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Title: Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104

Abstract

A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 {angstrom} resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 {angstrom}. Native 2 diffracted to 2.9 {angstrom} resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 {angstrom}. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.

Authors:
;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930006
Report Number(s):
BNL-80619-2008-JA
TRN: US200822%%957
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica Section F: Structural Biology and Crystallization Communications; Journal Volume: 63
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CRYSTALLIZATION; CRYSTALS; RESOLUTION; SACCHAROMYCES CEREVISIAE; SPACE GROUPS; YEASTS; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats

Lee,S., and Tsai, F. Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104. United States: N. p., 2007. Web. doi:10.1107/S1744309107038328.
Lee,S., & Tsai, F. Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104. United States. doi:10.1107/S1744309107038328.
Lee,S., and Tsai, F. Mon . "Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104". United States. doi:10.1107/S1744309107038328.
@article{osti_930006,
title = {Crystallization and Preliminary X-ray Crystallographic Analysis of a 40 kDa N-Terminal Fragment of the Yeast Prion-Remodeling Factor Hsp104},
author = {Lee,S. and Tsai, F.},
abstractNote = {A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 {angstrom} resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 {angstrom}. Native 2 diffracted to 2.9 {angstrom} resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 {angstrom}. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.},
doi = {10.1107/S1744309107038328},
journal = {Acta Crystallographica Section F: Structural Biology and Crystallization Communications},
number = ,
volume = 63,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • An N-terminal fragment of S. cerevisiae Hsp104 has been crystallized. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones. A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 Å. Native 2 diffracted to 2.9 Å resolution and belonged to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = 179.1, b = 179.1,more » c = 69.7 Å. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones.« less
  • The crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of FadD28, a fatty-acyl AMP ligase from M. tuberculosis, are reported. FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the first 460 residues, was crystallized by the hanging-drop vapour-diffusion method at 295 K. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 50.97, b = 60.74, c =more » 136.54 Å. The crystal structure of the N-terminal domain of FadD28 at 2.35 Å resolution has been solved using the MAD method.« less
  • An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively. N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring in approximately 80–90% of cytosolic mammalian proteins and about 50% of yeast proteins. ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1) and possibly NAT5, is responsible for the NatA activity in Saccharomyces cerevisiae. In mammals, ARD1 is involved in cell proliferation, neuronal development and cancer. Interestingly, it has been reported that mouse ARD1 (mARD1{sup 225}) mediatesmore » ∊-acetylation of hypoxia-inducible factor 1α (HIF-1α) and thereby enhances HIF-1α ubiquitination and degradation. Here, the preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins encoded by the Ta0058 and Ta1140 genes of Thermoplasma acidophilum are reported. The Ta0058 protein is related to an N-terminal acetyltransferase complex ARD1 subunit, while Ta1140 is a putative N-terminal acetyltransferase-related protein. Ta0058 shows 26% amino-acid sequence identity to both mARD1{sup 225} and human ARD1{sup 235}.The sequence identity between Ta0058 and Ta1140 is 28%. Ta0058 and Ta1140 were overexpressed in Escherichia coli fused with an N-terminal purification tag. Ta0058 was crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) polyethylene glycol 4000 and 35%(v/v) glycerol. X-ray diffraction data were collected to 2.17 Å. The Ta0058 crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 49.334, c = 70.384 Å, α = β = γ = 90°. The asymmetric unit contains a monomer, giving a calculated crystal volume per protein weight (V{sub M}) of 2.13 Å{sup 3} Da{sup −1} and a solvent content of 42.1%. Ta1140 was also crystallized at 297 K using a reservoir solution consisting of 0.1 M trisodium citrate pH 5.6, 20%(v/v) 2-propanol, 20%(w/v) polyethylene glycol 4000 and 0.2 M sodium chloride. X-ray diffraction data were collected to 2.40 Å. The Ta1140 crystals belong to space group R3, with hexagonal unit-cell parameters a = b = 75.174, c = 179.607 Å, α = β = 90, γ = 120°. Two monomers are likely to be present in the asymmetric unit, with a V{sub M} of 2.51 Å{sup 3} Da{sup −1} and a solvent content of 51.0%.« less
  • Yeast NAD{sup +}-isocitrate dehydrogenase has been purified and crystallized using sodium citrate, a competitive inhibitor of the enzyme, as a precipitant. Preliminary X-ray analyses indicate the molecular boundaries of the molecule and large continuous solvent channels in the crystal. NAD{sup +}-specific isocitrate dehydrogenase (IDH; EC 1.1.1.41) is a complex allosterically regulated enzyme in the tricarboxylic acid cycle. Yeast IDH is believed to be an octamer containing four catalytic IDH2 and four regulatory IDH1 subunits. Crystals of yeast IDH have been obtained and optimized using sodium citrate, a competitive inhibitor of the enzyme, as the precipitating agent. The crystals belong tomore » space group R3, with unit-cell parameters a = 302.0, c = 112.1 Å. Diffraction data were collected to 2.9 Å from a native crystal and to 4.0 Å using multiwavelength anomalous diffraction (MAD) methods from an osmium derivative. Initial electron-density maps reveal large solvent channels and the molecular boundaries of the allosteric IDH multimer.« less
  • Crystals of a complex of the E. coli proteins BtuB (outer membrane cobalamin transporter) and TonB (carboxy-terminal domain) diffracting to 2.1 Å resolution have been obtained. The energy-dependent uptake of organometallic compounds and other micronutrients across the outer membranes of Gram-negative bacteria is carried out by outer membrane active-transport proteins that utilize the proton-motive force of the inner membrane via coupling to the TonB protein. The Escherichia coli outer membrane cobalamin transporter BtuB and a carboxy-terminal domain of the TonB protein, residues 147–239 of the wild-type protein, were expressed and purified individually. A complex of BtuB and TonB{sup 147–239} wasmore » formed in the presence of the substrate cyanocobalamin (CN-Cbl; vitamin B{sub 12}) and calcium and was crystallized. BtuB was purified in the detergent LDAO (n-dodecyl-N,N-dimethylamine-N-oxide) and the complex was formed in a detergent mixture of LDAO and C{sub 8}E{sub 4} (tetraethylene glycol monooctylether). Crystals were obtained by sitting-drop vapor diffusion, with the reservoir containing 30%(v/v) polyethylene glycol (PEG 300) and 100 mM sodium acetate pH 5.2. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1} (unit-cell parameters a = 74.3, b = 82.4, c = 122.6 Å). The asymmetric unit consists of a single BtuB–TonB complex. Data sets have been collected to 2.1 Å resolution at a synchrotron beamline (APS SER-CAT 22-ID)« less