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Yorodumi- EMDB-9190: Cell-free-expressed Pyridoxal 5'-phosphate synthase-like subunit ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-9190 | |||||||||
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Title | Cell-free-expressed Pyridoxal 5'-phosphate synthase-like subunit (PDX1.2) from Arabidopsis thaliana | |||||||||
Map data | Cell-free-expressed PDX1.2 single particle cryo-EM map | |||||||||
Sample |
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Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 15.0 Å | |||||||||
Authors | Novikova IV / Evans JE / Hellmann H / Sharma N | |||||||||
Citation | Journal: Adv Struct Chem Imaging / Year: 2018 Title: Protein structural biology using cell-free platform from wheat germ. Authors: Irina V Novikova / Noopur Sharma / Trevor Moser / Ryan Sontag / Yan Liu / Michael J Collazo / Duilio Cascio / Tolou Shokuhfar / Hanjo Hellmann / Michael Knoblauch / James E Evans / Abstract: One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are ...One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_9190.map.gz | 59.3 MB | EMDB map data format | |
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Header (meta data) | emd-9190-v30.xml emd-9190.xml | 15.5 KB 15.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_9190_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_9190.png | 154.9 KB | ||
Others | emd_9190_half_map_1.map.gz emd_9190_half_map_2.map.gz | 5.4 MB 5.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-9190 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-9190 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_9190.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cell-free-expressed PDX1.2 single particle cryo-EM map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.172 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Cell-free-expressed PDX1.2 single particle cryo-EM even half-map
File | emd_9190_half_map_1.map | ||||||||||||
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Annotation | Cell-free-expressed PDX1.2 single particle cryo-EM even half-map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Cell-free-expressed PDX1.2 single particle cryo-EM odd half-map
File | emd_9190_half_map_2.map | ||||||||||||
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Annotation | Cell-free-expressed PDX1.2 single particle cryo-EM odd half-map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...
Entire | Name: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag |
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Components |
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-Supramolecule #1: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...
Supramolecule | Name: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Complex assembled from 12 monomers in D6 symmetry |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Recombinant expression | Organism: Triticum (plant) |
Molecular weight | Theoretical: 440 KDa |
-Macromolecule #1: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...
Macromolecule | Name: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO EC number: pyridoxal 5'-phosphate synthase (glutamine hydrolysing) |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Recombinant expression | Organism: Triticum (plant) |
Sequence | String: MDYKDHDGDY KDHDIDYKDD DDKLAADQAM TDQDQGAVTL YSGTAITDAK KNHPFSVKVG LAQVLRGGA IVEVSSVNQA KLAESAGACS VIVSDPVRSR GGVRRMPDPV LIKEVKRAVS V PVMARARV GHFVEAQILE SLAVDYIDES EIISVADDDH FINKHNFRSP ...String: MDYKDHDGDY KDHDIDYKDD DDKLAADQAM TDQDQGAVTL YSGTAITDAK KNHPFSVKVG LAQVLRGGA IVEVSSVNQA KLAESAGACS VIVSDPVRSR GGVRRMPDPV LIKEVKRAVS V PVMARARV GHFVEAQILE SLAVDYIDES EIISVADDDH FINKHNFRSP FICGCRDTGE AL RRIREGA AMIRIQGDLT ATGNIAETVK NVRSLMGEVR VLNNMDDDEV FTFAKKISAP YDL VAQTKQ MGRVPVVQFA SGGITTPADA ALMMQLGCDG VFVGSEVFDG PDPFKKLRSI VQAV QHYND PHVLAEMSSG LENAMESLNV RGDRIQDFGQ GSV |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL | |||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026 kPa Details: Glow discharging conditions on EasyGlow: 10 mA, 30 sec, 0.26 mbar | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: LEICA EM GP / Details: deposition: 3 uL sample, 3 sec blot. |
-Electron microscopy
Microscope | JEOL 3000SFF |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm |
Image recording | Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Number real images: 42 / Average electron dose: 2.0 e/Å2 |