scholarly journals An overview of heavy-atom derivatization of protein crystals

2016 ◽  
Vol 72 (3) ◽  
pp. 303-318 ◽  
Author(s):  
Ashley C. W. Pike ◽  
Elspeth F. Garman ◽  
Tobias Krojer ◽  
Frank von Delft ◽  
Elisabeth P. Carpenter
Keyword(s):  
Heavy Atom ◽  
Protein Crystals ◽  
Data Sets ◽  
Low Resolution ◽  
Heavy Atoms ◽  
Density Maps ◽  
Resolution Electron ◽  
First Choice ◽  
First Time ◽  
Resolution Data

Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

Use of Patterson-based methods automatically to determine the structures of heavy-atom-containing proteins with up to 6000 non-hydrogen atoms in the asymmetric unit

2006 ◽  
Vol 39 (5) ◽  
pp. 728-734 ◽  
Author(s):  
Maria Cristina Burla ◽  
Rocco Caliandro ◽  
Benedetta Carrozzini ◽  
Giovanni Luca Cascarano ◽  
Liberato De Caro ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Protein Data Bank ◽  
Heavy Atom ◽  
Data Bank ◽  
Atomic Resolution ◽  
Asymmetric Unit ◽  
Hydrogen Atoms ◽  
Heavy Atoms ◽  
Density Maps ◽  
Resolution Data

The Patterson superposition methods described by Burlaet al.[J. Appl. Cryst.(2006),39, 527–535], based on the use of the `multiple implication functions', have been enriched by supplementary filtering techniques based on some general (resolution-dependent) features of both the Patterson and the electron density maps. The method has been implemented in a modified version of the programSIR2004and tested using a set of 20 crystal structures selected from the Protein Data Bank, having a number of non-hydrogen atoms in the asymmetric unit larger than 2000, atomic resolution data and some heavy atoms (equal to or heavier than Ca). The new phasing procedure is able to solve most of the test structures, among which there are two proteins with more than 6000 non-hydrogen atoms in the asymmetric unit, so extending by far the complexity today commonly considered as the limit for Patterson-based methods (i.e.about 2000 non-hydrogen atoms).

scholarly journals Satellite tobacco mosaic virusrefined to 1.4 Å resolution

2014 ◽  
Vol 70 (9) ◽  
pp. 2316-2330 ◽  
Author(s):  
Steven B. Larson ◽  
John S. Day ◽  
Alexander McPherson
Keyword(s):  
Icosahedral Symmetry ◽  
Structural Detail ◽  
Virus Structure ◽  
Density Maps ◽  
Satellite Tobacco Mosaic Virus ◽  
Anisotropic Temperature ◽  
First Time ◽  
Temperature Factors ◽  
Precise Assessment ◽  
Resolution Data

Satellite tobacco mosaic virus(STMV) is among the smallest viruses, having 60 identical subunits arranged withT= 1 icosahedral symmetry. Its crystal structure was solved at 290 K and was refined using, in part, crystals grown in microgravity. Electron-density maps revealed nearly 57% of the genomic ssRNA. Using six flash-cooled crystals, diffraction data were recorded to 1.4 Å resolution and independent refinements of the STMV model were carried outversusthe previous 1.8 Å resolution data representing merged data from 21 crystals (271 689 unique reflections), data consisting of corresponding reflections to 1.8 Å resolution from the cooled crystals and 1.4 Å resolution data from the cooled crystals comprised of 570 721 unique reflections. Models were independently refined with full NCS constraints using the programCNSand in restrained mode using the programsCNS,REFMAC5 andSHELX-97, with the latter two procedures including anisotropic temperature factors. Significant additional structural detail emerged from the analyses, including a unique cation and anion arrangement on fivefold axes and a precise assessment of icosahedral symmetry exactness in the crystal lattice. STMV represents the highest resolution native virus structure currently known by a substantial margin, and it permits the evaluation of a precise atomic model of a spherical virus at near-atomic resolution for the first time.

scholarly journals High-throughput in situ experimental phasing

2020 ◽  
Vol 76 (8) ◽  
pp. 790-801 ◽  
Author(s):  
Joshua M. Lawrence ◽  
Julien Orlans ◽  
Gwyndaf Evans ◽  
Allen M. Orville ◽  
James Foadi ◽  
...  
Keyword(s):  
Heavy Atom ◽  
Data Sets ◽  
Human Intervention ◽  
Macromolecular Crystallography ◽  
New Approach ◽  
Partial Data ◽  
Experimental Phasing ◽  
Anomalous Signal ◽  
First Time

In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.

scholarly journals Paired refinement under the control of PAIREF

IUCrJ ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 681-692
Author(s):  
Martin Malý ◽  
Kay Diederichs ◽  
Jan Dohnálek ◽  
Petr Kolenko
Keyword(s):  
High Resolution ◽  
Diffraction Data ◽  
Structure Refinement ◽  
Data Sets ◽  
High Resolution Data ◽  
Density Maps ◽  
New Feature ◽  
And Control ◽  
The Impact ◽  
Resolution Data

Crystallographic resolution is a key characteristic of diffraction data and represents one of the first decisions an experimenter has to make in data evaluation. Conservative approaches to the high-resolution cutoff determination are based on a number of criteria applied to the processed X-ray diffraction data only. However, high-resolution data that are weaker than arbitrary cutoffs can still result in the improvement of electron-density maps and refined structure models. Therefore, the impact of reflections from resolution shells higher than those previously used in conservative structure refinement should be analysed by the paired refinement protocol. For this purpose, a tool called PAIREF was developed to provide automation of this protocol. As a new feature, a complete cross-validation procedure has also been implemented. Here, the design, usage and control of the program are described, and its application is demonstrated on six data sets. The results prove that the inclusion of high-resolution data beyond the conventional criteria can lead to more accurate structure models.

A simple clean vacuum system for carbon-film evaporation

1984 ◽  
Vol 42 ◽  
pp. 630-631
Author(s):  
M.K. Lamvik ◽  
A. LeFurgey
Keyword(s):  
Heavy Atom ◽  
Carbon Film ◽  
High Resolution Electron Microscopy ◽  
Vacuum System ◽  
Heavy Atoms ◽  
Resolution Electron ◽  
Electron Microscopes ◽  
Free Environment ◽  
Vacuum Systems ◽  
Electron Energy Loss

Electron microscopes with clean vacuum systems provide a contaminant-free environment for the specimen, but some of this advantage is lost if the substrate acts as a source of surface-bound contaminants. Particularly for microanalysis of thin specimens, whether by energy-dispersive x-ray spectroscopy or by electron energy-loss spectroscopy, substrates should be free of both organic and inorganic contaminants that might mask elements under Investigation. If specific heavy atom markers are to be studied by high resolution electron microscopy, the substrate should be free of extraneous heavy atoms. The substrate should be as thin and smooth as possible, and it is often helpful that the substrate films be hydrophilic, to assist the binding of some biological particles and improve the spreading of negative stains, when such stains are used.

scholarly journals ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps

2018 ◽  
Vol 74 (6) ◽  
pp. 519-530 ◽  
Author(s):  
Tristan Ian Croll
Keyword(s):  
Electron Density ◽  
Model Building ◽  
Low Resolution ◽  
Minichromosome Maintenance ◽  
Molecular Graphics ◽  
Final Model ◽  
Density Maps ◽  
Resolution Electron ◽  
Resolution Model

This paper introducesISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps.ISOLDEcombines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints. In addition, common validation tasks are accelerated and visualized in real time. Using the recently described 3.8 Å resolution cryo-EM structure of the eukaryotic minichromosome maintenance (MCM) helicase complex as a case study, it is demonstrated howISOLDEcan be used alongside other modern refinement tools to avoid common pitfalls of low-resolution modelling and improve the quality of the final model. A detailed analysis of changes between the initial and final model provides a somewhat sobering insight into the dangers of relying on a small number of validation metrics to judge the quality of a low-resolution model.

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