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- PDB-6si8: Escherichia coli AGPase in complex with AMP. -

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Basic information

Entry
Database: PDB / ID: 6si8
TitleEscherichia coli AGPase in complex with AMP.
ComponentsGlucose-1-phosphate adenylyltransferase
KeywordsTRANSFERASE / ADP-glucose pyrophosphorilase Complex with AMP inhibitor
Function / homology
Function and homology information


glucose-1-phosphate adenylyltransferase complex / glucose-1-phosphate adenylyltransferase / glucose-1-phosphate adenylyltransferase activity / glycogen biosynthetic process / AMP binding / protein homotetramerization / magnesium ion binding / ATP binding / identical protein binding
Similarity search - Function
Glucose-1-phosphate adenylyltransferase GlgC, bacterial / ADP-glucose pyrophosphorylase, conserved site / Glucose-1-phosphate adenylyltransferase / ADP-glucose pyrophosphorylase signature 1. / ADP-glucose pyrophosphorylase signature 2. / ADP-glucose pyrophosphorylase signature 3. / Nucleotidyl transferase domain / Nucleotidyl transferase / Hexapeptide repeat proteins / UDP N-Acetylglucosamine Acyltransferase; domain 1 ...Glucose-1-phosphate adenylyltransferase GlgC, bacterial / ADP-glucose pyrophosphorylase, conserved site / Glucose-1-phosphate adenylyltransferase / ADP-glucose pyrophosphorylase signature 1. / ADP-glucose pyrophosphorylase signature 2. / ADP-glucose pyrophosphorylase signature 3. / Nucleotidyl transferase domain / Nucleotidyl transferase / Hexapeptide repeat proteins / UDP N-Acetylglucosamine Acyltransferase; domain 1 / Trimeric LpxA-like superfamily / 3 Solenoid / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Nucleotide-diphospho-sugar transferases / Alpha-Beta Complex / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / Glucose-1-phosphate adenylyltransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsCifuente, J.O. / Comino, N. / D'Angelo, C. / Marina, A. / Gil-Carton, D. / Albesa-Jove, D. / Guerin, M.E.
Funding support Spain, 1items
OrganizationGrant numberCountry
Spanish Ministry of Economy and Competitiveness Spain
Citation
Journal: Curr Res Struct Biol / Year: 2020
Title: The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM.
Authors: Javier O Cifuente / Natalia Comino / Cecilia D'Angelo / Alberto Marina / David Gil-Carton / David Albesa-Jové / Marcelo E Guerin /
Abstract: Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for ...Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from (AGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a "locked" to a "free" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of AGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of AGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail.
#1: Journal: Biorxiv / Year: 2020
Title: The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM
Authors: Cifuente, J.O. / Comino, N. / D'Angelo, C. / Marina, A. / Gil-Carton, D. / Albesa-Jove, D. / Guerin, M.E.
History
DepositionAug 9, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 21, 2021Group: Database references / Category: citation / citation_author
Revision 1.2May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Glucose-1-phosphate adenylyltransferase
B: Glucose-1-phosphate adenylyltransferase
D: Glucose-1-phosphate adenylyltransferase
C: Glucose-1-phosphate adenylyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,4238
Polymers195,0344
Non-polymers1,3894
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18590 Å2
ΔGint-59 kcal/mol
Surface area63260 Å2
MethodPISA

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Components

#1: Protein
Glucose-1-phosphate adenylyltransferase / / ADP-glucose pyrophosphorylase / ADPGlc PPase / ADP-glucose synthase


Mass: 48758.590 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: glgC, b3430, JW3393 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A6V1, glucose-1-phosphate adenylyltransferase
#2: Chemical
ChemComp-AMP / ADENOSINE MONOPHOSPHATE / Adenosine monophosphate


Mass: 347.221 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
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: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP
Type: COMPLEX / Details: Homotetrameric enzyme / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.194 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMTris/Cl1
2100 mMNaClSodium chloride1
30.05 mMAdenosine Mono Phosphate1
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample with single particles and some linear chains of particles
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Titan Krios I - Ebic - Diamond Light Source
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11cryoSPARCv2final Euler assignment
13cryoSPARCv23D reconstructionNon-uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94769 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 5L6V

5l6v


Accession code: 5L6V / Source name: PDB / Type: experimental model

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