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- PDB-9b0i: Cryo-EM Structure of Sf9 produced recombinant N-acetyltransferase... -

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Basic information

Entry
Database: PDB / ID: 9b0i
TitleCryo-EM Structure of Sf9 produced recombinant N-acetyltransferase 10 (NAT10) in complex with cytidine-amide-CoA bisubstrate probe and ADP/Mg2+.
ComponentsRNA cytidine acetyltransferase
KeywordsRNA BINDING PROTEIN / ac4C modification / RNA acetyltransferase
Function / homology
Function and homology information


rRNA acetylation involved in maturation of SSU-rRNA / tRNA N-acetyltransferase activity / tRNA acetylation / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / nucleolus / ATP binding
Similarity search - Function
Possible tRNA binding domain / RNA cytidine acetyltransferase NAT10 / Possible tRNA binding domain / Helicase domain / tRNA(Met) cytidine acetyltransferase TmcA, N-terminal / TmcA/NAT10/Kre33 / Helicase / tRNA(Met) cytidine acetyltransferase TmcA, N-terminal / GNAT acetyltransferase 2 / GNAT domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / ADENOSINE-5'-DIPHOSPHATE / RNA cytidine acetyltransferase
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsZhou, M. / Marmorstein, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM11890 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)ZIABC011488 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01AG31862 United States
CitationJournal: bioRxiv / Year: 2024
Title: Molecular Basis for RNA Cytidine Acetylation by NAT10.
Authors: Mingyang Zhou / Supuni Thalalla Gamage / Khoa A Tran / David Bartee / Xuepeng Wei / Boyu Yin / Shelley Berger / Jordan L Meier / Ronen Marmorstein
Abstract: Human NAT10 acetylates the N4 position of cytidine in RNA, predominantly on rRNA and tRNA, to facilitate ribosome biogenesis and protein translation. NAT10 has been proposed as a therapeutic target ...Human NAT10 acetylates the N4 position of cytidine in RNA, predominantly on rRNA and tRNA, to facilitate ribosome biogenesis and protein translation. NAT10 has been proposed as a therapeutic target in cancers as well as aging-associated pathologies such as Hutchinson-Gilford Progeria Syndrome (HGPS). The ∼120 kDa NAT10 protein uses its acetyl-CoA-dependent acetyltransferase, ATP-dependent helicase, and RNA binding domains in concert to mediate RNA-specific N4-cytidine acetylation. While the biochemical activity of NAT10 is well known, the molecular basis for catalysis of eukaryotic RNA acetylation remains relatively undefined. To provide molecular insights into the RNA-specific acetylation by NAT10, we determined the single particle cryo-EM structures of NAT10 ( NAT10) bound to a bisubstrate cytidine-CoA probe with and without ADP. The structures reveal that NAT10 forms a symmetrical heart-shaped dimer with conserved functional domains surrounding the acetyltransferase active sites harboring the cytidine-CoA probe. Structure-based mutagenesis with analysis of mutants supports the catalytic role of two conserved active site residues (His548 and Tyr549 in NAT10), and two basic patches, both proximal and distal to the active site for RNA-specific acetylation. Yeast complementation analyses and senescence assays in human cells also implicates NAT10 catalytic activity in yeast thermoadaptation and cellular senescence. Comparison of the NAT10 structure to protein lysine and N-terminal acetyltransferase enzymes reveals an unusually open active site suggesting that these enzymes have been evolutionarily tailored for RNA recognition and cytidine-specific acetylation.
History
DepositionMar 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA cytidine acetyltransferase
B: RNA cytidine acetyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,9568
Polymers242,9522
Non-polymers3,0056
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Mass photometry and gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA cytidine acetyltransferase / 18S rRNA cytosine acetyltransferase


Mass: 121475.914 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermochaetoides thermophila DSM 1495 (fungus)
Gene: NAT10, CTHT_0016220 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: G0S273, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-A1AH4 / [[(2~{R},3~{S},4~{S},5~{R})-5-(6-aminopurin-9-yl)-4-oxidanyl-3-phosphonooxy-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] [(3~{R})-4-[[3-[2-[2-[[1-[(2~{R},3~{S},4~{R},5~{R})-5-(hydroxymethyl)-3,4-bis(oxidanyl)oxolan-2-yl]-2-oxidanylidene-pyrimidin-4-yl]amino]-2-oxidanylidene-ethyl]sulfanylethylamino]-3-oxidanylidene-propyl]amino]-2,2-dimethyl-3-oxidanyl-4-oxidanylidene-butyl] hydrogen phosphate


Mass: 1050.772 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H49N10O22P3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemically synthetic cytidine-amide-CoA bisubstrate probe
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 244 kDa/nm / Experimental value: YES
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 42.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.9.8.7model fitting
13PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162769 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: The unpublished cryo-EM structure in complex with cytidine-amide-CoA bisubstrate probe and ADP
Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313308
ELECTRON MICROSCOPYf_angle_d0.59318048
ELECTRON MICROSCOPYf_dihedral_angle_d5.9961802
ELECTRON MICROSCOPYf_chiral_restr0.0412060
ELECTRON MICROSCOPYf_plane_restr0.0052278

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