Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli

Avinash S. Punekar; Maria Selmer

doi:10.1107/S1744309113020289

Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli

Avinash S. Punekar, Maria Selmer (Lead / Corresponding author)

Drug Discovery Unit

Research output: Contribution to journal › Article › peer-review

2 Citations (Scopus)

Abstract

Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni²⁺-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å³Da^-1.

Original language	English
Pages (from-to)	1001-1003
Number of pages	3
Journal	Acta Crystallographica F-Structural Biology and Crystallization Communications
Volume	69
Issue number	Pt 9
DOIs	https://doi.org/10.1107/S1744309113020289
Publication status	Published - Sept 2013

Keywords

Amino Acid Sequence
Crystallography, X-Ray
Escherichia coli/chemistry
Escherichia coli Proteins/chemistry
Gene Expression
Methyltransferases/chemistry
Molecular Sequence Data
Protein Interaction Domains and Motifs
RNA, Ribosomal, 23S/chemistry
Recombinant Fusion Proteins/chemistry
Ribosomes/genetics
S-Adenosylmethionine/chemistry

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10.1107/S1744309113020289

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title = "Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli",

abstract = "Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 {\AA} resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 {\AA}, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 {\AA}3 Da-1.",

keywords = "Amino Acid Sequence, Crystallography, X-Ray, Escherichia coli/chemistry, Escherichia coli Proteins/chemistry, Gene Expression, Methyltransferases/chemistry, Molecular Sequence Data, Protein Interaction Domains and Motifs, RNA, Ribosomal, 23S/chemistry, Recombinant Fusion Proteins/chemistry, Ribosomes/genetics, S-Adenosylmethionine/chemistry",

author = "Punekar, {Avinash S.} and Maria Selmer",

year = "2013",

month = sep,

doi = "10.1107/S1744309113020289",

language = "English",

volume = "69",

pages = "1001--1003",

journal = "Acta Crystallographica F-Structural Biology and Crystallization Communications",

issn = "1744-3091",

publisher = "Wiley-Blackwell",

number = "Pt 9",

}

Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli. / Punekar, Avinash S.; Selmer, Maria (Lead / Corresponding author).
In: Acta Crystallographica F-Structural Biology and Crystallization Communications, Vol. 69, No. Pt 9, 09.2013, p. 1001-1003.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli

AU - Punekar, Avinash S.

AU - Selmer, Maria

PY - 2013/9

Y1 - 2013/9

N2 - Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å3 Da-1.

AB - Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å3 Da-1.

KW - Amino Acid Sequence

KW - Crystallography, X-Ray

KW - Escherichia coli/chemistry

KW - Escherichia coli Proteins/chemistry

KW - Gene Expression

KW - Methyltransferases/chemistry

KW - Molecular Sequence Data

KW - Protein Interaction Domains and Motifs

KW - RNA, Ribosomal, 23S/chemistry

KW - Recombinant Fusion Proteins/chemistry

KW - Ribosomes/genetics

KW - S-Adenosylmethionine/chemistry

U2 - 10.1107/S1744309113020289

DO - 10.1107/S1744309113020289

M3 - Article

C2 - 23989148

SN - 1744-3091

VL - 69

SP - 1001

EP - 1003

JO - Acta Crystallographica F-Structural Biology and Crystallization Communications

JF - Acta Crystallographica F-Structural Biology and Crystallization Communications

IS - Pt 9

ER -

Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli

Abstract

Keywords

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