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- PDB-8c5i: Cyanide dihydratase from Bacillus pumilus C1 variant - Q86R,H305K... -

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Basic information

Entry
Database: PDB / ID: 8c5i
TitleCyanide dihydratase from Bacillus pumilus C1 variant - Q86R,H305K,H308K,H323K
ComponentsCyanide dihydratase
KeywordsHYDROLASE / Helical / homo-oligomeric / cyanide dihydratase
Function / homology
Function and homology information


nitrilase activity / :
Similarity search - Function
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 speciesBacillus pumilus (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMulelu, A.E. / Reitz, J. / van Rooyen, J. / Scheffer, M. / Frangakis, A.S. / Dlamini, L.S. / Woodward, J.D. / Benedik, M.J. / Sewell, B.T.
Funding support South Africa, 1items
OrganizationGrant numberCountry
National Research Foundation in South Africa South Africa
CitationJournal: To Be Published
Title: The Role of Histidine Residues in the Oligomerization of Cyanide Dihydratase from Bacillus pumilus C1
Authors: Mulelu, A.E. / Reitz, J. / van Rooyen, J. / Scheffer, M. / Frangakis, A.S. / Dlamini, L.S. / Woodward, J.D. / Benedik, M.J. / Sewell, B.T.
History
DepositionJan 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cyanide dihydratase
B: Cyanide dihydratase
C: Cyanide dihydratase
E: Cyanide dihydratase
F: Cyanide dihydratase
G: Cyanide dihydratase
H: Cyanide dihydratase
I: Cyanide dihydratase
J: Cyanide dihydratase
K: Cyanide dihydratase
L: Cyanide dihydratase
M: Cyanide dihydratase
N: Cyanide dihydratase
O: Cyanide dihydratase
P: Cyanide dihydratase
Q: Cyanide dihydratase
R: Cyanide dihydratase
S: Cyanide dihydratase


Theoretical massNumber of molelcules
Total (without water)675,06118
Polymers675,06118
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cyanide dihydratase


Mass: 37503.363 Da / Num. of mol.: 18 / Mutation: Q86R,H305K,H308K,H323K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus pumilus (bacteria) / Gene: cynD / Plasmid: pET26b / Production host: Escherichia coli (E. coli) / References: UniProt: Q8GGL4

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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 homo-oligomer of cyanide dihydratase from Bacillus pumilus C1 variant (Q86R/H305K/H308K/H323K)
Type: COMPLEX
Details: Cyanide dihydratase from Bacillus pumilus C1 variant generated by site-directed mutagenesis.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus pumilus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5.4 / Details: 150 mM NaCl, 50 mM Tris-HCl pH 5.4
Buffer component
IDConc.NameFormulaBuffer-ID
10.150 Msodium chlorideNaClSodium chloride1
20.05 Mtrizma baseTris1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Homogeneous protein sample.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: A 2.5 microlitre sample was applied onto a glow-discharged grid, blotted and plunged without incubation.

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4RELION2.1CTF correctionCTFFIND 4.1used to calculate CTF of motion-corrected images
7UCSF Chimera1.15model fitting
12RELION2.13D reconstruction
13PHENIX1.20.1-4487model refinement
14ISOLDE1.1model refinement
15Coot0.9.8.1model refinement
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: -77 ° / Axial rise/subunit: 16.7 Å / Axial symmetry: C2
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 103000 / Algorithm: FOURIER SPACE / Num. of class averages: 92000 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00346620
ELECTRON MICROSCOPYf_angle_d0.49863252
ELECTRON MICROSCOPYf_dihedral_angle_d5.126228
ELECTRON MICROSCOPYf_chiral_restr0.0426660
ELECTRON MICROSCOPYf_plane_restr0.0048226

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