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- EMDB-4407: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4407 | |||||||||
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Title | Cryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure. | |||||||||
![]() | Synechocystis sp. PCC6803 (BAA10717.1) nitrilase truncated at residue 291 | |||||||||
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Biological species | ![]() ![]() | |||||||||
Method | helical reconstruction / negative staining / Resolution: 20.0 Å | |||||||||
![]() | Yamkela Q / Mulelu AE / Woodward JD | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 299.1 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.7 KB 11.7 KB | Display Display | ![]() |
Images | ![]() | 61.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Synechocystis sp. PCC6803 (BAA10717.1) nitrilase truncated at residue 291 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.11 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Synechocystis sp. PCC6803 nitrilase truncated at residue 291
Entire | Name: Synechocystis sp. PCC6803 nitrilase truncated at residue 291 |
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Components |
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-Supramolecule #1: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
Supramolecule | Name: Synechocystis sp. PCC6803 nitrilase truncated at residue 291 type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #1: Synechocystis sp. PCC6803 nitrilase truncated at residue 291
Macromolecule | Name: Synechocystis sp. PCC6803 nitrilase truncated at residue 291 type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nitrilase |
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Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 0.3 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Staining | Type: NEGATIVE / Material: Uranyl acetate |
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Electron microscopy
Microscope | FEI TECNAI 20 |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: PHILIPS ROTATION HOLDER |