Novel approach to phasing proteins: derivatization by short cryo-soaking with halides

Dauter, Zbigniew; Dauter, Mirosława; Rajashankar, K.R.

doi:10.1107/s0907444999016352

Cited by 274 publications

(288 citation statements)

References 27 publications

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“…Zn, Mg, Se, they can usually be located by inserting anomalous difference magnitudes into a direct-methods procedure. With very precise data, even lighter atoms such as S and Cl can be picked up in this way (Dauter et al, 1999(Dauter et al, , 2000. The separations of the anomalous scatterers are usually large enough for them to be located with less than atomic resolution data.…”

Section: Experimentally Positioned Anomalous Scatterersmentioning

confidence: 99%

ACORN: a review

Yao

Dodson

Wilson

et al. 2006

Acta Crystallogr D Biol Crystallogr

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The ACORN system was originally developed as a means of ab initio solution of protein structures when atomic resolution data were available. The first step is to obtain a starting set of phases, which must be at least slightly better than random. These may be calculated from a fragment of the structure, which can be anything from a single metal atom to a complete molecular-replacement model. A number of standard procedures are available in ACORN to orientate and position such a fragment. The fragment provides initial phases that give the first of a series of maps that are iteratively refined by a dynamic density-modification (DDM) process. Another FFTbased procedure is Sayre-equation refinement (SER), which modifies phases better to satisfy the Sayre equation. With good-quality atomic resolution data, the final outcome of applying DDM and SER is a map similar in appearance to that found from a refined structure, which is readily interpreted by automated procedures. Further development of ACORN now enables structures to be solved with less than atomic resolution data. A critical part of this development is the artificial extension of the data from the observed limit to 1 Å resolution. These extended reflections are allocated unit normalized structure amplitudes and then treated in a similar way to observed reflections except that they are downweighted in the calculation of maps. ACORN maps, especially at low resolution, tend to show C atoms less well, in particular C atoms which fall within the first diffraction minimum of their three neighbours. Two new density-modification procedures (DDM1 and DDM2) and a density-enhancement procedure (ENH) have been devised to counter this problem. It is demonstrated that high-quality maps showing individual atoms can be produced with the new ACORN. ACORN has also been demonstrated to be very effective in refining phase sets derived from physical processes such as those using anomalous scattering or isomorphous derivative data. Future work will be directed towards applying ACORN to resolutions down to 2 Å .

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Section: Experimentally Positioned Anomalous Scatterersmentioning

confidence: 99%

ACORN: a review

Yao

Dodson

Wilson

et al. 2006

Acta Crystallogr D Biol Crystallogr

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“…Phase information was obtained by multiple wavelength anomalous diffraction using NaBr-soaked crystals (12). Positions of five bromine atoms were determined, and phases were calculated to a 2.8-Å resolution applying the software SOLVE (13).…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Pereda

Waas

Jan

et al. 2004

Journal of Biological Chemistry

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Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-Å resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an ␣/␤ fold with a four-stranded antiparallel ␤-sheet in its core surrounded by two ␣-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.During the protein translation process, a significant proportion of the ribosomes that initiate mRNA readout do not reach the stop codon. Peptidyl-tRNA molecules may dissociate from the mRNA template causing a premature end of the process (1, 2). Accumulation of peptidyl-tRNAs reduces the efficiency of translation by sequestering tRNAs and impairing the initiation (3). Prokaryotic and eukaryotic cells show an enzymatic activity that releases the peptidyl moiety from the tRNA, called peptidyl-tRNA hydrolase (Pth), 1 allowing the free tRNA and peptide to be reused in protein synthesis (4, 5). First identified in Escherichia coli, genes encoding Pth have been found in bacteria and eukaryotes but not in archaea. E. coli Pth crystal structure has been solved and exhibits an ␣/␤ fold formed by three layers with a mixed five-strand ␤-sheet at the core. Its catalytic parameters have been measured, and several residues affecting the 5Ј-phosphate and 3Ј-end recognition of the peptidyl-tRNA substrate have been mapped (6, 7).Recently, a Pth-like activity has been identified in Methanocaldococcus jannaschii and characterized in Sulfolobus solfataricus, both archaebacteria (8, 9). The new enzyme called Pth2 is shorter in length and lacks any significant sequence homology to Pth. It coincides with the Pfam data base entry "Uncharacterized Protein Family 0099" (UPF0099) found in archaea and eukaryotes but not in bacterial genomes. Pth2 lacks any detectable homology to solved three-dimensional structures. S. solfataricus Pth2 displays a greater catalytic efficiency toward E. coli diacetyl-lysyl-tRNA Lys than E. coli Pth (9, 6). In addition, both enzymes differ in their mode of recognition of several tRNA features, like the 1-72-base pair mismatch in E. coli tRNA fMet or the presence of a phosphorylated 5Ј-end.Human Pth2, also called Bcl-2 inhibitor of transcription 1 (Bit1), possesses an ...

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“…strontium for calcium, (iii) selenium in selenomethionine, (iv) metal compounds which require a chemical reaction to take place, (v) multi-metal cluster complexes used for large proteins and multi-protein assemblies (Thygesen et al, 1996), (vi) the noble gases xenon (Scheonberg et al, 1965) and krypton , and (vii) halides (Dauter et al, 2000) and triiodide (Evans & Bricogne, 2002), including brominated nucleic acids (e.g. Ennifar et al, 2002).…”

Section: Classes Of Heavy Atom (Ha) Availablementioning

confidence: 99%

Heavy-atom derivatization

Garman

Murray

2003

Acta Crystallogr D Biol Cryst

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Most of the standard methods of solving macromolecular structures involve producing a protein crystal that is derivatized by an anomalous scatterer or heavy atom (MIR, SIRAS, MAD, SAD etc.). The theoretical methodology which underpins the extraction of phase information from such derivatives is widely available in the literature. In addition, there are comprehensive sources of information on the chemistry of heavy‐atom compounds and the ligands with which they are known to interact, as well as the Heavy Atom Databank accessible on the World Wide Web. This contribution therefore aims to provide some information on the less well documented practical problems of firstly deciding on an overall strategy for derivatization and secondly performing the physical manipulations involved in producing heavy‐atom derivatives from native protein crystals and then cryocooling them. Ways to optimize the chances of isomorphous unit cells are suggested. Methods of determining whether or not the heavy atom is bound are outlined, including the powerful technique of PIXE (particle‐induced X‐ray emission).

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Novel approach to phasing proteins: derivatization by short cryo-soaking with halides

Cited by 274 publications

References 27 publications

ACORN: a review

ACORN: a review

Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals a New Fold and Suggests Basis for a Bifunctional Activity

Heavy-atom derivatization

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