baniso -2Baniso -3
1-0.342 Å2-0 Å21.091 Å2
2---1.004 Å20 Å2
3---0.22 Å2
Refinement stepCycle: LAST / Resolution: 1.23→47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3761 0 0 513 4274
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0133856
X-RAY DIFFRACTIONr_bond_other_d0.0010.0173817
X-RAY DIFFRACTIONr_angle_refined_deg1.3711.6345222
X-RAY DIFFRACTIONr_angle_other_deg1.4861.5828799
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.885517
X-RAY DIFFRACTIONr_dihedral_angle_2_deg27.72822.229175
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.03915688
X-RAY DIFFRACTIONr_dihedral_angle_4_deg10.7631525
X-RAY DIFFRACTIONr_chiral_restr0.0720.2540
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.024364
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02810
X-RAY DIFFRACTIONr_nbd_refined0.2130.2810
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1770.23686
X-RAY DIFFRACTIONr_nbtor_refined0.1620.21938
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0810.21646
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1230.2399
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0950.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.280.215
X-RAY DIFFRACTIONr_nbd_other0.2210.257
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1770.232
X-RAY DIFFRACTIONr_mcbond_it1.2741.5892026
X-RAY DIFFRACTIONr_mcbond_other1.2691.5882025
X-RAY DIFFRACTIONr_mcangle_it1.7332.3922533
X-RAY DIFFRACTIONr_mcangle_other1.7342.3932534
X-RAY DIFFRACTIONr_scbond_it1.6241.8641830
X-RAY DIFFRACTIONr_scbond_other1.6241.8661831
X-RAY DIFFRACTIONr_scangle_it1.9522.6892681
X-RAY DIFFRACTIONr_scangle_other1.9522.6912682
X-RAY DIFFRACTIONr_lrange_it2.97221.0654522
X-RAY DIFFRACTIONr_lrange_other2.50220.1214371
X-RAY DIFFRACTIONr_rigid_bond_restr1.01437673
X-RAY DIFFRACTIONr_ncsr_local_group_10.0750.058171
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AAAX-RAY DIFFRACTIONLocal ncs0.075170.05008
12BBBX-RAY DIFFRACTIONLocal ncs0.075170.05008
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.23-1.2610.294070.2728540X-RAY DIFFRACTION91.9149
1.261-1.2950.2724630.2518692X-RAY DIFFRACTION95.5936
1.295-1.3330.2534410.2368440X-RAY DIFFRACTION95.7107
1.333-1.3740.2274590.2188324X-RAY DIFFRACTION96.8464
1.374-1.4190.224010.1978058X-RAY DIFFRACTION97.2187
1.419-1.4690.2084390.1717781X-RAY DIFFRACTION97.1172
1.469-1.5240.1834300.1537515X-RAY DIFFRACTION97.3652
1.524-1.5860.1623670.1417371X-RAY DIFFRACTION97.6034
1.586-1.6570.173850.1256943X-RAY DIFFRACTION97.4727
1.657-1.7370.1673350.1266677X-RAY DIFFRACTION97.0385
1.737-1.8310.1673380.1186346X-RAY DIFFRACTION97.236
1.831-1.9420.173120.1216059X-RAY DIFFRACTION97.8047
1.942-2.0760.1422920.1145706X-RAY DIFFRACTION98.4893
2.076-2.2430.1462950.1125311X-RAY DIFFRACTION98.3164
2.243-2.4560.1662560.1224876X-RAY DIFFRACTION97.9763
2.456-2.7460.1452460.1314423X-RAY DIFFRACTION97.9031
2.746-3.170.1352120.1433864X-RAY DIFFRACTION96.9553
3.17-3.8810.1491720.1453312X-RAY DIFFRACTION97.7279
3.881-5.480.191250.1532578X-RAY DIFFRACTION97.6165
5.48-470.184550.1921458X-RAY DIFFRACTION96.2468

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