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- EMDB-4407: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-4407
TitleCryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.
Map dataSynechocystis sp. PCC6803 (BAA10717.1) nitrilase truncated at residue 291
Sample
  • Complex: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
    • Protein or peptide: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodhelical reconstruction / negative staining / Resolution: 20.0 Å
AuthorsYamkela Q / Mulelu AE / Woodward JD
Funding support South Africa, 1 items
OrganizationGrant numberCountry
National Research Foundation in South AfricaResearch Career Advancement Fellowship South Africa
CitationJournal: Commun Biol / Year: 2019
Title: Cryo-EM and directed evolution reveal how nitrilase specificity is influenced by its quaternary structure.
Authors: Andani E Mulelu / Angela M Kirykowicz / Jeremy D Woodward /
Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced ...Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from . We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.
History
DepositionNov 9, 2018-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 7, 2019-
UpdateAug 7, 2019-
Current statusAug 7, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4407.png

Downloads & links

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Map

FileDownload / File: emd_4407.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSynechocystis sp. PCC6803 (BAA10717.1) nitrilase truncated at residue 291
Voxel sizeX=Y=Z: 4.11 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-3.1567473 - 4.447312
Average (Standard dev.)-0.000000004771252 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 263.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.114.114.11
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z263.040263.040263.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-15-9-23
NX/NY/NZ635180
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-3.1574.447-0.000

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Supplemental data

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Sample components

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Entire : Synechocystis sp. PCC6803 nitrilase truncated at residue 291

EntireName: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
Components
  • Complex: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
    • Protein or peptide: Synechocystis sp. PCC6803 nitrilase truncated at residue 291

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Supramolecule #1: Synechocystis sp. PCC6803 nitrilase truncated at residue 291

SupramoleculeName: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET-21b(+)

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Macromolecule #1: Synechocystis sp. PCC6803 nitrilase truncated at residue 291

MacromoleculeName: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHGSHM LGKIMLNYTK NIRAAAAQIS PVLFSQQGTM EKVLDAIANA AKKGVELIVF PETFVPYYPY FSFVEPPVLM GKSHLKLYQE AVTVPGKVTQ AIAQAAKTHG MVVVLGVNER EEGSLYNTQL IFDADGALVL KRRKITPTYH ERMVWGQGDG AGLRTVDTTV ...String:
HHHHHHGSHM LGKIMLNYTK NIRAAAAQIS PVLFSQQGTM EKVLDAIANA AKKGVELIVF PETFVPYYPY FSFVEPPVLM GKSHLKLYQE AVTVPGKVTQ AIAQAAKTHG MVVVLGVNER EEGSLYNTQL IFDADGALVL KRRKITPTYH ERMVWGQGDG AGLRTVDTTV GRLGALACWE HYNPLARYAL MAQHEQIHCG QFPGSMVGQI FADQMEVTMR HHALESGCFV INATGWLTAE QKLQITTDEK MHQALSGGCY TAIISPEGKH LCEPIAEGEG LAIADLDFSL IAKRKRMMDS

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HclTrisTris
200.0 mMNaClSodium chloridesodium chloride
StainingType: NEGATIVE / Material: Uranyl acetate

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: PHILIPS ROTATION HOLDER
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 50 / Average exposure time: 1.0 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Segment selectionNumber selected: 800 / Software - Name: EMAN
Software - details: Boxer was used in helical mode with 95% overlap
Details: Segments were picked with a 90% overlap
Startup modelType of model: INSILICO MODEL / In silico model: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 21.10) / Software - details: IHRSR
Final reconstructionNumber classes used: 90
Applied symmetry - Helical parameters - Δz: 13.02547 Å
Applied symmetry - Helical parameters - Δ&Phi: -61.44494 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER (ver. 21.10) / Software - details: IHRSR / Number images used: 800
DetailsThe images were low-pass filtered to 20 A and normalised.

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