Investigating Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods

2018 ◽  
Vol 8 (19) ◽  
pp. 5003-5016 ◽  
Author(s):  
Robert W. Powell, III ◽  
M. Pilar Buteler ◽  
Sunidhi Lenka ◽  
Michele Crotti ◽  
Sara Santangelo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Computational Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity

Saccharomyces cerevisiae OYE 3 and OYE 1 share 80% sequence identity, but sometimes differ in stereoselectivities.

Using cryo-electron microscopy maps for X-ray structure determination of homologues

2020 ◽  
Vol 76 (1) ◽  
pp. 63-72
Author(s):  
Lingxiao Zeng ◽  
Wei Ding ◽  
Quan Hao
Keyword(s):  
Electron Microscopy ◽  
Hybrid Method ◽  
Model Building ◽  
Test Cases ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Whole Process ◽  
Cryo Electron Microscopy

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable R work and R free values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

scholarly journals Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

2018 ◽  
Vol 19 (11) ◽  
pp. 3401 ◽  
Author(s):  
Ashutosh Srivastava ◽  
Tetsuro Nagai ◽  
Arpita Srivastava ◽  
Osamu Miyashita ◽  
Florence Tama
Keyword(s):  
Protein Dynamics ◽  
Computational Methods ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

scholarly journals Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

2008 ◽  
Vol 105 (46) ◽  
pp. 17682-17687 ◽  
Author(s):  
Jessica Bockhorn ◽  
Bharvi Balar ◽  
Dongming He ◽  
Eden Seitomer ◽  
Paul R. Copeland ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Cost Effective ◽  
Anomalous Dispersion ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Cost Effective Method ◽  
Strain Collection

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

scholarly journals Integrated X-ray crystallography, optical and computational methods in studies of structure and luminescence of new synthesized complexes of lanthanides with ligands derived from 2,6-diformylpyridine

Polyhedron ◽  
2011 ◽  
Vol 30 (5) ◽  
pp. 851-859 ◽  
Author(s):  
Gelson Manzoni de Oliveira ◽  
Aline Machado ◽  
Geraldo Wachholz Gomes ◽  
Jorge H.S.K. Monteiro ◽  
Marian R. Davolos ◽  
...  
Keyword(s):  
Computational Methods ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Diamagnetic cobalt(III)tris(o-ethylxanthate) and nickel(II)bis(o-ethylxanthate)

2017 ◽  
Vol 16 (2) ◽  
pp. 138-146 ◽  
Author(s):  
Filip Varga ◽  
Ján Titiš ◽  
Cyril Rajnák ◽  
Ján Moncoľ ◽  
Roman Boča
Keyword(s):  
Ir Spectroscopy ◽  
Single Crystal ◽  
Computational Methods ◽  
Magnetic Measurements ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ethyl Xanthate

Abstract Diamagnetic [Co(xanth)3] and [Ni(xanth)2] complexes have been prepared by reaction of Co(II) and Ni(II) salts with potassium O-ethyl xanthate (Kxanth). The isolated Co(III) and Ni(II) complexes have been characterized by single-crystal X-ray crystallography, UV-VIS and IR spectroscopy, computational methods, and magnetic measurements.

scholarly journals Principles and characteristics of biological assemblies in experimentally determined protein structures

2019 ◽  
Author(s):  
Qifang Xu ◽  
Roland L. Dunbrack
Keyword(s):  
Physical Properties ◽  
Computational Methods ◽  
Structural Information ◽  
Protein Structures ◽  
Sequence Conservation ◽  
Homologous Proteins ◽  
X Ray ◽  
Biologically Relevant ◽  
X Ray Crystallography

AbstractMore than half of all structures in the PDB are assemblies of two or more proteins, including both homooligomers and heterooligomers. Structural information on these assemblies comes from X-ray crystallography, NMR, and cryo-EM spectroscopy. The correct assembly in an X-ray structure is often ambiguous, and computational methods have been developed to identify the most likely biologically relevant assembly based on physical properties of assemblies and sequence conservation in interfaces. Taking advantage of the large number of structures now available, some of the most recent methods have relied on similarity of interfaces and assemblies across structures of homologous proteins.

scholarly journals Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

2010 ◽  
Vol 76 (19) ◽  
pp. 6351-6359 ◽  
Author(s):  
Toshihiko Kitajima ◽  
Yasunori Chiba ◽  
Yoshifumi Jigami
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Methylotrophic Yeast ◽  
Culture Conditions ◽  
Crystallographic Analysis ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Standard Culture ◽  
Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

scholarly journals Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography

2010 ◽  
Vol 71 (2) ◽  
pp. 207-223 ◽  
Author(s):  
Kathleen M. Clark ◽  
Nadia Fedoriw ◽  
Katrina Robinson ◽  
Sara M. Connelly ◽  
Joan Randles ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transmembrane Proteins ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals A new view on crystal harvesting

2014 ◽  
Vol 47 (3) ◽  
pp. 1158-1161 ◽  
Author(s):  
Joseph R. Luft ◽  
Thomas D. Grant ◽  
Jennifer R. Wolfley ◽  
Edward H. Snell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
High Throughput Screening ◽  
Scale Up ◽  
Trna Synthetase ◽  
Objective Lens ◽  
X Ray ◽  
X Ray Crystallography ◽  
Automated Process ◽  
Structural Solution

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain ofSaccharomyces cerevisiaeglutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

scholarly journals Factors influencing estimates of coordinate error for molecular replacement

2020 ◽  
Vol 76 (1) ◽  
pp. 19-27
Author(s):  
Kaushik S. Hatti ◽  
Airlie J. McCoy ◽  
Robert D. Oeffner ◽  
Massimo D. Sammito ◽  
Randy J. Read
Keyword(s):  
Error Estimates ◽  
Sequence Similarity ◽  
Protein Structures ◽  
Optimal Error Estimates ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Highly Correlated ◽  
Coordinate Error

Good prior estimates of the effective root-mean-square deviation (r.m.s.d.) between the atomic coordinates of the model and the target optimize the signal in molecular replacement, thereby increasing the success rate in difficult cases. Previous studies using protein structures solved by X-ray crystallography as models showed that optimal error estimates (refined after structure solution) were correlated with the sequence identity between the model and target, and with the number of residues in the model. Here, this work has been extended to find additional correlations between parameters of the model and the target and hence improved prior estimates of the coordinate error. Using a graph database, a curated set of 6030 molecular-replacement calculations using models that had been solved by X-ray crystallography was analysed to consider about 120 model and target parameters. Improved estimates were achieved by replacing the sequence identity with the Gonnet score for sequence similarity, as well as by considering the resolution of the target structure and the MolProbity score of the model. This approach was extended by analysing 12 610 additional molecular-replacement calculations where the model was determined by NMR. The median r.m.s.d. between pairs of models in an ensemble was found to be correlated with the estimated r.m.s.d. to the target. For models solved by NMR, the overall coordinate error estimates were larger than for structures determined by X-ray crystallography, and were more highly correlated with the number of residues.

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