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- PDB-8w2j: Human liver phosphofructokinase-1 filament in the T-state conformation -

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Basic information

Entry
Database: PDB / ID: 8w2j
TitleHuman liver phosphofructokinase-1 filament in the T-state conformation
ComponentsATP-dependent 6-phosphofructokinase, liver type
KeywordsTRANSFERASE / PFK / glycolysis
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
ADENOSINE-5'-TRIPHOSPHATE / 1,6-di-O-phosphono-beta-D-fructofuranose / ATP-dependent 6-phosphofructokinase, liver type
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLynch, E.M. / Kollman, J.M. / Webb, B.A.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Sep 25, 2024Group: Data collection / Database references / Category: citation_author / em_admin
Item: _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: ATP-dependent 6-phosphofructokinase, liver type
F: ATP-dependent 6-phosphofructokinase, liver type
G: ATP-dependent 6-phosphofructokinase, liver type
H: ATP-dependent 6-phosphofructokinase, liver type
A: ATP-dependent 6-phosphofructokinase, liver type
B: ATP-dependent 6-phosphofructokinase, liver type
C: ATP-dependent 6-phosphofructokinase, liver type
D: ATP-dependent 6-phosphofructokinase, liver type
hetero molecules


Theoretical massNumber of molelcules
Total (without water)696,05148
Polymers680,9638
Non-polymers15,08840
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ATP-dependent 6-phosphofructokinase, liver type / ATP-PFK / PFK-L / 6-phosphofructokinase type B / Phosphofructo-1-kinase isozyme B / PFK-B / Phosphohexokinase


Mass: 85120.375 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PFKL / Production host: unidentified baculovirus / References: UniProt: P17858, 6-phosphofructokinase
#2: Sugar
ChemComp-FBP / 1,6-di-O-phosphono-beta-D-fructofuranose / BETA-FRUCTOSE-1,6-DIPHOSPHATE / FRUCTOSE-1,6-BISPHOSPHATE / 1,6-di-O-phosphono-beta-D-fructose / 1,6-di-O-phosphono-D-fructose / 1,6-di-O-phosphono-fructose


Type: D-saccharide, beta linking / Mass: 340.116 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H14O12P2 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
b-D-Fruf1PO36PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
#3: Chemical...
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PFKL filament / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: unidentified baculovirus
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 90 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9ISOLDEmodel refinement
10PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45362 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: Modeller / Type: in silico model

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