Publications

@article{noKey,

title = {Automated Assembly of Standard Biological Parts},

url = {https://www.sciencedirect.com/science/article/abs/pii/B9780123851208000164?via%3Dihub},

doi = {https://doi.org/10.1016/B978-0-12-385120-8.00016-4},

year  = {2011},

date = {2011-01-01},

abstract = {The primary bottleneck in synthetic biology research today is the construction of physical DNAs, a process that is often expensive, time-consuming, and riddled with cloning difficulties associated with the uniqueness of each DNA sequence. We have developed a series of biological and computational tools that lower existing barriers to automation and scaling to enable affordable, fast, and accurate construction of large DNA sets. Here we provide detailed protocols for high-throughput, automated assembly of BglBrick standard biological parts using iterative 2ab reactions. We have implemented these protocols on a minimal hardware platform consisting of a Biomek 3000 liquid handling robot, a benchtop centrifuge and a plate thermocycler, with additional support from a software tool called AssemblyManager. This methodology enables parallel assembly of several hundred large error-free DNAs with a 96+% success rate.},

keywords = {TEMPEST},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The structure of LsrB from Yersinia pestis complexed with autoinducer-2},

author = {Kavanaugh, Jeffrey S., Gakhar, Lokesh},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3232125/},

doi = {https://doi.org/10.1107/S1744309111042953},

year  = {2011},

date = {2011-01-01},

abstract = {The crystal structure of LsrB from Yersinia pestis complexed with autoinducer-2 (AI-2; space group P212121, unit-cell parameters a = 40.61, b = 61.03, c = 125.23 �) has been solved by molecular replacement using the structure of LsrB from Salmonella typhimurium (PDB entry 1tjy) and refined to R = 0.180 (R free = 0.213) at 1.75 � resolution. The electron density for bound AI-2 and the stereochemistry of the AI-2-binding site are consistent with bound AI-2 adopting the (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran conformation, just as has been observed in the crystal structures of the Salmonella typhimurium and Sinorhizobium meliloti LsrB�AI-2 complexes.},

keywords = {ROCKMAKER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium},

author = {Jarrott, R., Shouldice, S. R.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864702/},

doi = {https://doi.org/10.1107/S1744309110011942},

year  = {2010},

date = {2010-01-01},

abstract = {Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-�12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 � and belonged to space groups P21, P21212 and C2, respectively.},

keywords = {ROCKMAKER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Activation of colicin M by the FkpA prolyl cis-trans isomerase/chaperone},

author = {Helbig, Stephanie, Patzer, Silke I.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057819/},

doi = {https://doi.org/10.1074/jbc.M110.165274},

year  = {2010},

date = {2010-01-01},

abstract = {Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Crystallization and preliminary X-ray data collection of the Escherichia coli lipoproteins BamC, BamD and BamE},

author = {Meriläinena, Gitte, Wierenga, Rik K.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998360/},

doi = {https://doi.org/10.1107/S1744309110034160},

year  = {2010},

date = {2010-01-01},

abstract = {In Escherichia coli, the �-barrel assembly machinery (or BAM complex) mediates the recognition, insertion and assembly of outer membrane proteins. The complex consists of the integral membrane protein BamA (an Omp85-family member) and the lipoproteins BamB, BamC, BamD and BamE. The purification and crystallization of BamC, BamD and BamE, each lacking the N-�terminal membrane anchor, is described. While the smallest protein BamE yielded crystals under conventional conditions, BamD only crystallized after stabilization with urea. Full-length BamC did not crystallize, but was cleaved by subtilisin into two domains which were subsequently crystallized independently. High-resolution data were acquired from all proteins.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P II signal transduction protein},

author = {Fokina, Oleksandra, Chellamuthu, Vasuki-Ranjani},

url = {https://www.pnas.org/content/107/46/19760},

doi = {https://doi.org/10.1073/pnas.1007653107},

year  = {2010},

date = {2010-01-01},

abstract = {P II proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P II and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P II target, whose activity responds to 2-OG via P II . Here we show structures of the Synechococcus elongatus P II protein in complex with ATP, Mg??, and 2-OG, which clarify how 2-OG affects P II -NAGK interaction. P II trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P II trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg?? ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P II target. Structures of P II trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P II senses effectors in a wide range of concentrations.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Crystallization and preliminary X-ray analysis of mycophenolic acid-resistant and mycophenolic acid-sensitive forms of IMP dehydrogenase from the human fungal pathogen Cryptococcus},

author = {Morrow, Carl A., Stamp, Anna},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935239/},

doi = {https://doi.org/10.1107/S1744309110031659},

year  = {2010},

date = {2010-01-01},

abstract = {Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals. The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components; however, the number of these targets is limited. In the search for new targets and lead compounds, C. neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase (IMPDH); in contrast, a rare subtype of the related C. gattii is naturally resistant. Here, the expression, purification, crystallization and preliminary crystallographic analysis of IMPDH complexed with IMP and NAD+ is reported for both of these Cryptococcus species. The crystals of IMPDH from both sources had the symmetry of the tetragonal space group I422 and diffracted to a resolution of 2.5 Å for C. neoformans and 2.6 Å for C. gattii.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK–PII Complex Formation},

author = {Fokina, Oleksandra, Chellamuthu, Vasuki-Ranjani},

url = {https://www.sciencedirect.com/science/article/abs/pii/S0022283610003797?via%3Dihub},

doi = {https://doi.org/10.1016/j.jmb.2010.04.018},

year  = {2010},

date = {2010-01-01},

abstract = {PII signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, PII binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, PII (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of PII binding to NAGK, we searched for PII variants with altered binding characteristics and found PII variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type PII protein. Analysis of interactions between these PII variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the PII I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the PII I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of PII proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus PII after having latched onto NAGK. Moreover, both PII I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of PII–NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of PII initiates the bending of the extended T-loop of PII, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals},

author = {Gilla, Harindarpal S.},

url = {https://journals.iucr.org/f/issues/2010/03/00/wd5124/index.html},

doi = {https://doi.org/10.1107/S1744309110002022},

year  = {2010},

date = {2010-01-01},

abstract = {The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with ~84% of their surface area buried) and to be surrounded by partially polar microenvironments (with ~43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (~18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases},

author = {Kissick, David J., Gualtieri, Ellen J.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825164/},

doi = {https://doi.org/10.1021/ac902139w},

year  = {2010},

date = {2010-01-01},

abstract = {Second order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 micron) with a minimal background due to the surrounding lipid mesophase (~1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Interaction between plate make and protein in protein crystallisation screening},

author = {King, Gordon J., Chen, Kai-En},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773427/},

doi = {https://doi.org/10.1371/journal.pone.0007851},

year  = {2009},

date = {2009-01-01},

abstract = {Background Protein crystallization screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. Methodology/Principal Findings We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. Conclusions/Significance Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallise, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Some lessons from the systematic production and structural analysis of soluble αβ T-cell receptors},

author = {Gillespiec, G.M.A., Harlos, Karl},

url = {https://www.sciencedirect.com/science/article/abs/pii/S0022175909002610?via%3Dihub},

doi = {https://doi.org/10.1016/j.jim.2009.08.008},

year  = {2009},

date = {2009-01-01},

abstract = {T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, αβ-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 microm size X-ray synchrotron beam},

author = {Cherezov, Vadim, Hanson, Michael A.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843980/},

doi = {https://doi.org/10.1098/rsif.2009.0142.focus},

year  = {2009},

date = {2009-01-01},

abstract = {Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 �m minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Crystallizing membrane proteins using lipidic mesophases},

author = {Caffrey, Martin, Cherezov, Vadim},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732203/},

doi = {https://doi.org/10.1038/nprot.2009.31},

year  = {2009},

date = {2009-01-01},

abstract = {A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and a-helical and �-barrel proteins. Its most recent successes are the human engineered �2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Expression, purification and preliminary X-ray diffraction studies of VERNALIZATION1(208-341) from Arabidopsis thaliana},

author = {King, Gordon, Hill, Justine M.},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650445/},

doi = {https://doi.org/10.1107/S1744309109004217},

year  = {2009},

date = {2009-01-01},

abstract = {VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1208�341 includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1208�341 was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1208�341 crystal diffracted to 2.1 � and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 �, a = 90.0, � = 115.4, ? = 90.0�. Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 �3 Da-1 and a solvent content of 40.1% were calculated.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Structural insights into mechanisms of the small RNA methyltransferase HEN1},

author = {Huang, Ying, Ji, Lijuan},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125239/},

doi = {https://doi.org/10.1038/nature08433},

year  = {2009},

date = {2009-01-01},

abstract = {RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ~20�30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide3�6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3�5,7,8. Here we report the 3.1 � crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2'-O-methylation mechanism.},

keywords = {ROCKMAKER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Design of Experiments useful statistical tool in assay development or vendor disconnect!},

url = {https://www.ddw-online.com/media/32/2273/winter-2009-design-of-experiments.pdf},

doi = {undefined},

year  = {2009},

date = {2009-01-01},

abstract = {The use of design of experiments (DOE) in assay development (AD) has the potential to speed up assay optimisation (ie reduce assay development bottlenecks) and to facilitate a more thorough evaluation of assay variables. Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays. Although standalone DOE software packages are available,  these were not written specifically for biological applications and they vary in their suitability for  AD.  DOE needs to be simpler to implement to make a major impact on  AD. A market opportunity exists for a turnkey solution that directly links statistical design with automated liquid handler programming and also feeds the assay readout directly into the statistical analysis,  to suggest and facilitate further iterative retesting.  Until new tools or more encompassing solutions emerge,  the full impact of DOE on  AD is unlikely to be realised.},

keywords = {FORMULATOR},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {The stimulatory RNA of the Visna-Maedi retrovirus ribosomal frameshifting signal is an unusual pseudoknot with an interstem element},

author = {Pennell, Simon, Manktelow, Emily},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441976/},

doi = {https://doi.org/10.1261/rna.1042108},

year  = {2008},

date = {2008-01-01},

abstract = {The stimulatory RNA of the Visna-Maedi virus (VMV) -1 ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix, reminiscent of the HIV-1 frameshift-stimulatory RNA, or as an RNA pseudoknot. The pseudoknot is unusual in that it would include a 7 nucleotide loop (termed here an interstem element [ISE]) between the two stems. In almost all frameshift-promoting pseudoknots, ISEs are absent or comprise a single adenosine residue. Using a combination of RNA structure probing, site directed mutagenesis, NMR, and phylogenetic sequence comparisons, we show here that the VMV stimulatory RNA is indeed a pseudoknot, conforming closely to the modeled structure, and that the ISE is essential for frameshifting. Pseudoknot function was predictably sensitive to changes in the length of the ISE, yet altering its sequence to alternate pyrimidine/purine bases was also detrimental to frameshifting, perhaps through modulation of local tertiary interactions. How the ISE is placed in the context of an appropriate helical junction conformation is not known, but its presence impacts on other elements of the pseudoknot, for example, the necessity for a longer than expected loop 1. This may be required to accommodate an increased flexibility of the pseudoknot brought about by the ISE. In support of this, 1H NMR analysis at increasing temperatures revealed that stem 2 of the VMV pseudoknot is more labile than stem 1, perhaps as a consequence of its connection to stem 1 solely via flexible single-stranded loops.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus},

author = {Evdokimova, Artem G., Mekel, Marlene},

url = {https://www.sciencedirect.com/science/article/abs/pii/S1047847707002882?via%3Dihub},

doi = {https://doi.org/10.1016/j.jsb.2007.11.002},

year  = {2008},

date = {2008-01-01},

abstract = {In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors.  The native enzyme crystals were of poor quality—they only diffracted X-rays to 3–5 Å resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 Å or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}

@article{noKey,

title = {In situ X-ray analysis of protein crystals in low-birefringent and X-ray transmissive plastic microchannels},

author = {Ng, Joseph D, Clark, Peter J},

url = {http://scripts.iucr.org/cgi-bin/paper?S0907444907060064},

doi = {https://doi.org/10.1107/S0907444907060064},

year  = {2008},

date = {2008-01-01},

abstract = {Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent, X-ray transmissive and compatible with microfluidic fabrication in restricted geometry. The model proteins thaumatin, lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration. Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing. Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination. Fluidic crystallization in the plastic platform was also coupled with a commercialized automated imager and an in situ X-ray scanner that allowed optical and X-ray inspection of crystallization hits. The results demonstrate the feasibility of rapid nanovolume counter-diffusion crystallization experiments without the need for additional instrumentation.},

keywords = {ROCKIMAGER},

pubstate = {published},

tppubtype = {article}

}