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- EMDB-9190: Cell-free-expressed Pyridoxal 5'-phosphate synthase-like subunit ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9190
TitleCell-free-expressed Pyridoxal 5'-phosphate synthase-like subunit (PDX1.2) from Arabidopsis thaliana
Map dataCell-free-expressed PDX1.2 single particle cryo-EM map
Sample
  • Complex: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag
    • Protein or peptide: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsNovikova IV / Evans JE / Hellmann H / Sharma N
CitationJournal: 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
DepositionOct 7, 2018-
Header (metadata) releaseOct 31, 2018-
Map releaseDec 26, 2018-
UpdateDec 26, 2018-
Current statusDec 26, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.368
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.368
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9190.png

Downloads & links

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Map

FileDownload / File: emd_9190.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCell-free-expressed PDX1.2 single particle cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 256 pix.
= 300.032 Å		1.17 Å/pix.
x 256 pix.
= 300.032 Å		1.17 Å/pix.
x 256 pix.
= 300.032 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.172 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.368 / Movie #1: 0.368
Minimum - Maximum-1.3590283 - 2.0836537
Average (Standard dev.)0.020332798 (±0.18892506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 300.032 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1721.1721.172
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z300.032300.032300.032
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.3592.0840.020

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Supplemental data

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Half map: Cell-free-expressed PDX1.2 single particle cryo-EM even half-map

Fileemd_9190_half_map_1.map
AnnotationCell-free-expressed PDX1.2 single particle cryo-EM even half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cell-free-expressed PDX1.2 single particle cryo-EM odd half-map

Fileemd_9190_half_map_2.map
AnnotationCell-free-expressed PDX1.2 single particle cryo-EM odd half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...

EntireName: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag
Components
  • Complex: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag
    • Protein or peptide: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-terminal 3XFLAG tag

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Supramolecule #1: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...

SupramoleculeName: 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
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Triticum (plant)
Molecular weightTheoretical: 440 KDa

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Macromolecule #1: Pyridoxal 5'-phosphate synthase-like subunit PDX1.2 with N-termin...

MacromoleculeName: 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)
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Triticum (plant)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
50.0 mMC4H12NO3CLTris hydrochloride
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: LEICA EM GP / Details: deposition: 3 uL sample, 3 sec blot.

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Electron microscopy

MicroscopeJEOL 3000SFF
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Number real images: 42 / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.6 mm

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Image processing

DetailsCisTEM software used for all image processing
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.33 CUT-OFF / Number images used: 5397
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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