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- EMDB-8542: Phosphofructokinase-1 Filamnet -

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Basic information

Entry
Database: EMDB / ID: EMD-8542
TitlePhosphofructokinase-1 Filamnet
Map datawildtype phosphofructokinase liver isoform filaments assembled with ATP and fructose-6-phosphate
Sample
  • Complex: Filament of phosphofructokinase-1 liver isoform
Methodhelical reconstruction / negative staining / Resolution: 25.0 Å
AuthorsDosey AM / Kollman JM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM118396 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA197855 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM047414 United States
CitationJournal: J Cell Biol / Year: 2017
Title: The glycolytic enzyme phosphofructokinase-1 assembles into filaments.
Authors: Bradley A Webb / Anne M Dosey / Torsten Wittmann / Justin M Kollman / Diane L Barber /
Abstract: Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that ...Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
History
DepositionDec 27, 2016-
Header (metadata) releaseJan 11, 2017-
Map releaseJan 24, 2018-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.163
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.163
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8542.png

Downloads & links

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Map

FileDownload / File: emd_8542.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationwildtype phosphofructokinase liver isoform filaments assembled with ATP and fructose-6-phosphate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.14 Å/pix.
x 120 pix.
= 496.8 Å		4.14 Å/pix.
x 120 pix.
= 496.8 Å		4.14 Å/pix.
x 120 pix.
= 496.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163 / Movie #1: 0.163
Minimum - Maximum-0.2782783 - 0.5283528
Average (Standard dev.)-0.00129166 (±0.059799593)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 496.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.144.144.14
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z496.800496.800496.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-0.2780.528-0.001

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Supplemental data

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Sample components

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Entire : Filament of phosphofructokinase-1 liver isoform

EntireName: Filament of phosphofructokinase-1 liver isoform
Components
  • Complex: Filament of phosphofructokinase-1 liver isoform

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Supramolecule #1: Filament of phosphofructokinase-1 liver isoform

SupramoleculeName: Filament of phosphofructokinase-1 liver isoform / type: complex / ID: 1 / Parent: 0
Details: PFKL tetramers assembled into filaments by addition of ATP and fructose-6-phosphate
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBac
Molecular weightTheoretical: 42.2 kDa/nm

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Material: uranyl formate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 120 / Average exposure time: 1.2 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 83.3 Å
Applied symmetry - Helical parameters - Δ&Phi: -140 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPIDER (ver. 21.00) / Number images used: 7695
CTF correctionSoftware - Name: CTFFIND (ver. 3.0)
Segment selectionNumber selected: 9324 / Software - Name: Appion Helix Boxer
Startup modelType of model: OTHER / Details: Featureless cylinder
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 21.00)
FSC plot (resolution estimation)

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