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- PDB-8ooh: Cryo-EM map of the focused refinement of the subfamily III haloal... -

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Basic information

Entry
Database: PDB / ID: 8ooh
TitleCryo-EM map of the focused refinement of the subfamily III haloalkane dehalogenase from Haloferax mediterranei dimer forming hexameric assembly.
ComponentsAlpha/beta fold hydrolase
KeywordsHYDROLASE / haloalkane dehalogenase
Function / homologyhaloalkane dehalogenase / haloalkane dehalogenase activity / Epoxide hydrolase-like / acyltransferase activity / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7 Å
AuthorsPolak, M. / Novacek, J. / Chmelova, K. / Marek, M.
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
DepositionApr 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha/beta fold hydrolase
B: Alpha/beta fold hydrolase


Theoretical massNumber of molelcules
Total (without water)71,4242
Polymers71,4242
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Alpha/beta fold hydrolase


Mass: 35711.797 Da / Num. of mol.: 2
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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Haloferax mediterranei (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 1 M NaCl, 10 mM Tris-HCl
Buffer componentConc.: 1 M / Name: Sodium Chloride / Formula: NaCl
SpecimenConc.: 0.035 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was purified by gel filtration.
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1148FEI FALCON II (4k x 4k)
2173GATAN K2 QUANTUM (4k x 4k)

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Processing

EM software
IDNameVersionCategory
12cryoSPARCv4classification
13cryoSPARCv43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55074
Details: 1)C1 Non-uniform Refinement 2)C3 Volume Alignment 3)C3 Symmetry Expansion (55074 particles expanded to 165 222 particles) 4)C1 Local Refinement
Symmetry type: POINT

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