The ribosome-associated chaperone trigger factor (TF) of Escherichia coli interacts with a variety of newly synthesized polypeptides to assist their correct folding. Here, we report that the TF of thermophilic eubacterium, Thermus thermophilus, arrested spontaneous folding of green fluorescent protein by forming a 1:1 binary complex. The complex was isolable by gel-filtration but was shown to be dynamic because green fluorescent protein was released by ␣-casein in large excess. Unexpectedly, EDTA completely abolished the foldingarrest activity of TF, and analysis revealed that the TF from our preparation contained ϳ0.5 mol Zn 2؉ /mol TF. The folding-arrest activity of TF that was saturated with Zn 2؉ (ϳ1 mol/mol TF) was twice as efficient as that of untreated TF. Thus, chaperone activity of thermophilic TF is Zn 2؉ -dependent.
IP is one of the most powerful detectors for collecting diffraction data in the sense that it has a wide detection area, high sensitivity, large dynamic range, high accuracy, and reasonable pixel size. In spite of such excellent features, a high-speed automatic data collection device that has enough speed to maximize use of SR X-rays is very difficult to make, due to the slow digitization speed of the IP. We have solved this difficulty by using a fully cylindrical IP cassette (radius = 400 mm, width = 450 mm) where several diffraction images can be recorded and be read by five IP reading heads. This devise called Galaxy has been developed and installed at BL6C in the PF. Net digitization time is 5 min and 2.5 min for pixel size being 100ƒÊm and 200ƒÊm, respectively. This is enough speed for a PF bending magnet beamline. This means, that when 2.88 Å resolution data using 1.0 Å x-rays are collected, a digitized speed for a diffraction image is 16.6sec for 100ƒÊm pixel size, because in this condition, 18 images can be recorded in an IP cassette. Additionally, the maximum resolution is 0.58° for 1.0 Å x-rays.Two examples follow;(1) sample crystal is Insulin, collected up to 1.0 Å collection range ; ƒ¢ƒ=63°, completeness=0.83, average redundancy= 1.42 and R-merge is 4.79%, (2) Citidine; data up to 0.7Å were recorded only 4 sheets using Weissenberg mode, with 80° on each sheet, completeness = 0.58, redundancy = 4.49 and R-merge = 3.2%. Keywords: IMAGING PLATE, PROTEIN CRYSTALLOGRAPHY, AUTOMATIC DIFFRACTION INTENSITY DATA COLLECTION SYSTEMActa Cryst. (2002). A58 (Supplement), C237 CCD-based detectors have become a key tool for x-ray crystallography, both at synchrotron beam lines and in the home laboratory. However, the performance of CCD-based detectors is fundamentally limited by the characteristics of the phosphor screens used to convert the incident x-ray flux to visible light. In particular, the spatial resolution of the detector is limited by photon scattering in the polycrystalline phosphor matrix. Also, the active areas which can be achieved are limited by the quantum efficiency of the phosphor. Here we describe a fundamentally new technology for the detection of x-ray quanta: the Quantum Resonance Converter (QRC). QRC technology allows quantum efficiencies several times higher than conventional phosphor screens and also eliminates the PSF degradation due to scattering. We describe the operation of these novel devices and report on their first experimental tests. Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a member of the newly classified Y-family of DNA polymerases. The Y-family polymerases are best characterized by their low-fidelity synthesis on undamaged DNA templates and propensity to traverse normally replication-blocking lesions. Crystal structures of Dpo4 in ternary complexes with DNA and an incoming nucleotide, either correct or incorrect, have been solved at 1.7 Å and 2.1 Å resolution, respectively. Despite a conserved active site and a hand-like configuration similar to all known polymeras...
Precursors for isoprenoid synthesis are essential in all organisms. These compounds are synthesized by one of two known routes: the well characterized mevalonate pathway [1] or a recently discovered non-mevalonate route which is used in many bacteria and human pathogens [2]. Since the second pathway is both vital and unlike any found in humans, enzymes catalysing reactions along this synthetic route are possible drug targets. The structure of one such enzyme from the thermophilic bacterium Thermus thermophilus has been solved to high resolution in the presence of substrate and with a substrate analogue. Enzyme co-crystallized with substrate shows only one product, cytosine monophosphate (CMP), in the active site. At the high resolution of the refinement (1.6 Å) the positions and coordination of the magnesium ions in the active site are clearly seen.Acetyl-CoA carboxylases (ACCs) catalyze the first committed step of fatty acid biosynthesis. Although ACC is an essential enzyme (complex) in every organism, the structure-function relationship of ACC remains to be unclear. As the first step for elucidating the structure-function relationship of ACC, we started the crystallographic analysis of DtsR1. DtsR1 is the -subunit of ACC multisubunit complex in Corynebacterium glutamicum, which catalyzes the transcarboxylation between biotin and acetyl-CoA.DtsR1 was over-expressed in E. coli, purified, and crystallized by the sitting-drop vapor diffusion method using PEG 6000 as a precipitant. The approximate dimensions of the obtained crystals were 0.07x0.07x0.03mm 3 . Diffraction data of the crystals were collected at NW12 of the Photon Factory (Tsukuba), revealing that the crystals belong to the space group R32. The crystal structure of DtsR1 was solved at 3.2Å resolution by the molecular replacement method using single-subunit coordinates of the 12S transcarboxylase (PDB ID: 1ON3) as a search model. The obtained structure suggests that the biological unit of DtsR1 is a ring-shaped hexamer with the 32-point group symmetry. Crystallographic refinement of DtsR1 is in progress at 2.7Å resolution.
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