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- EMDB-2017: Structure of the E. coli methyltransferase KsgA bound to the E. c... -

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Basic information

Entry
Database: EMDB / ID: EMD-2017
TitleStructure of the E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit
Map dataStructure of KsgA bound to the 30S ribosomal subunit
Sample
  • Sample: E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit
  • Complex: E. coli 30S ribosomal subunit
  • Protein or peptide: methyltransferase KsgA
KeywordsRibosome biogenesis / methyltransferase / KsgA / small ribosomal subunit
Function / homology
Function and homology information


16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase / 16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase activity / rRNA (adenine-N6,N6-)-dimethyltransferase activity / rRNA base methylation / rRNA methylation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / ribosomal small subunit binding ...16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase / 16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase activity / rRNA (adenine-N6,N6-)-dimethyltransferase activity / rRNA base methylation / rRNA methylation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / ribosomal small subunit binding / RNA folding / four-way junction DNA binding / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / transcription elongation factor complex / maturation of SSU-rRNA / regulation of DNA-templated transcription elongation / DNA endonuclease activity / transcription antitermination / DNA-templated transcription termination / maintenance of translational fidelity / mRNA 5'-UTR binding / rRNA processing / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosome biogenesis / regulation of translation / ribosomal small subunit assembly / small ribosomal subunit / double-stranded DNA binding / cytosolic small ribosomal subunit / cytoplasmic translation / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine methylase transferase, conserved site / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases signature. / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. ...rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine methylase transferase, conserved site / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases signature. / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S16 / Ribosomal protein S15, bacterial-type / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S12, bacterial-type / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S2 signature 2. / Ribosomal protein S18 / Ribosomal protein S18 / Ribosomal protein S18 superfamily / K Homology domain / K homology RNA-binding domain / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S14, conserved site / Ribosomal protein S14 signature. / Ribosomal protein S2 signature 1. / KH domain / Type-2 KH domain profile. / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S15/S19, conserved site / Ribosomal protein S19 signature. / Ribosomal protein S10 / : / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S19 / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S2, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein S7 signature. / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S5 / Ribosomal protein S17, conserved site / Ribosomal protein S17 signature. / S5 double stranded RNA-binding domain profile. / K homology domain superfamily, prokaryotic type / Ribosomal protein S5, N-terminal / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal / Ribosomal protein S5, C-terminal domain / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13/S18 / Ribosomal protein S13 family profile. / Ribosomal protein S8 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4, conserved site / Ribosomal protein S4 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / 30S ribosomal protein S14 / 30S ribosomal protein S15 / 30S ribosomal protein S17 / Ribosomal RNA small subunit methyltransferase A / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / 30S ribosomal protein S14 / 30S ribosomal protein S15 / 30S ribosomal protein S17 / Ribosomal RNA small subunit methyltransferase A / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein bS21
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.5 Å
AuthorsBoehringer D / O'Farrell HC / Rife JP / Ban N
CitationJournal: J Biol Chem / Year: 2012
Title: Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.
Authors: Daniel Boehringer / Heather C O'Farrell / Jason P Rife / Nenad Ban /
Abstract: The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal ...The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.
History
DepositionJan 3, 2012-
Header (metadata) releaseJan 27, 2012-
Map releaseFeb 15, 2012-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 39
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 39
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4adv
  • Surface level: 39
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2017.jpg

Downloads & links

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Map

FileDownload / File: emd_2017.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of KsgA bound to the 30S ribosomal subunit
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.07 Å/pix.
x 128 pix.
= 392.96 Å		3.07 Å/pix.
x 128 pix.
= 392.96 Å		3.07 Å/pix.
x 128 pix.
= 392.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 39.0 / Movie #1: 39
Minimum - Maximum-195.404663090000014 - 615.226074219999987
Average (Standard dev.)-10.220307350000001 (±23.594770430000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 392.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.073.073.07
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z392.960392.960392.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-195.405615.226-10.220

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Supplemental data

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Sample components

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Entire : E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal...

EntireName: E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit
Components
  • Sample: E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit
  • Complex: E. coli 30S ribosomal subunit
  • Protein or peptide: methyltransferase KsgA

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Supramolecule #1000: E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal...

SupramoleculeName: E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit
type: sample / ID: 1000
Details: 30S ribosomal subunits lack the methylation at A1518 and A1519
Oligomeric state: One monomer of KsgA binds to one monomer of the 30S ribosomal subunit
Number unique components: 2
Molecular weightExperimental: 820 KDa / Theoretical: 820 KDa

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Supramolecule #1: E. coli 30S ribosomal subunit

SupramoleculeName: E. coli 30S ribosomal subunit / type: complex / ID: 1 / Name.synonym: Small ribosomal subunit
Details: 30S ribosomal subunits lack the methylation at A1518 and A1519
Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 790 KDa / Theoretical: 790 KDa

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Macromolecule #1: methyltransferase KsgA

MacromoleculeName: methyltransferase KsgA / type: protein_or_peptide / ID: 1 / Name.synonym: methyltransferase KsgA / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: cytoplasma
Molecular weightExperimental: 30.42 KDa / Theoretical: 30.42 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET15b
SequenceInterPro: Ribosomal RNA adenine dimethylase

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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

BufferpH: 7.6
Details: 40 mM KCl, 4 mM MgCl2, 20 mM HEPES/KOH pH 7.6, 6 mM 2-mercaptoethanol
GridDetails: Quantifoil 200 mesh copper
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Plunger

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Electron microscopy

MicroscopeFEI TECNAI 20
DetailsSemi-automatic data acquisition with SerialEM script
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 82000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Per image, ctffind3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic-5, Spider / Number images used: 23343

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Atomic model buiding 1

Initial modelPDB ID:

2avy
PDB Unreleased entry


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. 30S ribosomal subunit head and body were fitted separately
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation
Output model

PDB-4adv:
Structure of the E. coli methyltransferase KsgA bound to the E. coli 30S ribosomal subunit

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