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- PDB-8k20: Cryo-EM structure of KEOPS complex from Arabidopsis thaliana -

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Basic information

Entry
Database: PDB / ID: 8k20
TitleCryo-EM structure of KEOPS complex from Arabidopsis thaliana
Components
  • At4g34412
  • At5g53043
  • Probable tRNA N6-adenosine threonylcarbamoyltransferase
  • non-specific serine/threonine protein kinaseSerine/threonine-specific protein kinase
KeywordsTRANSFERASE / KEOPS complex / tRNA t6A synthase / RNA-binding protein
Function / homology
Function and homology information


N(6)-L-threonylcarbamoyladenine synthase activity / EKC/KEOPS complex / tRNA threonylcarbamoyladenosine modification / tRNA processing / non-specific serine/threonine protein kinase / phosphorylation / protein serine/threonine kinase activity / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / CTAG/Pcc1 family / Transcription factor Pcc1 / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase ...tRNA N6-adenosine threonylcarbamoyltransferase Kae1, archaea and eukaryote / Serine/threonine-protein kinase Bud32 / CGI121/TPRKB / CGI121/TPRKB superfamily / Kinase binding protein CGI-121 / CTAG/Pcc1 family / Transcription factor Pcc1 / Kae1/TsaD family / Gcp-like domain / tRNA N6-adenosine threonylcarbamoyltransferase / ATPase, nucleotide binding domain / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
: / Glycoprotease 2 / At4g34412 / At5g53043 / non-specific serine/threonine protein kinase
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZheng, X.X. / Zhu, L. / Duan, L. / Zhang, W.H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32000847 China
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.
Authors: Xinxing Zheng / Chenchen Su / Lei Duan / Mengqi Jin / Yongtao Sun / Li Zhu / Wenhua Zhang /
Abstract: In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS ...In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.
History
DepositionJul 11, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2024Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Probable tRNA N6-adenosine threonylcarbamoyltransferase
B: non-specific serine/threonine protein kinase
C: At4g34412
D: At5g53043
F: At5g53043
E: Probable tRNA N6-adenosine threonylcarbamoyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,7187
Polymers145,6626
Non-polymers561
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Probable tRNA N6-adenosine threonylcarbamoyltransferase / Glycoprotease 2 / t(6)A37 threonylcarbamoyladenosine biosynthesis protein GCP2 / tRNA ...Glycoprotease 2 / t(6)A37 threonylcarbamoyladenosine biosynthesis protein GCP2 / tRNA threonylcarbamoyladenosine biosynthesis protein GCP2


Mass: 38813.570 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: GCP2, At4g22720, T12H17.110, T12H17_110 / Production host: Escherichia coli (E. coli)
References: UniProt: O49653, N6-L-threonylcarbamoyladenine synthase
#2: Protein non-specific serine/threonine protein kinase / Serine/threonine-specific protein kinase


Mass: 25170.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At5g26110, T1N24.12, T1N24_12 / Production host: Escherichia coli (E. coli)
References: UniProt: Q94K14, non-specific serine/threonine protein kinase
#3: Protein At4g34412 / EKC/KEOPS complex subunit tprkb-like protein


Mass: 19678.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At4g34412 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6NMZ4
#4: Protein At5g53043 / Transcription factor


Mass: 11592.994 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: At5g53045 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8GWD7
#5: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / 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: KEOPS complex from Arabidopsis thaliana / Type: COMPLEX
Details: The sample contains KAE1, BUD32, CGI121 and PCC1, which form a KEOPS complex.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 300 mM NaCl, 20 mM Tris-HCl pH 7.5,5 mM 2-mercaptoethanol
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was freshly purified by size exclusion chromatography.
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm / Cs: 0.27 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Temperature (max): 89.8 K / Temperature (min): 77.1 K
Image recordingAverage exposure time: 3.6 sec. / Electron dose: 64 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 232232 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039509
ELECTRON MICROSCOPYf_angle_d0.54912865
ELECTRON MICROSCOPYf_dihedral_angle_d4.2531302
ELECTRON MICROSCOPYf_chiral_restr0.0431508
ELECTRON MICROSCOPYf_plane_restr0.0051652

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