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- EMDB-44253: Human asparagine synthetase Arg-142 to Ile-142 (R142I) variant -

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Basic information

Entry
Database: EMDB / ID: EMD-44253
TitleHuman asparagine synthetase Arg-142 to Ile-142 (R142I) variant
Map dataSharpened EM map of ASNS_R142I variant
Sample
  • Complex: human asparagine synthetase dimer form
    • Protein or peptide: Asparagine synthetase [glutamine-hydrolyzing]
Keywordsasparagine / aspartic acid / glutamine / ammonia / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


asparagine synthase (glutamine-hydrolysing) / L-asparagine biosynthetic process / asparagine synthase (glutamine-hydrolyzing) activity / asparagine biosynthetic process / 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 / asparagine biosynthetic process / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsCoricello A / Nardone A / Lupia A / Gratteri C / Vos M / Chaptal V / Alcaro S / Zhu W / Takagi Y / Richards N
Funding support United States, France, United Kingdom, European Union, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM111695 United States
American Cancer SocietyDBG-23-1038947-01-IBCD 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
DepositionMar 25, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44253.png

Downloads & links

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Map

FileDownload / File: emd_44253.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map of ASNS_R142I variant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å		0.93 Å/pix.
x 256 pix.
= 238.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.121
Minimum - Maximum-0.03357301 - 2.0345755
Average (Standard dev.)0.0014055836 (±0.027438726)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 238.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44253_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_44253_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44253_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_44253_half_map_2.map
Projections & Slices
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Sample components

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Entire : human asparagine synthetase dimer form

EntireName: human asparagine synthetase dimer form
Components
  • Complex: human asparagine synthetase dimer form
    • Protein or peptide: Asparagine synthetase [glutamine-hydrolyzing]

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Supramolecule #1: human asparagine synthetase dimer form

SupramoleculeName: human asparagine synthetase dimer form / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 133 KDa

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Macromolecule #1: Asparagine synthetase [glutamine-hydrolyzing]

MacromoleculeName: Asparagine synthetase [glutamine-hydrolyzing] / type: protein_or_peptide / ID: 1
Details: The gaps in sequence represent area of EM map as either no density was found, mostly likely disordered, or/and density was not good enough to place amino acid residues.
Number of copies: 2 / Enantiomer: LEVO / EC number: asparagine synthase (glutamine-hydrolysing)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66.672117 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: CGIWALFGSD DCLSVQCLSA MKIAHRGPDA FRFENVNGYT NCCFGFHRLA VVDPLFGMQP IRVKKYPYLW LCYNGEIYNH KKMQQHFEF EYQTKVDGEI ILHLYDKGGI EQTICMLDGV FAFVLLDTAN KKVFLGRDTY GVIPLFKAMT EDGFLAVCSE A KGLVTLKH ...String:
CGIWALFGSD DCLSVQCLSA MKIAHRGPDA FRFENVNGYT NCCFGFHRLA VVDPLFGMQP IRVKKYPYLW LCYNGEIYNH KKMQQHFEF EYQTKVDGEI ILHLYDKGGI EQTICMLDGV FAFVLLDTAN KKVFLGRDTY GVIPLFKAMT EDGFLAVCSE A KGLVTLKH SATPFLKVEP FLPGHYEVLD LKPNGKVASV EMVKYHHCRD VPLHALYDNV EKLFPGFEIE TVKNNLRILF NN AVKKRLM TDRRIGCLLS GGLDSSLVAA TLLKQLKEAQ VQYPLQTFAI GMEDSPDLLA ARKVADHIGS EHYEVLFNSE EGI QALDEV IFSLETYDIT TVRASVGMYL ISKYIRKNTD SVVIFSGEGS DELTQGYIYF HKAPSPEKAE EESERLLREL YLFD VLRAD RTTAAHGLEL RVPFLDHRFS SYYLSLPPEM RIPKNGIEKH LLRETFEDSN LIPKEILWRP KEAFSDGITS VKNSW FKIL QEYVEHQVDD AMMANAAQKF PFNTPKTKEG YYYRQVFERH YPGRADWLSH YWMPKWINAT DPSARTLTHY KSAVKA AGE NLYFQSHHHH HHHHHH

UniProtKB: Asparagine synthetase [glutamine-hydrolyzing]

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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

Concentration3.2 mg/mL
BufferpH: 8
Details: 25 mM Tris-HCl, pH 8.0, containing 200 mM NaCl, 5 % glycerol and 5 mM beta-mercaptoethanol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm

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Image processing

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

8sue

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 147711
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8sue


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Overall B value: 118.5
Output model

PDB-9b6c:
Human asparagine synthetase Arg-142 to Ile-142 (R142I) variant

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