scholarly journals A beginner’s guide to macromolecular crystallization

2021 ◽  
Vol 43 (1) ◽  
pp. 36-43
Author(s):  
Fabrice Gorrec
Keyword(s):  
Crystal Nucleation ◽  
Trial And Error ◽  
Bioinformatics Analyses ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

scholarly journals Iterative screen optimization maximizes the efficiency of macromolecular crystallization

2019 ◽  
Vol 75 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Harrison G. Jones ◽  
Daniel Wrapp ◽  
Morgan S. A. Gilman ◽  
Michael B. Battles ◽  
Nianshuang Wang ◽  
...  
Keyword(s):  
Narrow Range ◽  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Experiment ◽  
Automated Process ◽  
Macromolecular Crystallization ◽  
Rate Limiting

Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full advantage of this method, the value of ISO is demonstrated by using it to successfully crystallize a panel of six diverse proteins. The results suggest that ISO is an effective method to obtain macromolecular crystals, particularly for proteins that crystallize under a narrow range of precipitant concentrations.

scholarly journals Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

2021 ◽  
Author(s):  
Mrinal Shekhar ◽  
Chitrak Gupta ◽  
Kano Suzuki ◽  
Abhishek Singharoy ◽  
Takeshi Murata
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Structural Information ◽  
Hydrolysis Product ◽  
X Ray ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Dynamics Simulations ◽  
Rate Limiting

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or 'dwell' is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transfor-mations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain level interactions transcend into larger scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

scholarly journals Chemical clustering and visualization applied to macromolecular crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1145-C1145
Author(s):  
Andrew Bruno ◽  
Amanda Ruby ◽  
Joseph Luft ◽  
Thomas Grant ◽  
Jayaraman Seetharaman ◽  
...  
Keyword(s):  
Hierarchical Clustering ◽  
High Throughput ◽  
Active Sites ◽  
X Ray Crystallography ◽  
Rate Limiting Step ◽  
Potential Applications ◽  
Crystallization Conditions ◽  
Electron Density Map ◽  
Distance Coefficient ◽  
Rate Limiting

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and the insertion and correct placement of a sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient appears to be robust in this application and coupled with hierarchical clustering and the overlay of crystallization outcome reveals information of biological relevance. While tested with a single example the potential applications appear promising.

scholarly journals The influence of active site conformations on the hydride transfer step of the thymidylate synthase reaction mechanism

2015 ◽  
Vol 17 (46) ◽  
pp. 30793-30804 ◽  
Author(s):  
Katarzyna Świderek ◽  
Amnon Kohen ◽  
Vicent Moliner
Keyword(s):  
Reaction Mechanism ◽  
Thymidylate Synthase ◽  
Active Site ◽  
Hydride Transfer ◽  
Md Simulations ◽  
Concerted Mechanism ◽  
X Ray ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

QM/MM MD simulations from different X-ray structures support the concerted mechanism character in the rate limiting step of thymidylate synthase catalysis.

Crystallization of protein–protein complexes

2002 ◽  
Vol 35 (6) ◽  
pp. 674-676 ◽  
Author(s):  
Sergei Radaev ◽  
Peter D. Sun
Keyword(s):  
Sparse Matrix ◽  
Protein Complexes ◽  
Data Bank ◽  
Structure Characterization ◽  
Salt Crystallization ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Crystallization Conditions ◽  
The Stability ◽  
Rate Limiting

Crystallizing protein–protein complexes remains a rate-limiting step in their structure characterization. Crystallization conditions for the known protein–protein complexes have been surveyed in both the Protein Data Bank and the BMCD database. Compared with non-complexed proteins, crystallization conditions for protein–protein complexes are less diverse and heavily favor (71%versus27%) polyethylene glycols (PEG) rather than ammonium sulfate or other high-salt crystallization conditions. The results suggest that the stability of protein complexes limits their available crystallization configuration space. Based on the survey, a set of sparse-matrix screen conditions was designed.

Formation and Decomposition of Nitrides on Iron Surfaces

1979 ◽  
Vol 34 (1) ◽  
pp. 30-39 ◽  
Author(s):  
G. Ertl ◽  
M. Huber ◽  
N. Thiele
Keyword(s):  
Photoelectron Spectroscopy ◽  
Electron Spectroscopy ◽  
Auger Electron ◽  
Thermal Desorption Spectroscopy ◽  
Iron Nitrides ◽  
Kcal Mole ◽  
X Ray ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract The formation (by interaction with ammonia) and decomposition of nitrides on clean Fe surfaces was studied by means of Auger electron spectroscopy, x-ray photoelectron spectroscopy, thermal desorption spectroscopy, and scanning electron microscopy. The N atoms may exist in various forms with quite similar electronic properties, viz. as chemisorbed layer (= "surface nitride"), dissolved in α-Fe or γ-Fe, as γ′-nitride (= Fe4N) or as e-nitride, depending on temperature as well as pressure and duration of interaction with NH3. There is no noticeable chemical shift of the ionization energies of the Fe core levels, indicating that the bond is essentially covalent. The activation energy for the decomposition of e-nitride into Fe4N + N2 is about 27 kcal/mole, that for the decomposition of Fe4N into Fe+N2 ranges between 51 and 57 kcal/mole, depending on the mode of preparation. The latter values are identical to those found previously for the desorption of N2 from various Fe single crystal planes and indicate that the decomposition of the chemisorbed "surface nitrides" is the rate-limiting step which prevents the spontaneous decom-position of the metastable bulk iron nitrides.

Kinetics of the Decomposition of Crocidolite Asbestos: A Preliminary Real-Time X-Ray Powder Diffraction Study

2004 ◽  
Vol 443-444 ◽  
pp. 291-294 ◽  
Author(s):  
A.F. Gualtieri ◽  
D. Levy ◽  
M. Dapiaggi ◽  
E. Belluso
Keyword(s):  
Real Time ◽  
Powder Diffraction ◽  
Ion Diffusion ◽  
Kcal Mole ◽  
X Ray ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Kinetics Of

This work is a preliminary kinetic study of the crocidolite decomposition followed in situ at high temperature using real time conventional powder diffraction and DTA in the temperature range 720-795 °C. The data analysis using the Avrami models indicates that the rate limiting step of the reaction is monodimensional ion diffusion (n=0.5) with an activation energy of 129 (10)kcal/mole.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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