PDBsum entry: 1d1u

Hydrolase/DNA

PDB id: 1d1u

Contents

Protein chain

255 a.a. *

DNA/RNA

Waters ×175

* Residue conservation analysis

PDB id:

1d1u

Name:

Hydrolase/DNA

Title:

Use of an n-terminal fragment from moloney murine leukemia virus reverse transcriptase to facilitate crystallization and analysis of a pseudo-16-mer DNA molecule containing g- a mispairs

Structure:

DNA (5'-d( Cp Tp Cp Gp Tp G)-3'). Chain: b. Engineered: yes. Other_details: synthetic. DNA (5'-d( Ap Cp Gp Gp Cp Ap Cp Gp Ap G)-3'). Chain: c. Engineered: yes. Other_details: synthetic. Protein (reverse transcriptase).

Source:

Synthetic: yes. Other_details: the sequence 5'-ctcgtg-3' was synthesized on an applied biosystems 392 DNA/RNA synthesizer. Moloney murine leukemia virus. Organism_taxid: 11801. Gene: mmlv reverse transcriptase. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Hexamer (from PQS)

Resolution:

2.30Å R-factor: 0.230 R-free: 0.286

Authors:

M.L.Cote,S.Yohannan,M.M.Georgiadis

Key ref:

M.L.Coté et al. (2000). Use of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase to facilitate crystallization and analysis of a pseudo-16-mer DNA molecule containing G-A mispairs. Acta Crystallogr D Biol Crystallogr, 56, 1120-1131. PubMed id: 10957631 DOI: 10.1107/S0907444900008246

Date:

21-Sep-99 Release date: 02-Apr-00

Protein chain

P03355  (POL_MLVMS) -  Gag-Pol polyprotein

Seq: 1738 a.a.

Struc: 255 a.a.

Enzyme reactions

• Enzyme class 2: E.C.2.7.7.49 - RNA-directed Dna polymerase.
• Reaction: Deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1)
• Enzyme class 3: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: Deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1)
• Enzyme class 4: E.C.3.1.26.4 - Ribonuclease H.
• Reaction: Endonucleolytic cleavage to 5'-phosphomonoester.

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: RNA-dependent DNA replication (1 term)
• Biochemical function: RNA binding (2 terms)

reference

DOI no: 10.1107/S0907444900008246

Acta Crystallogr D Biol Crystallogr 56:1120-1131 (2000)

PubMed id: 10957631

Use of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase to facilitate crystallization and analysis of a pseudo-16-mer DNA molecule containing G-A mispairs.

M.L.Coté, S.J.Yohannan, M.M.Georgiadis.

ABSTRACT

Complexation with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase offers a novel method of obtaining crystal structures of nucleic acid duplexes, which can be phased by molecular replacement. This method is somewhat similar to the method of using a monoclonal antibody Fab fragment complexed to the molecule of interest in order to obtain crystals suitable for X-ray crystallographic analysis. Here a novel DNA structure including two G-A mispairs in a pseudo-hexadecamer determined at 2.3 A resolution in a complex with the N-terminal fragment is reported. This structure has an asymmetric unit consisting of the protein molecule bound to the blunt end of a DNA 6/10-mer, which is composed of a six-base strand (5'-CTCGTG-3') and a ten-base strand (3'-GAGCACGGCA-5'). The 6/10-mer is thus composed of a six-base-pair duplex with a four-base single-stranded overhang. In the crystal structure, the bases of the overhang are reciprocally paired (symmetry element -x - 1, -y, z), yielding a doubly nicked pseudo-hexadecamer primarily B-form DNA molecule, which has some interesting A-like structural features. The pairing between the single strands results in two standard (G-C) Watson-Crick pairs and two G-A mispairs. The structural DNA model can accommodate either a standard syn or a standard anti conformation for the 5'-terminal adenine of the ten-base strand of the DNA based on analysis of simulated-annealing omit maps. Although the DNA model here includes nicks in the phosphodiester backbone, modeling of an intact phosphodiester backbone results in a very similar DNA model and indicates that the structure is biologically relevant.

Selected figure(s)

Figure 5.
Figure 5 Schematic representation (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]; Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]) of the interactions in the protein-DNA binding site of form IV. The hydrogen-bonding distances between 2.4 and 3.3 Å are indicated with white dotted lines. The non-bonded contacts ranging from 3.3 to 3.7 Å are indicated with longer-dashed magenta lines. Also shown with black dotted lines is the ion-pair formed by D114 O 1 with R116 N and D114 O 2 and R116 N 2 as observed in form IV.

Figure 7.
Figure 7 The superpositioning (Jones et al., 1991[Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110-119.]) of the observed pseudo-hexadecamer model (containing the anti A7) onto a theoretical intact hexadecamer containing bridging phosphate groups. (a) The navy-blue stick model (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]) depicts the observed pseudo-hexadecamer. The gold stick model represents the intact hexadecamer model, with the bridging phosphate groups emphasized in red. (b) A stereodiagram (Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]) close-up of the region where the phosphate group would most likely occur in an intact hexadecamer. The color schemes shown are identical to that in (a). Clearly shown are the two chain termini in the navy-blue model (the observed structure), the contiguous chain represented by the intact gold model containing the red phosphate group and the close agreement between the two.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2000, 56, 1120-1131) copyright 2000.

Figures were selected by the author.