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- PDB-8v1y: Composite map of AMPylated GlnA bound to hinT -

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Basic information

Entry
Database: PDB / ID: 8v1y
TitleComposite map of AMPylated GlnA bound to hinT
Components
  • Glutamine synthetase
  • Purine nucleoside phosphoramidase
KeywordsLIGASE/HYDROLASE / AMPylation / adenyltransferase / nucleotide binding protein / LIGASE-HYDROLASE complex
Function / homology
Function and homology information


ammonia assimilation cycle / D-alanine catabolic process / nitrogen utilization / Hydrolases; Acting on phosphorus-nitrogen bonds / adenosine 5'-monophosphoramidase activity / glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / response to radiation / nucleotide binding ...ammonia assimilation cycle / D-alanine catabolic process / nitrogen utilization / Hydrolases; Acting on phosphorus-nitrogen bonds / adenosine 5'-monophosphoramidase activity / glutamine synthetase / glutamine biosynthetic process / glutamine synthetase activity / response to radiation / nucleotide binding / protein homodimerization activity / ATP binding / metal ion binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
Glutamine synthetase class-I, adenylation site / Glutamine synthetase class-I adenylation site. / Glutamine synthetase type I / Histidine triad (HIT) protein / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / HIT domain / Glutamine synthetase putative ATP-binding region signature. ...Glutamine synthetase class-I, adenylation site / Glutamine synthetase class-I adenylation site. / Glutamine synthetase type I / Histidine triad (HIT) protein / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / HIT domain / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Histidine triad, conserved site / HIT domain signature. / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain / Glutamine synthetase, catalytic domain / HIT domain profile. / Glutamine synthetase (GS) catalytic domain profile. / Glutamine synthetase, catalytic domain / HIT-like domain / HIT-like superfamily / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / Glutamine synthetase / Purine nucleoside phosphoramidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHan, Y. / Sreelatha, A. / Gonzalez, A. / Chen, Z.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK123194 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR190106 United States
Welch FoundationI-2046-20200401 United States
Welch FoundationI-2046-20230405 United States
CitationJournal: Nat Commun / Year: 2025
Title: A repurposed AMP binding domain reveals mitochondrial protein AMPylation as a regulator of cellular metabolism.
Authors: Abner Gonzalez / Alex Pon / Kelly Servage / Krzysztof Pawłowski / Yan Han / Anju Sreelatha /
Abstract: Protein AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates, has been known as a post translational modification for over 50 years. Research in this field is ...Protein AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates, has been known as a post translational modification for over 50 years. Research in this field is largely underdeveloped due to the lack of tools that enable the systematic identification of AMPylated substrates. Here, we address this gap by developing an enrichment technique to isolate and study AMPylated proteins using a nucleotide-binding protein, hinT. Cryo-EM reconstruction of an AMPylated protein bound to hinT provides a structural basis for AMP selectivity. Using structure guided mutagenesis, we optimize enrichment to identify novel substrates of the evolutionarily conserved AMPylase, Selenoprotein O. We show that mammalian Selenoprotein O regulates metabolic flux through AMPylation of key mitochondrial proteins including glutamate dehydrogenase and pyruvate dehydrogenase. Our findings highlight the broader significance of AMPylation, an emerging post translational modification with critical roles in signal transduction and disease pathology. Furthermore, we establish a powerful enrichment platform for the discovery of novel AMPylated proteins to study the mechanisms and significance of protein AMPylation in cellular function.
History
DepositionNov 21, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamine synthetase
B: Glutamine synthetase
C: Purine nucleoside phosphoramidase
D: Purine nucleoside phosphoramidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,1146
Polymers132,4204
Non-polymers6942
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 Glutamine synthetase / GS / Glutamate--ammonia ligase / Glutamine synthetase I beta / GSI beta


Mass: 52572.250 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: glnA, b3870, JW3841 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A9C5, glutamine synthetase
#2: Protein Purine nucleoside phosphoramidase / Histidine triad nucleotide binding protein HinT / HIT protein


Mass: 13637.674 Da / Num. of mol.: 2 / Mutation: H101N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: hinT, ycfF, b1103, JW1089 / Production host: Escherichia coli (E. coli)
References: UniProt: P0ACE7, Hydrolases; Acting on phosphorus-nitrogen bonds
#3: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
Has ligand of interestY
Has protein modificationY

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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: GlnA dimer binding to hinT dimer / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.125 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 900 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEM4image acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10RELION4.0.1initial Euler assignment
11RELION4.0.1final Euler assignment
12RELION4.0.1classification
13RELION4.0.13D reconstruction
14PHENIX1.20.1-4487model refinement
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225422 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementResolution: 2.7→2.7 Å / Cor.coef. Fo:Fc: 0.877 / SU B: 16.198 / SU ML: 0.292 / ESU R: 0.305
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.31449 --
obs0.31449 121185 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 81.607 Å2
Refinement stepCycle: 1 / Total: 8918
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0129116
ELECTRON MICROSCOPYr_bond_other_d00.0168543
ELECTRON MICROSCOPYr_angle_refined_deg1.2221.65512347
ELECTRON MICROSCOPYr_angle_other_deg0.3741.57619693
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.6451125
ELECTRON MICROSCOPYr_dihedral_angle_2_deg3.771560
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.867101516
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0620.21358
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0210729
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022055
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it13.0516.5014536
ELECTRON MICROSCOPYr_mcbond_other13.0516.5014536
ELECTRON MICROSCOPYr_mcangle_it19.65111.7585649
ELECTRON MICROSCOPYr_mcangle_other19.65211.7615650
ELECTRON MICROSCOPYr_scbond_it16.7799.0744580
ELECTRON MICROSCOPYr_scbond_other16.7779.0764581
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other27.98215.4126699
ELECTRON MICROSCOPYr_long_range_B_refined34.20787.7736661
ELECTRON MICROSCOPYr_long_range_B_other34.20687.7836662
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.448 8971 -
obs--100 %

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