scholarly journals An anticipated optimization approach to macromolecular crystallization

2019 ◽  
Author(s):  
Fabrice Gorrec
Keyword(s):  
Cost Effective ◽  
Optimization Approach ◽  
Biological Processes ◽  
Initial Sample ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
Macromolecular Crystallization ◽  
As Solubility

AbstractCrystallization is an essential step for determining macromolecular structures at atomic resolution with X-ray crystallography. Crystals of diffraction quality obtained from purified samples of proteins, RNAs, DNAs and their complexes enable our understanding of biological processes and structure-based design of drugs. Targets of interest for researchers are however increasingly challenging to produce and crystallise. Progress in crystallization methods applicable to limiting amount of sample while increasing the yield of useful crystals are hence urgently needed. In this context, an Anticipated Optimization Approach was investigated. For this approach, it is assumed that samples are highly unstable and will most probably not produce useful crystals (‘hits’). By selecting leads very early, what remains from the initial sample can be used for follow-up optimization experiments. Subsequently, the reproducibility issues caused by sample variability are bypassed. An initial crystallization screen that failed to produce hits becomes a well-suited solubility assay that is a starting point for optimization. The approach was tested with a straightforward and cost-effective protocol developed elsewhere. The yield of useful crystals was increased and accelerated for three targets of pharmacologic studies.SynopsisThis study suggests that the yield of useful crystals obtained from challenging protein samples can be increased by using an initial crystallization screen as solubility assay.

scholarly journals Structural and functional characterization of proteins from the fire blight pathogen Erwinia amylovora. A review on the state of the art

2020 ◽  
Author(s):  
Stefano Benini
Keyword(s):  
Fire Blight ◽  
Erwinia Amylovora ◽  
Functional Characterization ◽  
Data Bank ◽  
Knock Out ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
Good Starting Point

Abstract Together with genome analysis and knock-out mutants, structural and functional characterization of proteins provide valuable hints on the biology of the organism under investigation. Structural characterization can be achieved by techniques such as X-ray crystallography, NMR, Cryo-EM. The information derived from the structure are a good starting point to comprehend the details of the proteins molecular function for a better understanding of their biological role. This review aims at describing the progress in the structural and functional characterization of proteins from the plant pathogen Erwinia amylovora obtained by structural biology and currently deposited in the Protein Data Bank.

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.

PLP undergoes conformational changes during the course of an enzymatic reaction

2014 ◽  
Vol 70 (2) ◽  
pp. 596-606 ◽  
Author(s):  
Ho-Phuong-Thuy Ngo ◽  
Nuno M. F. S. A. Cerqueira ◽  
Jin-Kwang Kim ◽  
Myoung-Ki Hong ◽  
Pedro Alexandrino Fernandes ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Enzymatic Reaction ◽  
Nucleophilic Attack ◽  
The Novel ◽  
X Ray ◽  
X Ray Crystallography ◽  
Starting Point ◽  
High Level

Numerous enzymes, such as the pyridoxal 5′-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

scholarly journals A Simple, Efficient, and Eco-Friendly Method for the Preparation of 3-Substituted-2,3-dihydroquinazolin-4(1H)-one Derivatives

Molecules ◽  
2019 ◽  
Vol 24 (22) ◽  
pp. 4052 ◽  
Author(s):  
Zainab Almarhoon ◽  
Kholood A. Dahlous ◽  
Rakia Abd Alhameed ◽  
Hazem A. Ghabbour ◽  
Ayman El-Faham
Keyword(s):  
Molecular Structure ◽  
Cost Effective ◽  
Environmentally Benign ◽  
Conventional Heating ◽  
Second Step ◽  
The Novel ◽  
Monoclinic Crystal ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cost Effective Method

A simple, cost-effective method under environmentally benign conditions is a very important concept for the preparation of 2,3-dihydroquinazolin-4(1H)-one derivatives. The present work describes an efficient and eco-friendly protocol for the synthesis of 2-amino-N-(2-substituted-ethyl)benzamide and 3-substituted-2,3-dihydroquinazolin-4(1H)-one derivatives. The novel feature of this protocol is the use of 2-methyl tetrahydrofuran (2-MeTHF) as an eco-friendly alternative solvent to tetrahydrofuran (THF) in the first step. In the second step, methanol in the presence of potassium carbonate as a catalyst was used under conventional heating or microwave irradiation, which provided an eco-friendly method to afford the target products in excellent yields and purities. NMR (1H and 13C), elemental analysis, and LC-MS confirmed the structures of all compounds. X-ray crystallography further confirmed the structure of the intermediate 2-amino-N-(2-substituted-ethyl)benzamide 3a. The molecular structure of 3a was monoclinic crystal, with P21/c, a = 13.6879 (11) Å, b = 10.2118 (9) Å, c = 9.7884 (9) Å, β = 105.068 (7)°, V = 1321.2 (2) Å3, and Z = 4.

scholarly journals Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

2021 ◽  
Author(s):  
Geqing Wang ◽  
Biswaranjan Mohanty ◽  
Martin Williams ◽  
Bradley Doak ◽  
Rabeb Dhouib ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Crystal Structures ◽  
Small Molecule ◽  
Sodium Taurocholate ◽  
Structural Basis ◽  
Human Pathogens ◽  
X Ray ◽  
X Ray Crystallography ◽  
Hydrophobic Groove ◽  
Starting Point

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446 ± 10 µM. The same benzimidazole compound has ~8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD > 3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveals the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

scholarly journals A Ubiquitin-Binding Domain that Does Not Bind Ubiquitin

2018 ◽  
Author(s):  
Michael Lim ◽  
Joseph A. Newman ◽  
Hannah L. Williams ◽  
Hazel Aitkenhead ◽  
Opher Gileadi ◽  
...  
Keyword(s):  
Structural Model ◽  
Coiled Coil ◽  
Biological Processes ◽  
Hydrophobic Residues ◽  
X Ray ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
Ubiquitin Binding Domain ◽  
Cue Domain

Ubiquitylation, the post-translational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function exclusively by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. We show that the SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. A structural model of the minimal KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography analysis is presented. Remarkably, the CUE1 domain, which resembles a canonical CUE domain, recognizes 2 clusters of exposed hydrophobic residues on KAP1, but these are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data challenge the well-established dogma that CUE domains exclusively recognize the ubiquitin-fold.

scholarly journals The Diamond Light Source and the challenges ahead for structural biology: some informal remarks

2015 ◽  
Vol 373 (2036) ◽  
pp. 20130156 ◽  
Author(s):  
V. Ramakrishnan
Keyword(s):  
Synchrotron Radiation ◽  
Light Source ◽  
Structural Biology ◽  
Complex Structures ◽  
Biological Processes ◽  
Short Article ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past

The remarkable advances in structural biology in the past three decades have led to the determination of increasingly complex structures that lie at the heart of many important biological processes. Many of these advances have been made possible by the use of X-ray crystallography using synchrotron radiation. In this short article, some of the challenges and prospects that lie ahead will be summarized.

scholarly journals PHENIX: a comprehensive Python-based system for macromolecular structure solution

2010 ◽  
Vol 66 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Paul D. Adams ◽  
Pavel V. Afonine ◽  
Gábor Bunkóczi ◽  
Vincent B. Chen ◽  
Ian W. Davis ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Numerical Data ◽  
Crystallographic Structure ◽  
Biological Processes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Software Packages ◽  
Structure Solution ◽  
Repeated Use ◽  
Subjective Input

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics.PHENIXhas been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.

scholarly journals Isonitrile Ruthenium and Iron PNP Complexes: Synthesis, Characterization and Catalytic Assessment for Base-Free Dehydrogenative Coupling of Alcohols

2020 ◽  
Author(s):  
Duc Hanh Nguyen ◽  
Delphine Merel ◽  
Nicolas Merle ◽  
Xavier Trivelli ◽  
Frederic Capet ◽  
...  
Keyword(s):  
Ruthenium Complexes ◽  
Outer Sphere ◽  
Pincer Complexes ◽  
Initial Activity ◽  
Ancillary Ligands ◽  
X Ray ◽  
Bromide Anion ◽  
X Ray Crystallography ◽  
Dehydrogenative Coupling

Neutral and ionic ruthenium and iron aliphatic PNPH-type pincer complexes (PNPH= NH(CH2CH2PiPr2)2) bearing benzyl, n-butyl or tert-butyl isocyanide ancillary ligands have been prepared and characterized. Reaction of [RuCl2(PNPH)]2 with one equivalent CN-R per ruthe-nium center affords complexes [Ru(PNPH)Cl2(CNR)] (R= benzyl, 1a, R= n-butyl, 1b, R= t-butyl, 1c), with cationic [Ru(PNPH)(Cl)(CNR)2]Cl 2a-c as side-products. Complexes 2a-c are selectively prepared upon reaction of [RuCl2(PNPH)]2 with 2 equiva-lents of isonitrile per ruthenium center. Dichloride species 1a-c react with excess NaBH4 to afford [Ru(PNPH)(H)(BH4)(CN-R)] 3a-c, analogues to benchmark Takasago catalyst [Ru(PNP)(H)(BH4)(CO)]. Reaction of 1a-c with a single equivalent of NaBH4 under protic conditions results in formation of hydrido chloride derivatives [Ru(PNPH)(H)(Cl)(CN-R)] (4a-c), from which 3a-c can be prepared upon reaction with excess NaBH4. Use of one equivalent of NaHBEt3 with 4a and 4c affords bishydrides [Ru(PNPH)(H)2(CN-R)] 5a and 5c. In the case of bulkier t-butylisonitrile, two isomers were observed by NMR, with the PNP framework in either meridional or facial confor-mation. Deprotonation of 4c by KOtBu generates amido derivative [Ru(PNP’)(H)(CN-t-Bu)] (6, PNP’= -N(CH2CH2PiPr2)2), unstable in solution. Addition of excess benzylisonitrile to 4a provides cationic hydride [Ru(PNPH)(H)(CN-CH2Ph)2]Cl (7). Concerning iron chemis-try, [Fe(PNPH)Br2] reacts one equivalent benzylisonitrile to afford [Fe(PNPH)(Br)(CNCH2Ph)2]Br (8). The outer-sphere bromide anion can be exchanged by salt metathesis with NaBPh4 to generate [Fe(PNPH)(Br)(CNCH2Ph)2](BPh4) (9). Cationic hydride species [Fe(PNPH)(H)(CN-t-Bu)2](BH4) (10) is prepared from consecutive addition of excess CN-t-Bu and NaBH4 on [Fe(PNPH)Br2]. Ruthenium complexes 3a-c are active in acceptorless alcohol dehydrogenative coupling into ester under base-free conditions. From kinetic follow-up, the trend in initial activity is 3a ≈ 3b > [Ru(PNPH)(H)(BH4)(CO)] >> 3c; for robustness, [Ru(H)(BH4)(CO)(PNPH)] > 3a > 3b >> 3c. Hy-potheses are given to account for the observed deactivation. Complexes 3b, 3c, 4a, 4c, 5c, 7, cis-8 and 9 were characterized by X-ray crystallography.

scholarly journals Achieving a Good Crystal System for Crystallographic X-ray Fragment Screening

2018 ◽  
Author(s):  
Patrick M. Collins ◽  
Alice Douangamath ◽  
Romain Talon ◽  
Alexandre Dias ◽  
José Brandão-Neto ◽  
...  
Keyword(s):  
User Program ◽  
X Ray ◽  
Crystal System ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Surface Sites ◽  
Screening Experiment ◽  
Active User ◽  
Density Maps

The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program.  The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites:  this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user programme that is generating large amounts of data:  in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals.  It has also generated a large number of learnings concerning the main remaining bottleneck, namely obtaining a suitable crystal system that will support a successful fragment screen.  Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab.

Sign in / Sign up

Export Citation Format

Share Document