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- EMDB-17015: Cryo-EM map of the focused refinement of the subfamily III haloal... -

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Entry
Database: EMDB / ID: EMD-17015
TitleCryo-EM map of the focused refinement of the subfamily III haloalkane dehalogenase from Haloferax mediterranei dimer forming hexameric assembly.
Map dataCryo-EM map of the focused refinement of the subfamily III haloalkane dehalogenase from Haloferax mediterranei dimer forming hexameric assembly.
Sample
  • Complex: The subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
    • Protein or peptide: Alpha/beta fold hydrolase
Keywordshaloalkane dehalogenase / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsPolak M / Novacek J / Chmelova K / Marek M
Funding support Czech Republic, 1 items
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, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17015.png

Downloads & links

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Map

FileDownload / File: emd_17015.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the focused refinement of the subfamily III haloalkane dehalogenase from Haloferax mediterranei dimer forming hexameric assembly.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.616 Å		1.06 Å/pix.
x 256 pix.
= 271.616 Å		1.06 Å/pix.
x 256 pix.
= 271.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.061 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-0.6139831 - 1.5464419
Average (Standard dev.)0.00018453453 (±0.074437104)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.616 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Second half map.

Fileemd_17015_half_map_1.map
AnnotationSecond half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First half map.

Fileemd_17015_half_map_2.map
AnnotationFirst half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The subfamily III haloalkane dehalogenase DhmeA from Haloferax me...

EntireName: The subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
Components
  • Complex: The subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
    • Protein or peptide: Alpha/beta fold hydrolase

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Supramolecule #1: The subfamily III haloalkane dehalogenase DhmeA from Haloferax me...

SupramoleculeName: The subfamily III haloalkane dehalogenase DhmeA from Haloferax mediterranei
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Haloferax mediterranei (archaea)

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Macromolecule #1: Alpha/beta fold hydrolase

MacromoleculeName: Alpha/beta fold hydrolase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Haloferax mediterranei (archaea)
Molecular weightTheoretical: 35.711797 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSASSNARD EVIAAIHEEA DWVDRTVYPF ESRCIGLSSG AVHYIDEGPD DGGRETLLML HGNPTWSFLY RHLVRDLRDE YRCVALDYL GFGLSERPTD FSYRPEDHAD VVEEFIDELG LEDVVLVGHD WGGPIGFSYA IDHPENVGGL VVMNTWMWPV S DDKHFSRF ...String:
MSSASSNARD EVIAAIHEEA DWVDRTVYPF ESRCIGLSSG AVHYIDEGPD DGGRETLLML HGNPTWSFLY RHLVRDLRDE YRCVALDYL GFGLSERPTD FSYRPEDHAD VVEEFIDELG LEDVVLVGHD WGGPIGFSYA IDHPENVGGL VVMNTWMWPV S DDKHFSRF SKLLGGRIGR ELCERYDLFT RVIMPMGFAD RSRFTESARE QYRAANRGDR TGTGIFPQAI LGSRAWLSSL WE QRDNIAD IPARIIWGME DSAFRPAELR TFEALFEDSS TVRLYGVGHY VPEEFGSDLV PLVREFLEEV LEVLFQ

UniProtKB: Alpha/beta fold hydrolase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.035 mg/mL
BufferpH: 8 / Component - Concentration: 1.0 M / Component - Formula: NaClSodium chloride / Component - Name: Sodium Chloride / Details: 1 M NaCl, 10 mM Tris-HCl
VitrificationCryogen name: ETHANE
DetailsThis sample was purified by gel filtration.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: FEI FALCON II (4k x 4k) / #0 - Average electron dose: 48.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 QUANTUM (4k x 4k) / #1 - Average electron dose: 73.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4)
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
Number images used: 55074
Image recording ID1
FSC plot (resolution estimation)

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