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- EMDB-44042: Cryo-EM Structure of Sf9 produced recombinant N-acetyltransferase... -

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Entry
Database: EMDB / ID: EMD-44042
TitleCryo-EM Structure of Sf9 produced recombinant N-acetyltransferase 10 (NAT10) in complex with cytidine-amide-CoA bisubstrate probe and ADP/Mg2+.
Map data
Sample
  • Complex: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemically synthetic cytidine-amide-CoA bisubstrate probe
    • Protein or peptide: RNA cytidine acetyltransferase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: [[(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
KeywordsRNA binding protein / ac4C modification / RNA acetyltransferase
Function / homology
Function and homology information


tRNA wobble cytosine modification / rRNA acetylation involved in maturation of SSU-rRNA / rRNA cytidine N-acetyltransferase activity / tRNA N-acetyltransferase activity / tRNA acetylation / 90S preribosome / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / tRNA binding / 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
RNA cytidine acetyltransferase
Similarity search - Component
Biological speciesThermochaetoides thermophila DSM 1495 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsZhou M / Marmorstein R
Funding support United States, 3 items
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, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44042.png

Downloads & links

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Map

FileDownload / File: emd_44042.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 309.6 Å		0.86 Å/pix.
x 360 pix.
= 309.6 Å		0.86 Å/pix.
x 360 pix.
= 309.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.192
Minimum - Maximum-2.209698 - 3.418405
Average (Standard dev.)-0.0009052919 (±0.068004616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 309.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44042_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_44042_half_map_2.map
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Histogram (log scale)

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Sample components

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Entire : Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemi...

EntireName: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemically synthetic cytidine-amide-CoA bisubstrate probe
Components
  • Complex: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemically synthetic cytidine-amide-CoA bisubstrate probe
    • Protein or peptide: RNA cytidine acetyltransferase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: [[(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

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Supramolecule #1: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemi...

SupramoleculeName: Sf9 produced recombinant NAT10 in complex with ADP/Mg2+ and chemically synthetic cytidine-amide-CoA bisubstrate probe
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 244 kDa/nm

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Macromolecule #1: RNA cytidine acetyltransferase

MacromoleculeName: RNA cytidine acetyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Thermochaetoides thermophila DSM 1495 (fungus)
Molecular weightTheoretical: 121.475914 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HHHHHHENLY FQGMTVQKTV DSRIPTLIRN GLQTKKRSFF VVVGDHAKEA IVHLYYIMSS MDVRQNKSVL WAYKKELLGF TSHRKKREA KIKKEIKRGI REPNQADPFE LFISLNDIRY CYYKETDKIL GNTYGMCILQ DFEAITPNIL ARTIETVEGG G LVVLLLKG ...String:
HHHHHHENLY FQGMTVQKTV DSRIPTLIRN GLQTKKRSFF VVVGDHAKEA IVHLYYIMSS MDVRQNKSVL WAYKKELLGF TSHRKKREA KIKKEIKRGI REPNQADPFE LFISLNDIRY CYYKETDKIL GNTYGMCILQ DFEAITPNIL ARTIETVEGG G LVVLLLKG MTSLKQLYTM TMDVHARYRT EAHDDVIARF NERFLLSLGS CESCLVIDDE LNVLPISGGK GVKPLPPPDE DE ELSPAAK ELKKIKDELE DTQPIGSLIK LARTVDQAKA LLTFVDAIAE KTLRNTVTLT AARGRGKSAA MGVAIAAAVA YGY SNIFIT SPSPENLKTL FEFVFKGFDA LDYKDHADYT IIQSTNPEFN KAIVRVNIHR NHRQTIQYIR PQDAHVLGQA ELVV IDEAA AIPLPLVKKL MGPYLVFMAS TISGYEGTGR SLSLKLIKQL REQSRAGANP NGGNAVEVDR STLKATKETT SVGGR SLKE ITLSEPIRYA QGDNVEKWLN TLLCLDATLP RSKISTTGCP DPSQCELLHV NRDTLFSFHP VSEKFLQQMV ALYVAS HYK NSPNDLQLMS DAPAHELFVL TGPIQEGRLP EPLCVIQVSL EGKISKQSIL KSLSRGQQPA GDLIPWLVSQ QFQDDEF AS LSGARIVRIA TNPDYMSMGY GSKALQLLVD YYEGKFADLS EDAAAEVPRS IPRVTDAELS KGSLFDDIKV RDMHELPP L FSKLSERRPE KLDYVGVSYG LTQQLHKFWK RAQFVPVYLR QTANDLTGEH TCVMIRPLQD GNDPSWLGAF AADFHKRFL SLLSYKFREF PSILALTIEE SANAGAMLDP SNAPTELTKA ELDQLFTPFD HKRLESYANG LLDYHVVLDL MPTIAQLYFT GRLREAVKL SGLQQAILLA LGLQRKDIDT LATELNLPGS QVLAIFMKIM RKVTQHFGAL VSGAIAAELP DPNKTVGVSK E NAMGIHDD EVVGLKFEAL EQRLEDELDE GGDEALRELR KKQRELIDSL PLDQYEIDGD DDAWKEAEKR VASAAKSGKK VD GTLVSVP SAKAAKRKAE EMAALRDELE KMEKGKERGS KKAKKEKRR

UniProtKB: RNA cytidine acetyltransferase

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: [[(2~{R},3~{S},4~{S},5~{R})-5-(6-aminopurin-9-yl)-4-oxidanyl-3-ph...

MacromoleculeName: [[(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- ...Name: [[(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
type: ligand / ID: 4 / Number of copies: 2 / Formula: A1AH4
Molecular weightTheoretical: 1.050772 KDa

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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

Concentration0.8 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 280.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 42.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 162769
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model
Details: The unpublished cryo-EM structure in complex with cytidine-amide-CoA bisubstrate probe and ADP
SoftwareName: Coot (ver. 0.9.8.7)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9b0i:
Cryo-EM Structure of Sf9 produced recombinant N-acetyltransferase 10 (NAT10) in complex with cytidine-amide-CoA bisubstrate probe and ADP/Mg2+.

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