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- PDB-9z2w: CryoEM structure of human NSUN2(C271A) with SAH cross-linked to t... -

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Basic information

Entry
Database: PDB / ID: 9z2w
TitleCryoEM structure of human NSUN2(C271A) with SAH cross-linked to tRNA Lys(TTT) (No D-arm conformation)
Components
  • RNA cytosine C(5)-methyltransferase NSUN2
  • tRNA Lys(TTT)
KeywordsTRANSFERASE / Methyltransferase / tRNA / Complex
Function / homology
Function and homology information


tRNA (cytosine34-C5)-methyltransferase / meiotic cell cycle checkpoint signaling / tRNA (cytidine-N5)-methyltransferase activity / tRNA stabilization / mRNA (cytidine-5-)-methyltransferase activity / regulation of mRNA export from nucleus / tRNA modification in the nucleus and cytosol / chromatoid body / hair follicle maturation / tRNA modification ...tRNA (cytosine34-C5)-methyltransferase / meiotic cell cycle checkpoint signaling / tRNA (cytidine-N5)-methyltransferase activity / tRNA stabilization / mRNA (cytidine-5-)-methyltransferase activity / regulation of mRNA export from nucleus / tRNA modification in the nucleus and cytosol / chromatoid body / hair follicle maturation / tRNA modification / tRNA methylation / regulation of stem cell differentiation / spermatid development / Transferases; Transferring one-carbon groups; Methyltransferases / spindle / mRNA processing / in utero embryonic development / tRNA binding / cell division / nucleolus / mitochondrion / RNA binding / extracellular exosome / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
tRNA (C5-cytosine) methyltransferase, NCL1 / : / : / NSUN2 Pre-PUA domain / NSUN2 PUA domain / SAM-dependent methyltransferase RsmB/NOP2-type / RNA (C5-cytosine) methyltransferase / : / 16S rRNA methyltransferase RsmB/F / SAM-dependent MTase RsmB/NOP-type domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / RNA / RNA (> 10) / RNA cytosine C(5)-methyltransferase NSUN2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCanepa, J. / Ruiz-Arroyo, V.M. / Nam, Y.
Funding support United States, 8items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122960 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA258589 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)R01ES038329 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM131963 United States
Welch FoundationI-2115-20220331 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP210041 United States
American Cancer SocietyPF-24-1308804-01-RMC United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP170644 United States
CitationJournal: Nature / Year: 2026
Title: Substrate selectivity of the human RNA mC methyltransferase NSUN2.
Authors: Jacob Canepa / Victor M Ruiz-Arroyo / Netanya S Schlamowitz / Yunsun Nam /
Abstract: Specific deposition of RNA modifications is important for regulating gene expression. 5-Methylcytosine (mC) is a common epitranscriptomic modification, and NSUN2 is a key enzyme responsible for mC ...Specific deposition of RNA modifications is important for regulating gene expression. 5-Methylcytosine (mC) is a common epitranscriptomic modification, and NSUN2 is a key enzyme responsible for mC methylation of various types of RNA. Dysregulation of NSUN2 is associated with numerous diseases, including cancers and neurological disorders. The versatility of NSUN2 complicates our understanding of its substrate specificity and molecular roles in biology and disease. Here we show how NSUN2 interacts with RNA substrates at distinct stages of its catalytic cycle to modify cytidines. Furthermore, we show the role of RNA structure in facilitating NSUN2 activity at multiple tRNA positions. We identify RNA duplexes surrounding the mC modification site as crucial recognition elements for methylation, which enabled us to derive a minimized substrate that captures the preferred features of an NSUN2 substrate-a dual-stem structure containing the CNNRR motif at the 5' end of the first stem. Insights into the mechanisms underlying substrate-specific NSUN2 enzymatic activity provide opportunities for understanding and therapeutically targeting NSUN2-dependent methylation. Overall, our work highlights the roles of RNA structure and sequence in defining substrate specificity and regulating RNA-modifying enzymes.
History
DepositionNov 5, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 10, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jun 10, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA cytosine C(5)-methyltransferase NSUN2
B: tRNA Lys(TTT)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,2013
Polymers112,8162
Non-polymers3841
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA cytosine C(5)-methyltransferase NSUN2 / Myc-induced SUN domain-containing protein / Misu / NOL1/NOP2/Sun domain family member 2 / Substrate ...Myc-induced SUN domain-containing protein / Misu / NOL1/NOP2/Sun domain family member 2 / Substrate of AIM1/Aurora kinase B / mRNA cytosine C(5)-methyltransferase / tRNA cytosine C(5)-methyltransferase / tRNA methyltransferase 4 homolog / hTrm4


Mass: 88382.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NSUN2, SAKI, TRM4 / Production host: Escherichia coli (E. coli)
References: UniProt: Q08J23, Transferases; Transferring one-carbon groups; Methyltransferases, tRNA (cytosine34-C5)-methyltransferase
#2: RNA chain tRNA Lys(TTT)


Mass: 24433.492 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S
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: NSUN2(C271A) and tRNA Lys(TTT) adduct with SAH / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
10.02 MTris(HOCH2)3CNH21
20.1 MSodium ChlorideNaCl1
30.002 MMagnesium ChlorideMgCl21
40.005 MDithiothreitol(CH(OH)CH2SH)21
50.02 % (w/v)NP40H(C2H4O)nO(C6H4)C9H191
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 magnification: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.1 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6704
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM4.1image acquisition
4cryoSPARCCTF correction
7Coot0.9.8.7model fitting
9PHENIX1.21.1_5286model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 738000
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173000 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1 / Chain-ID: A

IDPDB-IDPdb chain-IDAccession codeChain residue rangeInitial refinement model-IDSource nameTypePdb chain residue range
1AF-Q08J23-F1-v436-7061AlphaFoldin silico model
21FIR

1fir

A1FIR1-732PDBexperimental model1-73
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026500
ELECTRON MICROSCOPYf_angle_d0.4829126
ELECTRON MICROSCOPYf_dihedral_angle_d19.0511611
ELECTRON MICROSCOPYf_chiral_restr0.0381065
ELECTRON MICROSCOPYf_plane_restr0.004918

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