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- PDB-7l1k: Cryo-EM structure of S. Pombe NatC complex with a Bisubstrate inh... -

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Basic information

Entry
Database: PDB / ID: 7l1k
TitleCryo-EM structure of S. Pombe NatC complex with a Bisubstrate inhibitor and inositol hexaphosphate
Components
  • (N-alpha-acetyltransferase ...) x 3
  • MLGP peptide
KeywordsTRANSFERASE / NatB / NAA20 / NAA25
Function / homology
Function and homology information


N-terminal methionine Nalpha-acetyltransferase NatC / NatC complex / peptide alpha-N-acetyltransferase activity / inositol hexakisphosphate binding / protein maturation / endoplasmic reticulum membrane / endoplasmic reticulum / RNA binding / nucleus / cytosol
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 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 / INOSITOL HEXAKISPHOSPHATE / N-alpha-acetyltransferase 38, NatC auxiliary subunit / N-alpha-acetyltransferase 30 / N-alpha-acetyltransferase 35, NatC auxiliary subunit
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 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: 2021
Title: Molecular mechanism of N-terminal acetylation by the ternary NatC complex.
Authors: Sunbin Deng / Leah Gottlieb / Buyan Pan / Julianna Supplee / Xuepeng Wei / E James Petersson / Ronen Marmorstein /
Abstract: Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic ...Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.
History
DepositionDec 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 12, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 20, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)113,4236
Polymers111,9384
Non-polymers1,4862
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10430 Å2
ΔGint-53 kcal/mol
Surface area36330 Å2

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Components

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N-alpha-acetyltransferase ... , 3 types, 3 molecules ABC

#1: Protein N-alpha-acetyltransferase 30 / N-terminal acetyltransferase C complex catalytic subunit mak3 homolog / NatC catalytic subunit


Mass: 17721.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843 / Gene: naa30, mak3, SPBC15D4.06 / Production host: Escherichia coli (E. coli)
References: UniProt: O74311, N-terminal methionine Nalpha-acetyltransferase NatC
#2: Protein N-alpha-acetyltransferase 35, NatC auxiliary subunit / N-terminal acetyltransferase C complex subunit mak10 / NatC complex subunit mak10


Mass: 80541.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843 / Gene: mak10, naa35, SPBC1861.03 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9USY3
#3: Protein N-alpha-acetyltransferase 38, NatC auxiliary subunit / N-terminal acetyltransferase C complex subunit naa38


Mass: 13258.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Strain: 972 / ATCC 24843 / Gene: naa38, mak31, SPBC947.03c / Production host: Escherichia coli (E. coli) / References: UniProt: O43080

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Protein/peptide , 1 types, 1 molecules D

#4: Protein/peptide MLGP peptide


Mass: 416.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6 / Feature type: SUBJECT OF INVESTIGATION
#6: 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

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Details

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 S.pombe NatC complex with a bisubstrate inhibitor and inositol hexaphosphateCOMPLEX#1-#40MULTIPLE SOURCES
2N-alpha-acetyltransferase NatC, catalytic subunit NAA30, auxiliary subunits NAA35 and NAA38COMPLEX#1-#31RECOMBINANT
3CoA-Ac-MLGP bi-substrateCOMPLEX#41SYNTHETIC
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium clorideNaCl1
225 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes1
31 mMDithiothreitolDTT1
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 FIELD
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

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: 5397 images
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3289528
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 607131 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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