PDBsum entry 2roe

Metal binding protein

PDB id: 2roe

Contents

Protein chain

66 a.a. *

* Residue conservation analysis

PDB id:

2roe

Name:

Metal binding protein

Title:

Solution structure of thermus thermophilus hb8 ttha1718 protein in vitro

Structure:

Heavy metal binding protein. Chain: a. Synonym: ttha1718 heavy metal binding protein. Engineered: yes

Source:

Thermus thermophilus. Organism_taxid: 274. Strain: hb8. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

D.Sakakibara,A.Sasaki,T.Ikeya,J.Hamatsu,H.Koyama,M.Mishima,T.Mikawa, M.Waelchli,B.O.Smith,M.Shirakawa,P.Guentert,Y.Ito

Key ref:

D.Sakakibara et al. (2009). Protein structure determination in living cells by in-cell NMR spectroscopy. Nature, 458, 102-105. PubMed id: 19262674 DOI: 10.1038/nature07814

Date:

20-Mar-08 Release date: 03-Mar-09

Protein chain

Q5SHL2  (Q5SHL2_THET8) -  Heavy metal binding protein from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 66 a.a.

Struc: 66 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1038/nature07814

Nature 458:102-105 (2009)

PubMed id: 19262674

Protein structure determination in living cells by in-cell NMR spectroscopy.

D.Sakakibara, A.Sasaki, T.Ikeya, J.Hamatsu, T.Hanashima, M.Mishima, M.Yoshimasu, N.Hayashi, T.Mikawa, M.Wälchli, B.O.Smith, M.Shirakawa, P.Güntert, Y.Ito.

ABSTRACT

Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Selected figure(s)

Figure 2.
Figure 2: Rapid acquisition of 3D NMR spectra of TTHA1718 in living E. coli cells. a, Rapid acquisition of 3D NMR spectra using a nonlinear sampling scheme. DFT, discrete Fourier transform; dim, dimension; MaxEnt, maximum-entropy processing. b, Repeated observation of 3D NMR spectra with intermittent monitoring of the sample condition by short 2D ^1H–^15N HSQC experiments.

Figure 3.
Figure 3: Collection of NOE-derived distance restraints in TTHA1718 in living E. coli cells. a, Methyl region of the ^1H–^13C heteronuclear multiple-quantum coherence (HMQC) spectrum of the selectively methyl-protonated sample. Assignments of the methyl groups of Ala, Leu and Val residues are indicated, if available. b, ^13C–^13C cross-sections corresponding to the ^1H frequencies of representative methyl groups extracted from the 3D ^13C/^13C-separated HMQC-NOE-HMQC spectrum. The cross-peaks due to interresidual NOEs are assigned in red. Intraresidual NOEs are indicated by blue boxes and annotated. c, ^1H–^1H cross-sections corresponding to the ^15N frequencies of selected backbone amide groups extracted from the 3D ^15N-separated NOESY-HSQC spectrum. The inter- and intraresidue NOEs are indicated as in b. d, Topology diagram of the -sheet structure in TTHA1718. Interstrand backbone NOEs are depicted as double-headed arrows.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2009, 458, 102-105) copyright 2009.

Figures were selected by an automated process.