Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization

Stewart, Patrick D. Shaw; Kolek, Stefan; Briggs, Richard A.; Chayen, Naomi E.; Baldock, Peter F. M.

doi:10.1021/cg2001442

Cited by 70 publications

(69 citation statements)

References 26 publications

(48 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Extensive screening with an Oryx8 robot (Douglas Instruments) [22] yielded a suitable solution composed of 100 mM Hepes/NaOH (pH 7.5) and 1.5 M Li 2 SO 4 . For freezing, single crystals were passed through a small drop of paratone (Hampton Research) and plunged in liquid N 2 .…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

et al. 2013

View full text Add to dashboard Cite

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

show abstract

Section: Methodsmentioning

confidence: 99%

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

et al. 2013

View full text Add to dashboard Cite

show abstract

“…A micro-seeding approach [19] has produced crystals in many more conditions in a range of precipitants and at various pH values with different additives. These crystals were used for substrate/inhibitor soaks.…”

Section: Active Sitementioning

confidence: 99%

Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis

et al. 2015

View full text Add to dashboard Cite

Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters, with optimal activity for pNPpropionate. The enzyme retained 95% activity after incubation for 1 h at 80°C. The crystal structures of the native TtEst and its complexes with the substrate analogue D-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNPesters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate.

show abstract

“…This removes misoriented macromolecules or other molecules that may have poisoned the crystal surface. The microseeding method [10,14,36,124] involves the transfer of very small nuclei to solutions with metastable supersaturation (Figure 11B). …”

Section: Approaches To Induce Nucleationmentioning

confidence: 99%

An Overview of Biological Macromolecule Crystallization

Krauss

Merlino

Vergara

et al. 2013

IJMS

123

View full text Add to dashboard Cite

The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.

show abstract

Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization

Cited by 70 publications

References 26 publications

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis

An Overview of Biological Macromolecule Crystallization

Contact Info

Product

Resources

About