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- PDB-8uxu: Cryo-EM structure of a bacterial nitrilase filament with a covale... -

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Basic information

Entry
Database: PDB / ID: 8uxu
TitleCryo-EM structure of a bacterial nitrilase filament with a covalent adduct derived from benzonitrile hydrolysis
ComponentsNitrilase
KeywordsHYDROLASE / Aromatic-nitrilase / helical-filament / benzaldehyde covalent-adduct intermediate / truncated mutant
Function / homology
Function and homology information


nitrilase activity / :
Similarity search - Function
Nitrilases / cyanide hydratase active site signature. / Nitrilase/Cyanide hydratase / Nitrilases / cyanide hydratase signature 1. / Nitrilase/cyanide hydratase, conserved site / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
benzaldehyde / Nitrilase
Similarity search - Component
Biological speciesRhodococcus sp. (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsAguirre-Sampieri, S. / Casanal, A. / Emsley, P. / Garza-Ramos, G.
Funding support Mexico, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitrilase
B: Nitrilase
C: Nitrilase
D: Nitrilase
E: Nitrilase
F: Nitrilase
G: Nitrilase
H: Nitrilase
I: Nitrilase
J: Nitrilase
K: Nitrilase
L: Nitrilase
M: Nitrilase
N: Nitrilase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)509,05328
Polymers507,56814
Non-polymers1,48614
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Nitrilase


Mass: 36254.840 Da / Num. of mol.: 14 / Fragment: C-terminal truncated mutant
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria)
Strain: V51B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A4LA85
#2: Chemical
ChemComp-HBX / benzaldehyde


Mass: 106.122 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C7H6O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Bacterial nitrilase filament with covalent adduct derived from benzonitrile hydrolysis.
Type: COMPLEX / Details: C-terminal truncated mutant. / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria)
Strain: V51B
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET24a (+)
Buffer solutionpH: 7.8
Details: Monobasic and dibasic potassium phosphate mixed at 7.8 pH.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMPotassium phosphateKHPO1
2100 mMPotassium clorideKCl1
SpecimenConc.: 3.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified protein was centrifuged and incubated with 100 mM benzonitrile for 15 min and applied to the grid.
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
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.

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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 magnification: 75000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.04081633 sec. / Electron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2358

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Processing

EM software
IDNameVersionCategoryFitting-ID
1RELION3.0.8particle selection
4GctfCTF correction
7Coot0.9.8.7model fitting1
9REFMAC5model refinement1
10Coot0.9.8.7model fitting2
11ISOLDEmodel fitting2
12PHENIX1.19.2model refinement2
13RELION3.0.8initial Euler assignment
14RELION3.0.8final Euler assignment
16RELION3.0.83D reconstruction
CTF correctionType: NONE
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
11-7218.2573D1
21-7218.2573D1
Particle selectionNum. of particles selected: 140381
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56369 / Num. of class averages: 45 / Symmetry type: HELICAL
Atomic model building
IDProtocolSpaceB value
1BACKBONE TRACEREAL
240
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12VHH

2vhh

12VHH1PDBexperimental model
22
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 37.82 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006334399
ELECTRON MICROSCOPYf_angle_d0.536646947
ELECTRON MICROSCOPYf_chiral_restr0.04625296
ELECTRON MICROSCOPYf_plane_restr0.00436118
ELECTRON MICROSCOPYf_dihedral_angle_d5.15694929

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