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- PDB-7mx2: Cryo-EM structure of human ternary NatC complex with a Bisubstrat... -

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Basic information

Entry
Database: PDB / ID: 7mx2
TitleCryo-EM structure of human ternary NatC complex with a Bisubstrate inhibitor
Components
  • N-alpha-acetyltransferase 30
  • N-alpha-acetyltransferase 35, NatC auxiliary subunit
  • N-alpha-acetyltransferase 38, NatC auxiliary subunit
  • peptide portion of bisubstrate inhibitor
KeywordsTRANSFERASE / NatC / NAA30 / NAA35 / NAA38
Function / homology
Function and homology information


N-terminal methionine Nalpha-acetyltransferase NatC / NatC complex / smooth muscle cell proliferation / N-terminal peptidyl-methionine acetylation / Retrograde transport at the Trans-Golgi-Network / peptide alpha-N-acetyltransferase activity / negative regulation of apoptotic process / nucleoplasm / nucleus / plasma membrane ...N-terminal methionine Nalpha-acetyltransferase NatC / NatC complex / smooth muscle cell proliferation / N-terminal peptidyl-methionine acetylation / Retrograde transport at the Trans-Golgi-Network / peptide alpha-N-acetyltransferase activity / negative regulation of apoptotic process / nucleoplasm / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
LSM domain containing 1 / -alpha-acetyltransferase 35, NatC auxiliary subunit / N-alpha-acetyltransferase 30-like / Mak10 subunit, NatC N(alpha)-terminal acetyltransferase / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / Sm domain profile. / LSM domain superfamily / Acetyltransferase (GNAT) family ...LSM domain containing 1 / -alpha-acetyltransferase 35, NatC auxiliary subunit / N-alpha-acetyltransferase 30-like / Mak10 subunit, NatC N(alpha)-terminal acetyltransferase / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / Sm domain profile. / LSM domain superfamily / Acetyltransferase (GNAT) family / Gcn5-related N-acetyltransferase (GNAT) domain profile. / GNAT domain / Acyl-CoA N-acyltransferase
Similarity search - Domain/homology
CARBOXYMETHYL COENZYME *A / N-alpha-acetyltransferase 30 / N-alpha-acetyltransferase 35, NatC auxiliary subunit / N-alpha-acetyltransferase 38, NatC auxiliary subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsDeng, S. / Marmorstein, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118090 United States
CitationJournal: Structure / Year: 2023
Title: Molecular role of NAA38 in thermostability and catalytic activity of the human NatC N-terminal acetyltransferase.
Authors: Sunbin Deng / Sarah M Gardner / Leah Gottlieb / Buyan Pan / E James Petersson / Ronen Marmorstein /
Abstract: N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a ...N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a large auxiliary subunit that facilitates catalysis and ribosome targeting for co-translational acetylation. NatC is one of the major NATs containing an NAA30 catalytic subunit, but uniquely contains two auxiliary subunits, large NAA35 and small NAA38. Here, we report the cryo-EM structures of human NatC (hNatC) complexes with and without NAA38, together with biochemical studies, to reveal that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide binding loop for optimal catalysis, respectively. We also note important differences in engagement with a stabilizing inositol hexaphosphate molecule between human and yeast NatC. These studies provide new insights for the function and evolution of the NatC complex.
History
DepositionMay 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Data collection / Database references / Category: citation / citation_author / em_imaging
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: N-alpha-acetyltransferase 35, NatC auxiliary subunit
C: N-alpha-acetyltransferase 38, NatC auxiliary subunit
A: N-alpha-acetyltransferase 30
D: peptide portion of bisubstrate inhibitor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)116,6105
Polymers115,7844
Non-polymers8261
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein N-alpha-acetyltransferase 35, NatC auxiliary subunit / Embryonic growth-associated protein homolog / Protein MAK10 homolog


Mass: 83754.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NAA35, EGAP, MAK10 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q5VZE5
#2: Protein N-alpha-acetyltransferase 38, NatC auxiliary subunit / LSM domain-containing protein 1 / Phosphonoformate immuno-associated protein 2


Mass: 13530.276 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NAA38, LSMD1, MAK31, PFAAP2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BRA0
#3: Protein N-alpha-acetyltransferase 30 / N-acetyltransferase 12 / N-acetyltransferase MAK3 homolog / NatC catalytic subunit


Mass: 18083.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NAA30, C14orf35, MAK3, NAT12 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q147X3, N-terminal methionine Nalpha-acetyltransferase NatC
#4: Protein/peptide peptide portion of bisubstrate inhibitor


Mass: 416.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-CMC / CARBOXYMETHYL COENZYME *A


Mass: 825.570 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H38N7O18P3S / 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

Component
IDNameTypeEntity IDParent-IDSource
1heterotimeric human NatC complex with a bisubstrate inhibitorCOMPLEX#1-#30MULTIPLE SOURCES
2N-alpha-acetyltransferase NatC complexCOMPLEX#1-#31RECOMBINANT
3bisubstrate inhibitorCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.114 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
31 mMDithiothreitol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
111.6GATAN K3 (6k x 4k)
211.3GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFINDCTF correction
7Coot0.8.9.2model fitting
9PHENIX1.17.1-3660model refinement
10cryoSPARCv2initial Euler assignment
11cryoSPARCv2final Euler assignment
12cryoSPARCv2classification
13cryoSPARCv23D reconstruction
Image processingDetails: 5372 images
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172041 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036242
ELECTRON MICROSCOPYf_angle_d0.5468537
ELECTRON MICROSCOPYf_dihedral_angle_d3.515926
ELECTRON MICROSCOPYf_chiral_restr0.0391028
ELECTRON MICROSCOPYf_plane_restr0.0051091

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