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- EMDB-74594: Cryo-EM structure of NRAS(Q61K)-BRIL fusion in complex with Fab(B... -

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Basic information

Entry
Database: EMDB / ID: EMD-74594
TitleCryo-EM structure of NRAS(Q61K)-BRIL fusion in complex with Fab(BAG2) and Monobody(Mb24)
Map datastructure of NRAS(Q61K)-BRIL fusion in complex with Fab(BAG2) and Monobody(Mb24)
Sample
  • Complex: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)
    • Protein or peptide: anti-BRIL Fab, BAG2, heavy chain
    • Protein or peptide: anti-BRIL Fab, BAG2, light chain
  • Protein or peptide: GTPase NRas,Soluble cytochrome b562
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
KeywordsNRAS / BRIL-fusion / ONCOPROTEIN / ONCOPROTEIN-Immune System complex
Function / homology
Function and homology information


positive regulation of endothelial cell proliferation / small monomeric GTPase / electron transport chain / G protein activity / Ras protein signal transduction / periplasmic space / electron transfer activity / iron ion binding / Golgi membrane / GTPase activity ...positive regulation of endothelial cell proliferation / small monomeric GTPase / electron transport chain / G protein activity / Ras protein signal transduction / periplasmic space / electron transfer activity / iron ion binding / Golgi membrane / GTPase activity / heme binding / GTP binding / protein-containing complex binding / plasma membrane
Similarity search - Function
Small GTPase, Ras-type / Small GTPase Ras domain profile. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases ...Small GTPase, Ras-type / Small GTPase Ras domain profile. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Soluble cytochrome b562 / GTPase NRas
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsHu Z / Koide S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: J Mol Biol / Year: 2026
Title: Protein Engineering-Enabled Cryo-EM Investigation of Small GTPases.
Authors: Zhengshan Hu / Unnatiben Rajeshbhai Patel / Eliezra Glasser / Akiko Koide / Shohei Koide /
Abstract: Small GTPases play important roles in cellular signaling. Due to their small sizes (∼21 kDa), structural studies of small GTPases have been predominantly performed using x-ray crystallography in ...Small GTPases play important roles in cellular signaling. Due to their small sizes (∼21 kDa), structural studies of small GTPases have been predominantly performed using x-ray crystallography in which crystal lattice contacts made it challenging to define unperturbed conformations of the key switch regions. Here, we developed a protein-engineering strategy that enables cryo-EM analysis of small soluble proteins and applied to RAS. We fused the C-terminal α5 helix of the RAS globular domain to a small protein BRIL by forming a continuous helix, which leaves most RAS surfaces exposed to the solvent and unperturbed, followed by the complex formation with an anti-BRIL Fab. This engineered complex with an increased molecular weight, termed "RAS-lollipop", enabled single-particle cryo-EM of RAS. Using this approach, we determined the cryo-EM structure of NRAS, whose structural studies using crystallography have been the least successful among the RAS isoforms. We revealed the conformations of the switch region and α 5 helix that differ from those observed in published crystal structures, and also defined the binding site of an NRAS-specific monobody. We uncovered an unexpected surfactant-like property of this monobody, which reduces orientation biases of particles on cryo-EM grids. Together, this work establishes a platform for visualizing small GTPases and potentially other small proteins with minimal perturbation of their surfaces.
History
DepositionDec 19, 2025-
Header (metadata) releaseJun 10, 2026-
Map releaseJun 10, 2026-
UpdateJun 10, 2026-
Current statusJun 10, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_74594.png

Downloads & links

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Map

FileDownload / File: emd_74594.map.gz / Format: CCP4 / Size: 113.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of NRAS(Q61K)-BRIL fusion in complex with Fab(BAG2) and Monobody(Mb24)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 310 pix.
= 255.75 Å		0.83 Å/pix.
x 310 pix.
= 255.75 Å		0.83 Å/pix.
x 310 pix.
= 255.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0985
Minimum - Maximum-0.6873789 - 1.1509557
Average (Standard dev.)0.00009865826 (±0.012224767)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions310310310
Spacing310310310
CellA=B=C: 255.75 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_74594_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_74594_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)

EntireName: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)
Components
  • Complex: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)
    • Protein or peptide: anti-BRIL Fab, BAG2, heavy chain
    • Protein or peptide: anti-BRIL Fab, BAG2, light chain
  • Protein or peptide: GTPase NRas,Soluble cytochrome b562
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)

SupramoleculeName: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GTPase NRas,Soluble cytochrome b562

MacromoleculeName: GTPase NRas,Soluble cytochrome b562 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.441625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKHHHHHHSS GLNDIFEAQK IEWHEENLYF QGSMTEYKLV VVGAGGVGKS ALTIQLIQNH FVDEYDPTIE DSYRKQVVID GETSLLDIL DTAGKEEYSA MRDQYMRTGE GFLLVFAINN SKSFADINLY REQIKRVKDS DDVPMVLVGN KSDLPTRTVD T KQAHELAK ...String:
MKHHHHHHSS GLNDIFEAQK IEWHEENLYF QGSMTEYKLV VVGAGGVGKS ALTIQLIQNH FVDEYDPTIE DSYRKQVVID GETSLLDIL DTAGKEEYSA MRDQYMRTGE GFLLVFAINN SKSFADINLY REQIKRVKDS DDVPMVLVGN KSDLPTRTVD T KQAHELAK SYGIPFIETS AKTRQGVEDA FYTLVREIRQ YRMADLEDNW ETLNDNLKVI EKADNAAQVK DALTKMRAAA LD AQKATPP KLEDKSPDSP EMKDFRHGFD ILVGQIDDAL KLANEGKVKE AQAAAEQLKT TRNAYIQ

UniProtKB: GTPase NRas, Soluble cytochrome b562

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Macromolecule #2: anti-BRIL Fab, BAG2, heavy chain

MacromoleculeName: anti-BRIL Fab, BAG2, heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.390232 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVFNQI KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVFNQI KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS

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Macromolecule #3: anti-BRIL Fab, BAG2, light chain

MacromoleculeName: anti-BRIL Fab, BAG2, light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.266871 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRG

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Macromolecule #4: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.22 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 64660
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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