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- EMDB-42779: Cryo-EM structure of a bacterial nitrilase filament with a covale... -

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Basic information

Entry
Database: EMDB / ID: EMD-42779
TitleCryo-EM structure of a bacterial nitrilase filament with a covalent adduct derived from benzonitrile hydrolysis
Map data
Sample
  • Complex: Bacterial nitrilase filament with covalent adduct derived from benzonitrile hydrolysis.
    • Protein or peptide: Nitrilase
  • Ligand: benzaldehyde
KeywordsAromatic-nitrilase / helical-filament / benzaldehyde covalent-adduct intermediate / truncated mutant / HYDROLASE
Function / homology
Function and homology information


Nitrilases / cyanide hydratase active site signature. / Nitrilase/Cyanide hydratase / Nitrilases / cyanide hydratase signature 1. / Nitrilase/cyanide hydratase, conserved site / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Biological speciesRhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsAguirre-Sampieri S / Casanal A / Emsley P / Garza-Ramos G
Funding support Mexico, 1 items
OrganizationGrant numberCountry
Other governmentDGAPA-PAPIIT UNAM IN218318 Mexico
CitationJournal: J Struct Biol / Year: 2024
Title: Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.
Authors: Sergio Aguirre-Sampieri / Ana Casañal / Paul Emsley / Georgina Garza-Ramos /
Abstract: Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo- ...Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.
History
DepositionNov 10, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42779.png

Downloads & links

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Map

FileDownload / File: emd_42779.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 360 pix.
= 374.4 Å		1.04 Å/pix.
x 360 pix.
= 374.4 Å		1.04 Å/pix.
x 360 pix.
= 374.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.08324552 - 0.18638894
Average (Standard dev.)-0.00015633294 (±0.0067941733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42779_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_42779_half_map_1.map
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Half map: #1

Fileemd_42779_half_map_2.map
Projections & Slices
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Sample components

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Entire : Bacterial nitrilase filament with covalent adduct derived from be...

EntireName: Bacterial nitrilase filament with covalent adduct derived from benzonitrile hydrolysis.
Components
  • Complex: Bacterial nitrilase filament with covalent adduct derived from benzonitrile hydrolysis.
    • Protein or peptide: Nitrilase
  • Ligand: benzaldehyde

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Supramolecule #1: Bacterial nitrilase filament with covalent adduct derived from be...

SupramoleculeName: Bacterial nitrilase filament with covalent adduct derived from benzonitrile hydrolysis.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: C-terminal truncated mutant.
Source (natural)Organism: Rhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria)
Strain: V51B

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Macromolecule #1: Nitrilase

MacromoleculeName: Nitrilase / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Rhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria)
Strain: V51B
Molecular weightTheoretical: 36.25484 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVEYTNTFKV AAVQAQPVWF DAAKTVDKTV SNIAEAARNG CELVAFPEVF IPGYPYHIWV DSPLAGMAKF AVRYHENSLT MDSPHVQRL LDAARDHNIA VVVGISERDG GSLYMTQLII DADGQLVARR RKLKPTHVER SVYGEGNGSD ISVYDMPFAR L GALNCWEH ...String:
MVEYTNTFKV AAVQAQPVWF DAAKTVDKTV SNIAEAARNG CELVAFPEVF IPGYPYHIWV DSPLAGMAKF AVRYHENSLT MDSPHVQRL LDAARDHNIA VVVGISERDG GSLYMTQLII DADGQLVARR RKLKPTHVER SVYGEGNGSD ISVYDMPFAR L GALNCWEH FQTLTKYAMY SMHEQVHVAS WPGMSLYQPE VPAFGVDAQL TATRMYALEG QTFVVCTTQV VTPEAHEFFC EN EEQRKLI GRGGGFARII GPDGRDLATP LAEDEEGILY ADIDLSAITL AKQAADPVGH YSRPDVLSLN FNQRRTTPVN TPL STIHAT H

UniProtKB: Nitrilase

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Macromolecule #2: benzaldehyde

MacromoleculeName: benzaldehyde / type: ligand / ID: 2 / Number of copies: 14 / Formula: HBX
Molecular weightTheoretical: 106.122 Da
Chemical component information

ChemComp-HBX:
benzaldehyde

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration3.8 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
100.0 mMKHPOPotassium phosphate
100.0 mMKClPotassium cloride

Details: Monobasic and dibasic potassium phosphate mixed at 7.8 pH.
GridModel: Quantifoil / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were glow-discharged for 60 sec before deposition of 3 ul sample, blotted for 4 s, and vitrified by plunging into liquid ethane and stored in a cryobox in liquid nitrogen..
DetailsPurified protein was centrifuged and incubated with 100 mM benzonitrile for 15 min and applied to the grid.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 2358 / Average exposure time: 2.04081633 sec. / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 45
Applied symmetry - Helical parameters - Δz: 18.2573 Å
Applied symmetry - Helical parameters - Δ&Phi: -72 °
Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 56369
Segment selectionNumber selected: 140381 / Software - Name: RELION (ver. 3.0.8)
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2vhh


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-8uxu:
Cryo-EM structure of a bacterial nitrilase filament with a covalent adduct derived from benzonitrile hydrolysis

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Atomic model buiding 2

RefinementOverall B value: 40
Output model

PDB-8uxu:
Cryo-EM structure of a bacterial nitrilase filament with a covalent adduct derived from benzonitrile hydrolysis

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