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- EMDB-18687: Cryo-EM Structure of Regulated CBS Domain-Containing Pyrophosphat... -

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Basic information

Entry
Database: EMDB / ID: EMD-18687
TitleCryo-EM Structure of Regulated CBS Domain-Containing Pyrophosphatase without One Catalytic Domain
Map data
Sample
  • Complex: Homotetrameric CBS-PPase_delta_DHHA2-domain
    • Protein or peptide: pyrophosphatase containing cystathionine beta-synthase domains and lacking one catalytic domain
Keywordscystathionine beta-synthase domain / DHH-domain / domain swapping / HYDROLASE
Function / homology
Function and homology information


inorganic diphosphatase / inorganic diphosphate phosphatase activity / metal ion binding / cytoplasm
Similarity search - Function
DRTGG / DRTGG domain / HPr(Ser) kinase/phosphorylase-like, N-terminal domain superfamily / DHHA2 domain / DHHA2 domain superfamily / DHHA2 domain / DHHA2 / DDH domain / DHH family / DHH phosphoesterase superfamily ...DRTGG / DRTGG domain / HPr(Ser) kinase/phosphorylase-like, N-terminal domain superfamily / DHHA2 domain / DHHA2 domain superfamily / DHHA2 domain / DHHA2 / DDH domain / DHH family / DHH phosphoesterase superfamily / CBS domain superfamily / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
inorganic diphosphatase
Similarity search - Component
Biological speciesDesulfitobacterium hafniense DCB-2 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.3 Å
AuthorsMoiseenko AV / Anashkin VA / Zamakhov IM / Sokolova OS / Baykov AA
Funding support Russian Federation, 1 items
OrganizationGrant numberCountry
Russian Science Foundation22-74-00031 Russian Federation
CitationJournal: Int J Mol Sci / Year: 2023
Title: The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.
Authors: Ilya M Zamakhov / Viktor A Anashkin / Andrey V Moiseenko / Victor N Orlov / Natalia N Vorobyeva / Olga S Sokolova / Alexander A Baykov /
Abstract: Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is ...Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.
History
DepositionOct 19, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18687.png

Downloads & links

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Map

FileDownload / File: emd_18687.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.33 Å
Density
Contour LevelBy AUTHOR: 0.925
Minimum - Maximum-0.93799376 - 5.981531
Average (Standard dev.)-0.0004022014 (±0.078003906)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 699.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_18687_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18687_half_map_2.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 : Homotetrameric CBS-PPase_delta_DHHA2-domain

EntireName: Homotetrameric CBS-PPase_delta_DHHA2-domain
Components
  • Complex: Homotetrameric CBS-PPase_delta_DHHA2-domain
    • Protein or peptide: pyrophosphatase containing cystathionine beta-synthase domains and lacking one catalytic domain

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Supramolecule #1: Homotetrameric CBS-PPase_delta_DHHA2-domain

SupramoleculeName: Homotetrameric CBS-PPase_delta_DHHA2-domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: 2,5 mg/ml 0.1 M MOPS/KOH, 2 mM MgCl2, 150 mM KCl, pH 7.2
Source (natural)Organism: Desulfitobacterium hafniense DCB-2 (bacteria)

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Macromolecule #1: pyrophosphatase containing cystathionine beta-synthase domains an...

MacromoleculeName: pyrophosphatase containing cystathionine beta-synthase domains and lacking one catalytic domain
type: protein_or_peptide / ID: 1 / Details: CBS-PPase without DHHA2-domain. / Enantiomer: LEVO / EC number: inorganic diphosphatase
Source (natural)Organism: Desulfitobacterium hafniense DCB-2 (bacteria) / Strain: DCB-2
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKKIHVVGH RNPDTDSICA AIAYARLKQR LGMDHVIPYR AGKINRETEF VLNAFGVEAP ELLKDLHLR VKDMLNGPIP AVKPRTSLLE AWKIMKESNQ KTLPVVDHTE QMIGMITVGD L SGSYIESM ADHELESLHI PVENVIRTLN GRLLVGSAEQ DLKGNVYVGA ...String:
MSKKIHVVGH RNPDTDSICA AIAYARLKQR LGMDHVIPYR AGKINRETEF VLNAFGVEAP ELLKDLHLR VKDMLNGPIP AVKPRTSLLE AWKIMKESNQ KTLPVVDHTE QMIGMITVGD L SGSYIESM ADHELESLHI PVENVIRTLN GRLLVGSAEQ DLKGNVYVGA MRHETLEAYV GN GNIVLMG DRQTAQESVL RQGVSALILT GGATLAPELE DLARQQNCVV ISVPYDTFTA ARL LPMTAP VQNAMKTEGI VAFNEDDLIS EVKEKMLETR FRNYPVLDEQ GKVVGLISRY HLLS LSRKQ VILVDHNEFG QAVVGTDQAQ ILEVVDHHRV GGIQTGEPIL FRNEPVGSTC TIVAK CYRD WDVVPEKAIA GIMLGAILSD TVIFKSPTCT EIDRENATYL ASIAGVDPKE FGVQMF KAA

UniProtKB: inorganic diphosphatase

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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

Concentration2.5 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
0.1 MMOPS/KOHMOPS/KOH
0.002 MMgCl2magnesiium chloride
0.15 MKClPotassium cloride
GridModel: EMS Lacey Carbon / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 3.3000000000000003 µm / Calibrated defocus min: 0.6 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.4 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: JEOL / Cooling holder cryogen: NITROGEN
TemperatureMin: 97.0 K / Max: 103.0 K
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber selected: 223576
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1) / Details: Cryosparc Ab initio
Final 3D classificationNumber classes: 50 / Avg.num./class: 840 / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 13.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 14850
FSC plot (resolution estimation)

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