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- PDB-8sue: Human asparagine synthetase (apo-ASNS) -

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Basic information

Entry
Database: PDB / ID: 8sue
TitleHuman asparagine synthetase (apo-ASNS)
ComponentsAsparagine synthetase [glutamine-hydrolyzing]
KeywordsBIOSYNTHETIC PROTEIN / asparagine / aspartic acid / glutamine / ammonia
Function / homology
Function and homology information


asparagine synthase (glutamine-hydrolysing) / L-asparagine biosynthetic process / asparagine synthase (glutamine-hydrolyzing) activity / : / Response of EIF2AK1 (HRI) to heme deficiency / Aspartate and asparagine metabolism / ATF4 activates genes in response to endoplasmic reticulum stress / Response of EIF2AK4 (GCN2) to amino acid deficiency / cellular response to glucose starvation / positive regulation of mitotic cell cycle ...asparagine synthase (glutamine-hydrolysing) / L-asparagine biosynthetic process / asparagine synthase (glutamine-hydrolyzing) activity / : / Response of EIF2AK1 (HRI) to heme deficiency / Aspartate and asparagine metabolism / ATF4 activates genes in response to endoplasmic reticulum stress / Response of EIF2AK4 (GCN2) to amino acid deficiency / cellular response to glucose starvation / positive regulation of mitotic cell cycle / negative regulation of apoptotic process / ATP binding / cytosol
Similarity search - Function
Asparagine synthase, glutamine-hydrolyzing / Asparagine synthase, N-terminal domain / : / Glutamine amidotransferase domain / Asparagine synthase / Asparagine synthase / Glutamine amidotransferase type 2 domain profile. / Glutamine amidotransferase type 2 domain / Nucleophile aminohydrolases, N-terminal / Rossmann-like alpha/beta/alpha sandwich fold
Similarity search - Domain/homology
Asparagine synthetase [glutamine-hydrolyzing]
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsCoricello, A. / Zhu, W. / Lupia, A. / Gratteri, C. / Vos, M. / Chaptal, V. / Alcaro, S. / Takagi, Y. / Richards, N.
Funding support United States, France, United Kingdom, European Union, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM111695 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD028723 United States
Centre National de la Recherche Scientifique (CNRS)ANR-19-CE11-0023-01 France
Biotechnology and Biological Sciences Research Council (BBSRC)P/0118017/1 United Kingdom
European CommissionFESR FSE 2014- 2020European Union
CitationJournal: Nat Commun / Year: 2024
Title: 3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase.
Authors: Adriana Coricello / Alanya J Nardone / Antonio Lupia / Carmen Gratteri / Matthijn Vos / Vincent Chaptal / Stefano Alcaro / Wen Zhu / Yuichiro Takagi / Nigel G J Richards /
Abstract: Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other ...Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.
History
DepositionMay 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
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: Asparagine synthetase [glutamine-hydrolyzing]
B: Asparagine synthetase [glutamine-hydrolyzing]


Theoretical massNumber of molelcules
Total (without water)128,6492
Polymers128,6492
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Asparagine synthetase [glutamine-hydrolyzing] / Cell cycle control protein TS11 / Glutamine-dependent asparagine synthetase


Mass: 64324.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ASNS, TS11
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P08243, asparagine synthase (glutamine-hydrolysing)
Has protein modificationN

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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: human asparagine synthetase dimer form / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.133 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 8
Details: 25 mM Tris-HCl, pH 8.0, containing 250 mM NaCl and 5 mM DTT.
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 9
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: dev_4893 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47929 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6GQ3

6gq3


Accession code: 6GQ3 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.66 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00258212
ELECTRON MICROSCOPYf_angle_d0.557411113
ELECTRON MICROSCOPYf_chiral_restr0.0411210
ELECTRON MICROSCOPYf_plane_restr0.00391430
ELECTRON MICROSCOPYf_dihedral_angle_d4.02641095

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