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- PDB-6zby: Cryo-EM structure of the nitrilase from Pseudomonas fluorescens E... -

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Basic information

Entry
Database: PDB / ID: 6zby
TitleCryo-EM structure of the nitrilase from Pseudomonas fluorescens EBC191 at 3.3 Angstroms
ComponentsNitA
KeywordsHYDROLASE / bacterial nitrilase / arylacetonitrilase
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 superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Biological speciesPseudomonas fluorescens (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsEppinger, E. / Stolz, A. / Sewell, B.T.
CitationJournal: To Be Published
Title: Cryo-EM structure of the nitrilase from Pseudomonas fluorescens EBC191 at 3.3 Angstroms
Authors: Eppinger, E. / Stolz, A. / Sewell, B.T.
History
DepositionJun 9, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Superimposition on EM map
  • EMDB-11158
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: NitA
B: NitA
C: NitA
D: NitA
E: NitA
F: NitA
G: NitA
H: NitA
I: NitA
J: NitA
K: NitA
L: NitA


Theoretical massNumber of molelcules
Total (without water)452,88912
Polymers452,88912
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53470 Å2
ΔGint-331 kcal/mol
Surface area114010 Å2
MethodPISA

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Components

#1: Protein
NitA


Mass: 37740.746 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas fluorescens (bacteria) / Gene: nitA / Plasmid: pIK9 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5EG61

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Active helical nitrilase homooligomer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas fluorescens (bacteria) / Strain: EBC191
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pIK9
Buffer solutionpH: 7.8
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %
Details: The sample (2.5 ul) was applied to the grid and incubated for 30 seconds at 100% humidity before blotting and plunging.

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 43.1 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2929
Image scansMovie frames/image: 30

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.1_3865refinement
PHENIX1.18.1_3865refinement
UCSF ChimeraX0.93/v8model building
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
9ISOLDEmodel refinement
10Cootmodel refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -67.31 ° / Axial rise/subunit: 16.36 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 278053
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91429 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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