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- EMDB-43747: Human liver phosphofructokinase-1 in the R-state conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-43747
TitleHuman liver phosphofructokinase-1 in the R-state conformation
Map dataPFKL R-state tetramer
Sample
  • Complex: PFKL tetramer
    • Protein or peptide: ATP-dependent 6-phosphofructokinase, liver type
  • Ligand: 6-O-phosphono-beta-D-fructofuranose
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 1,6-di-O-phosphono-beta-D-fructofuranose
KeywordsPFK / glycolysis / TRANSFERASE
Function / homology
Function and homology information


fructose binding / 6-phosphofructokinase complex / 6-phosphofructokinase / 6-phosphofructokinase activity / fructose-6-phosphate binding / fructose 6-phosphate metabolic process / monosaccharide binding / canonical glycolysis / fructose 1,6-bisphosphate metabolic process / Glycolysis ...fructose binding / 6-phosphofructokinase complex / 6-phosphofructokinase / 6-phosphofructokinase activity / fructose-6-phosphate binding / fructose 6-phosphate metabolic process / monosaccharide binding / canonical glycolysis / fructose 1,6-bisphosphate metabolic process / Glycolysis / AMP binding / negative regulation of insulin secretion / response to glucose / glycolytic process / kinase binding / secretory granule lumen / ficolin-1-rich granule lumen / Neutrophil degranulation / extracellular exosome / extracellular region / ATP binding / identical protein binding / membrane / metal ion binding / cytosol
Similarity search - Function
ATP-dependent 6-phosphofructokinase, vertebrate-type / ATP-dependent 6-phosphofructokinase, eukaryotic-type / Phosphofructokinase, conserved site / Phosphofructokinase signature. / Phosphofructokinase domain / ATP-dependent 6-phosphofructokinase / Phosphofructokinase superfamily / Phosphofructokinase
Similarity search - Domain/homology
ATP-dependent 6-phosphofructokinase, liver type
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLynch EM / Kollman JM / Webb BA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM149542 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for allosteric regulation of human phosphofructokinase-1.
Authors: Eric M Lynch / Heather Hansen / Lauren Salay / Madison Cooper / Stepan Timr / Justin M Kollman / Bradley A Webb /
Abstract: Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of ...Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
History
DepositionFeb 20, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43747.png

Downloads & links

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Map

FileDownload / File: emd_43747.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPFKL R-state tetramer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 400 pix.
= 337.2 Å		0.84 Å/pix.
x 400 pix.
= 337.2 Å		0.84 Å/pix.
x 400 pix.
= 337.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.843 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.8
Minimum - Maximum-10.088613 - 16.787642000000002
Average (Standard dev.)0.000011553825 (±0.334545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 337.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43747_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43747_half_map_2.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 : PFKL tetramer

EntireName: PFKL tetramer
Components
  • Complex: PFKL tetramer
    • Protein or peptide: ATP-dependent 6-phosphofructokinase, liver type
  • Ligand: 6-O-phosphono-beta-D-fructofuranose
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: 1,6-di-O-phosphono-beta-D-fructofuranose

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Supramolecule #1: PFKL tetramer

SupramoleculeName: PFKL tetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: ATP-dependent 6-phosphofructokinase, liver type

MacromoleculeName: ATP-dependent 6-phosphofructokinase, liver type / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: 6-phosphofructokinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 85.120375 KDa
Recombinant expressionOrganism: unidentified baculovirus
SequenceString: MAAVDLEKLR ASGAGKAIGV LTSGGDAQGM NAAVRAVTRM GIYVGAKVFL IYEGYEGLVE GGENIKQANW LSVSNIIQLG GTIIGSARC KAFTTREGRR AAAYNLVQHG ITNLCVIGGD GSLTGANIFR SEWGSLLEEL VAEGKISETT ARTYSHLNIA G LVGSIDND ...String:
MAAVDLEKLR ASGAGKAIGV LTSGGDAQGM NAAVRAVTRM GIYVGAKVFL IYEGYEGLVE GGENIKQANW LSVSNIIQLG GTIIGSARC KAFTTREGRR AAAYNLVQHG ITNLCVIGGD GSLTGANIFR SEWGSLLEEL VAEGKISETT ARTYSHLNIA G LVGSIDND FCGTDMTIGT DSALHRIMEV IDAITTTAQS HQRTFVLEVM GRHCGYLALV SALASGADWL FIPEAPPEDG WE NFMCERL GETRSRGSRL NIIIIAEGAI DRNGKPISSS YVKDLVVQRL GFDTRVTVLG HVQRGGTPSA FDRILSSKMG MEA VMALLE ATPDTPACVV TLSGNQSVRL PLMECVQMTK EVQKAMDDKR FDEATQLRGG SFENNWNIYK LLAHQKPPKE KSNF SLAIL NVGAPAAGMN AAVRSAVRTG ISHGHTVYVV HDGFEGLAKG QVQEVGWHDV AGWLGRGGSM LGTKRTLPKG QLESI VENI RIYGIHALLV VGGFEAYEGV LQLVEARGRY EELCIVMCVI PATISNNVPG TDFSLGSDTA VNAAMESCDR IKQSAS GTK RRVFIVETMG GYCGYLATVT GIAVGADAAY VFEDPFNIHD LKVNVEHMTE KMKTDIQRGL VLRNEKCHDY YTTEFLY NL YSSEGKGVFD CRTNVLGHLQ QGGAPTPFDR NYGTKLGVKA MLWLSEKLRE VYRKGRVFAN APDSACVIGL KKKAVAFS P VTELKKDTDF EHRMPREQWW LSLRLMLKML AQYRISMAAY VSGELEHVTR RTLSMDKGF

UniProtKB: ATP-dependent 6-phosphofructokinase, liver type

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Macromolecule #2: 6-O-phosphono-beta-D-fructofuranose

MacromoleculeName: 6-O-phosphono-beta-D-fructofuranose / type: ligand / ID: 2 / Number of copies: 4 / Formula: F6P
Molecular weightTheoretical: 260.136 Da
Chemical component information

ChemComp-F6P:
6-O-phosphono-beta-D-fructofuranose

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: 1,6-di-O-phosphono-beta-D-fructofuranose

MacromoleculeName: 1,6-di-O-phosphono-beta-D-fructofuranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: FBP
Molecular weightTheoretical: 340.116 Da
Chemical component information

ChemComp-FBP:
1,6-di-O-phosphono-beta-D-fructofuranose

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 63.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 82731
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC

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Atomic model buiding 1

Initial modelChain - Source name: Modeller / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8w2g:
Human liver phosphofructokinase-1 in the R-state conformation

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