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- EMDB-72476: His-tagged Glutamine Synthetase on a Ni-NTA lipid monolayer grid -

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Basic information

Entry
Database: EMDB / ID: EMD-72476
TitleHis-tagged Glutamine Synthetase on a Ni-NTA lipid monolayer grid
Map datadeepEMhancer sharpened map
Sample
  • Complex: Glutamine synthetase
    • Protein or peptide: Glutamine synthetase
KeywordsEnzyme / BIOSYNTHETIC PROTEIN
Function / homology
Function and homology information


glutamine synthetase / : / glutamine synthetase activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain ...Glutamine synthetase type I / Glutamine synthetase, N-terminal conserved site / Glutamine synthetase signature 1. / Glutamine synthetase, beta-Grasp domain / Glutamine synthetase, glycine-rich site / Glutamine synthetase putative ATP-binding region signature. / Glutamine synthetase (GS) beta-grasp domain profile. / Glutamine synthetase, N-terminal domain superfamily / Glutamine synthetase, catalytic domain / Glutamine synthetase, N-terminal domain / Glutamine synthetase, catalytic domain / Glutamine synthetase (GS) catalytic domain profile. / Glutamine synthetase, catalytic domain / Glutamine synthetase/guanido kinase, catalytic domain
Similarity search - Domain/homology
Glutamine synthetase
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsBaker RW / Strauss JD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM150960 United States
CitationJournal: J Struct Biol / Year: 2025
Title: Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM.
Authors: Aleksandra Skrajna / Clara Lenger / Emily Robinson / Kevin Cannon / Reta Sarsam / Richard G Ouellette / Alberta M Abotsi / Patrick Brennwald / Robert K McGinty / Joshua D Strauss / Richard W Baker /
Abstract: Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air-water interface often leads to denaturation, ...Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air-water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air-water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the "resolution revolution" of cryo-EM. Here, we provide a straightforward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found the lipid affinity grids concentrate particles away from the AWI in thin ice (∼30 nm). We determined three structures ranging from 2.4 to 3.0 Å resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 Å structure of a sub-100 kDa protein without symmetry, demonstrating the utility for a range of biological macromolecules. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air-water interface, and thicker ice.
History
DepositionSep 2, 2025-
Header (metadata) releaseApr 1, 2026-
Map releaseApr 1, 2026-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72476.png

Downloads & links

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Map

FileDownload / File: emd_72476.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 400 pix.
= 349.48 Å		0.87 Å/pix.
x 400 pix.
= 349.48 Å		0.87 Å/pix.
x 400 pix.
= 349.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8737 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0017932593 - 1.8215915
Average (Standard dev.)0.0020821886 (±0.033585384)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 349.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_72476_msk_1.map
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Mask #2

Fileemd_72476_msk_2.map
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Additional map: Locres file for coloring by local resolution

Fileemd_72476_additional_1.map
AnnotationLocres file for coloring by local resolution
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Additional map: Map filtered by local resolution

Fileemd_72476_additional_2.map
AnnotationMap filtered by local resolution
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Additional map: B-factor sharpened map

Fileemd_72476_additional_3.map
AnnotationB-factor sharpened map
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Additional map: Full map

Fileemd_72476_additional_4.map
AnnotationFull map
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Half map: Half-map 1

Fileemd_72476_half_map_1.map
AnnotationHalf-map 1
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Half map: Half-map 2

Fileemd_72476_half_map_2.map
AnnotationHalf-map 2
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Sample components

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Entire : Glutamine synthetase

EntireName: Glutamine synthetase
Components
  • Complex: Glutamine synthetase
    • Protein or peptide: Glutamine synthetase

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Supramolecule #1: Glutamine synthetase

SupramoleculeName: Glutamine synthetase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)

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Macromolecule #1: Glutamine synthetase

MacromoleculeName: Glutamine synthetase / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: glutamine synthetase
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 53.085914 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MPKRTFTKED IRKFAEEENV RYLRLQFTDI LGTIKNVEVP VSQLEKVLDN EMMFDGSSIE GFVRIEESD MYLHPDLDTW VIFPWTAGQG KVARLICDVY KTDGTPFEGD PRANLKRVLK EMEDLGFTDF NLGPEPEFFL F KLDEKGEP ...String:
MGSSHHHHHH SSGLVPRGSH MPKRTFTKED IRKFAEEENV RYLRLQFTDI LGTIKNVEVP VSQLEKVLDN EMMFDGSSIE GFVRIEESD MYLHPDLDTW VIFPWTAGQG KVARLICDVY KTDGTPFEGD PRANLKRVLK EMEDLGFTDF NLGPEPEFFL F KLDEKGEP TLELNDDGGY FDLAPTDLGE NCRRDIVLEL EDMGFDIEAS HHEVAPGQHE IDFKYADAVT ACDNIQTFKL VV KTIARKH NLHATFMPKP LFGVNGSGMH FNVSLFKGKE NAFFDPNTEM GLTETAYQFT AGVLKNARGF TAVCNPLVNS YKR LVPGYE APCYIAWSGK NRSPLIRVPS SRGLSTRIEV RSVDPAANPY MALAAILEAG LDGIKNKLKV PEPVNQNIYE MNRE EREAV GIQDLPSTLY TALKAMRENE VIKKALGNHI YNQFINSKSI EWDYYRTQVS EWERDQYMKQ Y

UniProtKB: Glutamine synthetase

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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

Concentration0.075 mg/mL
BufferpH: 8
GridModel: Quantifoil / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Homogenous refinement tool in cryoSPARC, with defocus and CTF refinement enabled.
Number images used: 96036
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7tf7


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsChimeraX
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9y4a:
His-tagged Glutamine Synthetase on a Ni-NTA lipid monolayer grid

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