A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

Brown, James; Walter, Thomas S.; Carter, L.; Abrescia, Nicola G. A.; Aricescu, A. Radu; Batuwangala, Thil; Bird, Louise E.; Brown, Nils; Chamberlain, Philip P.; Davis, Simon J.; Дубинина, Е. О.; Endicott, Jane; Fennelly, J A; Gilbert, R.J.C.; Harkiolaki, Maria; Hon, Wai-Ching; Kimberley, Fiona C.; Love, Christopher; Mancini, Erika J.; Manso-Sancho, Raquel; Nichols, C.E.; Robinson, R.A.; Sutton, Geoff; Schueller, N.; Sleeman, Mark C.; Stewart‐Jones, Guillaume; Vuong, Mai Tuyet; Welburn, Julie; Zhang, Z.; Stammers, D.K.; Owens, Ray; Jones, Emma; Harlos, K.; Stuart, David I.

doi:10.1107/s0021889803002012

Cited by 46 publications

(23 citation statements)

References 5 publications

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“…Hampton Research sparse matrix & GRID screens were then set up (i.e., Crystal Screen I, Crystal Screen II, Crystal Screen Cryo, PEG/Ion, Natrix, MembFac, Index, SaltRX, PEG/LiCl Grid, NaCl Grid, PEG 6000 Grid, and A/S Grid). All crystallizations were performed at 293 K and set up as sitting‐drop vapor‐diffusion experiments, initially at 100‐nL droplet size using a Cartesian robot system,31 and later by hand for crystal optimization with 6‐μL droplet size. For crystals that grew from the screens, single frames of X‐ray data were collected in house, using a MAR345 image plate system on a Rigaku generator equipped with a Cu anode and Osmic multilayer optics to give Kα radiation (λ = 1.5418 Å).…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

Nichols

Johnson

Lockyer³

et al. 2006

Proteins

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The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.

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Section: Methodsmentioning

confidence: 99%

Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

Nichols

Johnson

Lockyer³

et al. 2006

Proteins

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“…Crystallization experiments were initially carried out at the Oxford Protein Production Facility (Brown et al. , 2003; Walter et al.…”

Section: Methodsmentioning

confidence: 99%

An ATP‐binding cassette‐type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Müller¹,

Thomas

Horler

et al. 2005

Molecular Microbiology

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SummaryCampylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease GuillainBarré syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K d of ~ 10 ----7 M consistent with a role as a receptor for a high-affinity transporter. These data imply that Cj0982 is the binding protein component of an ABCtype cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni.

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“…Key foundations for structural genomics included developments in macromolecular X-ray crystallography and NMR along with general systems for cloning and expression of proteins. In X-ray crystallography the increasing use of tunable synchrotron radiation in collecting anomalous diffraction data on protein crystals, the use of selenomethionine in crystallographic phase determination (68), automation of nanodroplet crystallization (11, 52), cryo-cooling techniques that reduced radiation damage to those crystals (30, 49), and automation of the structure determination process for macromolecular crystallography (47, 62) suggested that determining a protein structure once crystals were available would be increasingly straightforward. In the NMR field, the availability of cryo-probe high-field NMR devices (32), the development of multidimensional techniques for data acquisition (60), and the introduction of automated procedures for data analysis (8, 28, 43) similarly suggested that NMR structure determination was headed toward ever-higher throughput.…”

Section: Introductionmentioning

confidence: 99%

Lessons from Structural Genomics

Terwilliger

Stuart²,

Yokoyama³

2009

Annu. Rev. Biophys.

120

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A decade of structural genomics, the large-scale determination of protein structures, has generated a wealth of data and many important lessons for structural biology and for future large-scale projects. These lessons include a confirmation that it is possible to construct large-scale facilities that can determine the structures of a hundred or more proteins per year, that these structures can be of high quality, and that these structures can have an important impact. Technology development has played a critical role in structural genomics, the difficulties at each step of determining a structure of a particular protein can be quantified, and validation of technologies is nearly as important as the technologies themselves. Finally, rapid deposition of data in public databases has increased the impact and usefulness of the data and international cooperation has advanced the field and improved data sharing.

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A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results

Abstract: Harkiolaki, M et al. (2003) A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results. Journal of Applied Crystallography, 36 (2). pp. 315-318.

Cited by 46 publications

References 5 publications

Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

An ATP‐binding cassette‐type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Lessons from Structural Genomics

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