Human putative tRNA(1-methyladenosine) methyltransferase

Target ID:

FLJ20628

Aliases:

DKFZp564I2178FLJ20628

Deposition Date:

Friday 16th September 2005

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2B25

Authors:

W.TEMPEL,H.WU,A.DONG,H.ZENG,P.LOPPNAU,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2B25

tabs

Structure Details

The methylation of a variety of biologically active molecules is a common theme in biology. The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repair, nucleic acid processing, gene silencing and imprinting. The majority of methylation reactions are carried out by the S-adenosylmethionine (SAM)-dependent methyltransferases.

We have solved the human putative tRNA(1-methyladenosine) methyltransferase (FLJ20628) structure in complex with S-adenosyl-L-methionine at 2.5 Ã… resolution. Modifications of RNAs at specific positions by RNA methyltransferases are involved in various aspects of RNA metabolism, including post-transcriptional maturation, pre-mRNA processing, translation initiation, and antibiotics resistance. Like in other methltransferase subfamilies, low level of sequence similarity was found between members of RNA methyltransferase sub-family. The human FLJ20628 gene is located on chromosome 2p23.2. Broad mRNA expression has been observed in human tissue, including brain, kidney, lung and pancreas.

Materials and Methods

FLJ20628

PDB:2B25

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:47271406
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gsSTSRERPFQAGELILAETGEGETKFKKLFRLNNFGLLNSNWGAVPFGKIVGKFPGQILRSSFGKQYMLRRPALEDYVVLMKRGTAITFPKDINMILSMMDINPGDTVLEAGSGSGGMSLFLSKAVGSQGRVISFEVRKDHHDLAKKNYKHWRDSWKLSHVEEWPDNVDFIHKDISGATEDIKSLTFDAVALDMLNPHVTLPVFYPHLKHGGVCAVYVVNITQVIELLDGIRTCELALSCEKISEVIVRDWLVCLAKQKNGILAQKVESKINTDVQLDSQEKIGVKGELFQEDDHEESHSDFPYGSFPYVARPVHWQPGHTAFLVKLRKVKPQLN
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:FLJ20628 was expressed in E.coli BL21 (DE3) codon plus RIL in M9 minimal medium in the presence of 50 µg/mL of kanamycin. Cell were grown at 37oC to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, in the presence of 50 mg/L of SeMet and incubated overnight at 15oC.

Purification

Procedure

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (20 mM Tris, pH 8.5, 0.5 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol, 0.1% CHAPS) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:
Ligand
MassSpec:
Crystallization:Purified FLJ20628 was complexed with S-adenosyl-L-methionine (SAM, Sigma) at 1:10 molar ratio of protein: SAM and crystallized using hanging drop vapor diffusion method drop at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 11% PEG3350, 0.2M Li Citrate, 0.1 M BisTris pH 6.5, 10% PEG400.
NMR Spectroscopy:
Data Collection:
Data Processing: