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- PDB-8ckp: X-ray structure of the crystallization-prone form of subfamily II... -

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Basic information

Entry
Database: PDB / ID: 8ckp
TitleX-ray structure of the crystallization-prone form of subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
ComponentsAlpha/beta fold hydrolase
KeywordsUNKNOWN FUNCTION / haloalkane dehalogenase-like / hydrolase
Function / homologyhaloalkane dehalogenase / haloalkane dehalogenase activity / acyltransferase activity / Epoxide hydrolase-like / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / Alpha/Beta hydrolase fold / Alpha/beta fold hydrolase
Function and homology information
Biological speciesHaloferax mediterranei (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.31 Å
AuthorsMarek, M. / Chmelova, K. / Schenkmayerova, A. / Croll, T. / Read, R.J. / Diederichs, K.
Funding support Czech Republic, 1items
OrganizationGrant numberCountry
Czech Science FoundationGA22-09853S Czech Republic
CitationJournal: Protein Sci / Year: 2023
Title: Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.
Authors: Klaudia Chmelova / Tadeja Gao / Martin Polak / Andrea Schenkmayerova / Tristan I Croll / Tanvir R Shaikh / Jana Skarupova / Radka Chaloupkova / Kay Diederichs / Randy J Read / Jiri Damborsky ...Authors: Klaudia Chmelova / Tadeja Gao / Martin Polak / Andrea Schenkmayerova / Tristan I Croll / Tanvir R Shaikh / Jana Skarupova / Radka Chaloupkova / Kay Diederichs / Randy J Read / Jiri Damborsky / Jiri Novacek / Martin Marek /
Abstract: Haloalkane dehalogenase (HLD) enzymes employ an S 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well- ...Haloalkane dehalogenase (HLD) enzymes employ an S 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeA ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeA forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.
History
DepositionFeb 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Alpha/beta fold hydrolase
F: Alpha/beta fold hydrolase
G: Alpha/beta fold hydrolase
H: Alpha/beta fold hydrolase
I: Alpha/beta fold hydrolase
J: Alpha/beta fold hydrolase
A: Alpha/beta fold hydrolase
B: Alpha/beta fold hydrolase
C: Alpha/beta fold hydrolase
E: Alpha/beta fold hydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,00311
Polymers356,96710
Non-polymers351
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19260 Å2
ΔGint-51 kcal/mol
Surface area107700 Å2
Unit cell
Length a, b, c (Å)172.760, 289.900, 168.310
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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Components

#1: Protein
Alpha/beta fold hydrolase


Mass: 35696.707 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haloferax mediterranei (archaea) / Gene: mhpC / Production host: Escherichia coli (E. coli) / References: UniProt: I3R766
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.95 Å3/Da / Density % sol: 58.33 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6.5 / Details: PEG 4000, MES/Imidazole buffer system, amino acids

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 0.9999 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: May 12, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9999 Å / Relative weight: 1
ReflectionResolution: 3.306→49.47 Å / Num. obs: 63328 / % possible obs: 99.55 % / Redundancy: 70.3 % / Biso Wilson estimate: 151.61 Å2 / CC1/2: 0.999 / Net I/σ(I): 10.76
Reflection shellResolution: 3.306→3.424 Å / Num. unique obs: 6044 / CC1/2: 0.241

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Processing

Software
NameVersionClassification
XDSdata scaling
XDSdata reduction
PHASERphasing
PHENIX1.20.1-4487refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.31→49.47 Å / SU ML: 0.6973 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 36.1909
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2748 3153 5 %
Rwork0.2358 59920 -
obs0.2379 63073 99.59 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 155.02 Å2
Refinement stepCycle: LAST / Resolution: 3.31→49.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms23882 0 1 1 23884
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002524550
X-RAY DIFFRACTIONf_angle_d0.691633342
X-RAY DIFFRACTIONf_chiral_restr0.04543439
X-RAY DIFFRACTIONf_plane_restr0.01194420
X-RAY DIFFRACTIONf_dihedral_angle_d13.28838833
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.31-3.360.50281240.54412394X-RAY DIFFRACTION93.26
3.36-3.410.53271360.46622569X-RAY DIFFRACTION99.41
3.41-3.460.4491360.41622590X-RAY DIFFRACTION99.96
3.46-3.520.43791370.41082602X-RAY DIFFRACTION99.96
3.52-3.590.43961360.35582585X-RAY DIFFRACTION99.96
3.59-3.660.38041370.34712602X-RAY DIFFRACTION99.93
3.66-3.730.45231360.35632583X-RAY DIFFRACTION99.78
3.73-3.810.34621350.292556X-RAY DIFFRACTION99.85
3.81-3.90.36371370.30552611X-RAY DIFFRACTION99.96
3.9-40.38261360.31592579X-RAY DIFFRACTION99.52
4-4.110.36311380.26322620X-RAY DIFFRACTION99.86
4.11-4.230.30911360.24552586X-RAY DIFFRACTION99.96
4.23-4.360.26211360.22532583X-RAY DIFFRACTION99.96
4.36-4.520.29121370.22312604X-RAY DIFFRACTION100
4.52-4.70.28821380.22222616X-RAY DIFFRACTION99.93
4.7-4.910.28181370.22292604X-RAY DIFFRACTION99.93
4.91-5.170.23851390.19482635X-RAY DIFFRACTION99.93
5.17-5.50.24891370.22182616X-RAY DIFFRACTION100
5.5-5.920.29431390.23382640X-RAY DIFFRACTION100
5.92-6.510.24011400.21982647X-RAY DIFFRACTION99.96
6.52-7.450.28051390.21872641X-RAY DIFFRACTION99.96
7.45-9.380.21161410.1792690X-RAY DIFFRACTION100
9.38-49.470.21461460.21192767X-RAY DIFFRACTION99.49

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