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- EMDB-10203: Escherichia coli AGPase in complex with AMP. Symmetry C2 -

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Basic information

Entry
Database: EMDB / ID: EMD-10203
TitleEscherichia coli AGPase in complex with AMP. Symmetry C2
Map dataSharp map of the full map of the single-particle reconstruction of Escherichia coli AGPase in complex with AMP
Sample
  • Complex: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP
    • Protein or peptide: Glucose-1-phosphate adenylyltransferase
  • Ligand: ADENOSINE MONOPHOSPHATE
KeywordsADP-glucose pyrophosphorilase Complex with AMP inhibitor / TRANSFERASE
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 / Trimeric LpxA-like superfamily / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Glucose-1-phosphate adenylyltransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsCifuente JO / Comino N
Funding support Spain, 1 items
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 JO / Comino N / D'Angelo C / Marina A / Gil-Carton D / Albesa-Jove D / Guerin ME
History
DepositionAug 7, 2019-
Header (metadata) releaseFeb 5, 2020-
Map releaseFeb 5, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6shq
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10203.png

Downloads & links

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Map

FileDownload / File: emd_10203.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharp map of the full map of the single-particle reconstruction of Escherichia coli AGPase in complex with AMP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 200 pix.
= 209.4 Å		1.05 Å/pix.
x 200 pix.
= 209.4 Å		1.05 Å/pix.
x 200 pix.
= 209.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.4
Minimum - Maximum-1.9837079 - 2.9478302
Average (Standard dev.)0.00087202317 (±0.1170972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 209.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z209.400209.400209.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-1.9842.9480.001

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Supplemental data

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Additional map: First half map of the single-particle reconstruction of...

Fileemd_10203_additional_1.map
AnnotationFirst half map of the single-particle reconstruction of Escherichia coli AGPase in complex with AMP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Second half map of the single-particle reconstruction of...

Fileemd_10203_additional_2.map
AnnotationSecond half map of the single-particle reconstruction of Escherichia coli AGPase in complex with AMP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map of the single-particle reconstruction of Escherichia...

Fileemd_10203_additional_3.map
AnnotationFull map of the single-particle reconstruction of Escherichia coli AGPase in complex with AMP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ADP.glucose pyrophosphorylase in complex with the inhibitor AMP

EntireName: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP
Components
  • Complex: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP
    • Protein or peptide: Glucose-1-phosphate adenylyltransferase
  • Ligand: ADENOSINE MONOPHOSPHATE

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Supramolecule #1: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP

SupramoleculeName: ADP.glucose pyrophosphorylase in complex with the inhibitor AMP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Homotetrameric enzyme
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 194 KDa

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Macromolecule #1: Glucose-1-phosphate adenylyltransferase

MacromoleculeName: Glucose-1-phosphate adenylyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: glucose-1-phosphate adenylyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 48.75859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPAVHFGGK FRIIDFALSN CINSGIRRMG VITQYQSHTL VQHIQRGWS FFNEEMNEFV DLLPAQQRMK GENWYRGTAD AVTQNLDIIR RYKAEYVVIL AGDHIYKQDY SRMLIDHVEK G ARCTVACM ...String:
MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPAVHFGGK FRIIDFALSN CINSGIRRMG VITQYQSHTL VQHIQRGWS FFNEEMNEFV DLLPAQQRMK GENWYRGTAD AVTQNLDIIR RYKAEYVVIL AGDHIYKQDY SRMLIDHVEK G ARCTVACM PVPIEEASAF GVMAVDENDK IIEFVEKPAN PPSMPNDPSK SLASMGIYVF DADYLYELLE EDDRDENSSH DF GKDLIPK ITEAGLAYAH PFPLSCVQSD PDAEPYWRDV GTLEAYWKAN LDLASVVPEL DMYDRNWPIR TYNESLPPAK FVQ DRSGSH GMTLNSLVSG GCVISGSVVV QSVLFSRVRV NSFCNIDSAV LLPEVWVGRS CRLRRCVIDR ACVIPEGMVI GENA EEDAR RFYRSEEGIV LVTREMLRKL GHKQER

UniProtKB: Glucose-1-phosphate adenylyltransferase

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Macromolecule #2: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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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

Concentration0.35 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
50.0 mMTris/Cl
100.0 mMNaCl
0.05 mMAdenosine Mono Phosphate
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK II
DetailsSample with single particles and some linear chains of particles

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMax: 80.0 K
DetailsTitan Krios I - Ebic - Diamond Light Source
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5l6s


Details: Calculated density map at 35A resolution
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v2) / Software - details: Non-uniform refinement / Number images used: 94769
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v2) / Software - details: non-uniform refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5l6v


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6shq:
Escherichia coli AGPase in complex with AMP. Symmetry C2

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