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- PDB-9zr7: Cryo-EM structure of NRAS(Q61K)-BRIL fusion in complex with Fab(B... -

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Basic information

Entry
Database: PDB / ID: 9zr7
TitleCryo-EM structure of NRAS(Q61K)-BRIL fusion in complex with Fab(BAG2) and Monobody(Mb24)
Components
  • GTPase NRas,Soluble cytochrome b562
  • anti-BRIL Fab, BAG2, heavy chain
  • anti-BRIL Fab, BAG2, light chain
KeywordsONCOPROTEIN/Immune System / NRAS / 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
GUANOSINE-5'-DIPHOSPHATE / Soluble cytochrome b562 / GTPase NRas
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsHu, Z. / Koide, S.
Funding support United States, 1items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 10, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GTPase NRas,Soluble cytochrome b562
H: anti-BRIL Fab, BAG2, heavy chain
L: anti-BRIL Fab, BAG2, light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)82,5424
Polymers82,0993
Non-polymers4431
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein GTPase NRas,Soluble cytochrome b562 / Transforming protein N-Ras / Cytochrome b-562


Mass: 34441.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NRAS, cybC / Production host: Escherichia coli (E. coli)
References: UniProt: P12825, UniProt: P0ABE7, small monomeric GTPase
#2: Antibody anti-BRIL Fab, BAG2, heavy chain


Mass: 24390.232 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody anti-BRIL Fab, BAG2, light chain


Mass: 23266.871 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestN
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: Complex of NRAS(Q61K)-BRIL, anti-BRIL Fab(BAG2), and Monobody(Mb24)
Type: COMPLEX / Entity ID: #2-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 47.22 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.17.1_3660model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64660 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0052009
ELECTRON MICROSCOPYf_angle_d0.9182725
ELECTRON MICROSCOPYf_dihedral_angle_d17.715281
ELECTRON MICROSCOPYf_chiral_restr0.053322
ELECTRON MICROSCOPYf_plane_restr0.008355

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