Protocols and tips in protein purification or How to purify protein in one day.
Protein purification is the first step of almost all in vitro protein studies. High quality of purified protein could be critical in respect to their activity and crystallization ability. Almost all proteins lose their activity during any manipulations. Even stored at 4°C protein can lose activity and crystallization ability. That is why it is important to purify protein as quick as possible. This guide give information how to purify protein in one day.
This is not a complete guide to protein purification and you still should to read serious books on theory of chromatography to become familiar with the subject.
2. Protein crystallization
The most important part of any protein crystallography studies is protein crystallization. Without perfect crystals of protein (or any other biological samples) it is impossible to carry out any crystallographic structural studies. The aim of protein crystallization is to produce well-ordered protein mono-crystals without any inclusion and large enough to diffract X-Ray beam. You can also find some techniques for low molecular weight peptide crystallization. Despite very wide knowledge about protein crystallization it is still impossible to predict any conditions for protein crystallization. The protein crystallization process is still empiric and the biggest part of success is hidden in the hand and experience of the scientist who performed the protein crystallization and pure luck. So... Read this chapter and don't shy to do the first step in protein crystallography!
- Peptide crystallization
- Requirements for Protein crystallization
- Crystallization by Vapor Diffusion
- Crystallization by Dialysis
- Crystallization in space
- Automated Protein crystallization
- Crystallization conditions
3. Crystal mounting
Usually, protein crystals are grown in some solutions and before further analysis, protein crystals must be mounted and placed on the detector under the X-Ray beam. Protein Crystallography deals with crystals about 0.1 mm in size, that's why crystal mounting requires a very high coordination of all motions and reasonable practice. The best advice is to spend 1-2 days practising with cheap protein crystals, for example Lysozyme. Currently a lot of efforts are targeted to automate crystal mounting process, but despite this the manual crystal mounting is a still very important part of protein crystallography. Here you can learn some base techniques of crystal mounting, suitable for almost all protein crystallography needs.
- Crystal separating
- Crystal mounting in capillary
- Crystal mounting in the loop
- Crystals Freezing/refreezing
- Crystal storage in liquid nitrogen
- Automated crystal mounting
- Dry crystals
4. Data collection
X-ray Data collection is a central part of all protein crystallography. This chapter describes all modern equipment for X-ray data collection: detectors and X-ray radiation sources. A very important part of X-ray data collection is the geometry of the experiment. Correct alignment of the crystal sometimes allows improving completeness on the final data set. X-ray data collection is a time consuming procedure and is very expensive. It is possible to significantly minimize the time of X-ray data collection by correct prediction of strategy of data collection. Multi-wavelength X-ray data collection, as a part of anomalous data collection will be discussed as a most popular technique of crystal structure solution in protein crystallography.
- X-ray radiation source
- Detector types
- Radiation damage
- Strategy prediction
- Anomalous diffraction
- Geometry of the data collection
5. Data processing
X-ray data processing is a key stage for protein crystallography. The final quality of X-Ray data is related with the data processing procedure, which includes integration of the crystallographic data and scaling. In this chapter the basic programs for data processing will be discussed. You also can find manuals for most popular programs written by our specialists with set of samples of data processing for many different cases, from easy to very difficult ones. Also the role of some parameters widely used for data processing, such as integration spot radius or estimated error, will be discussed together with number of examples showing the impact of these parameters of data processing on the final model and the final statistics.
6. Structure solution
The first step between experimental X-ray data and protein structure is the structure solution procedure. Structure solution can be based on the many different techniques, depending on the data available. Many different programs can be used for the structure solution and in this chapter you can read descriptions and instructions for many of them. Selection between different techniques and between different programs is a very important step for protein structure solution and with this chapter you can choose the correct choice. Many structures was never been solved due to mistakes in structure solution procedure, which in fact ruins all protein crystallography experiments.
- Direct methods
- Molecular replacement
- Electrone microscopy
7. Structure refinement
The final stage of protein crystallography is the structure refinement. At this stage it is necessary to do a lot of visual graphics work with the model together with structure refinement. Choosing the program for structure refinement is a matter of personal preferences, but nevertheless existing programs are different in the range of functionality. The major criteria for selection between these programs are the quality of X-ray data and of course the resolution of data. Furthermore, not many programs are able to do structure refinement for data collected in crystal space groups with mirror symmetry. In this chapter the major programs for structure refinement are discussed.
- Molecular mechanics
- Molecular dynamics
- B-factor optimization
- Final statistic
8. Structure analysis
Protein structure representation with subsequent analysis of all features related with this particular structure is a main goal of all protein crystallography studies. The amount of all programs, tools and utilities for protein structure analysis is significantly greater than those available for protein crystallography itself. Usually it is almost impossible to quickly select the best tools for protein structure analysis. For our studies we've not only used a lot of different programs but even developed some of them. Our guide will help you to select the perfect tools for protein structure analysis and read simple manuals and step-by-step algorithm for achieving requested results. All our programs described here are distributed for free.
9. Crystallographic societies
Crystallographic associations and societies are very popular within molecular biologists. These groups are usually formed around interesting research projects with reasonable impact to all protein crystallographers in the world. These groups organise meetings and conferences, publish their newsletter and magazines, support young scientists etc. Here you can find a comprehensive list of crystallographic societies, sometimes with the archives of their newsletters and abstracts.
10. Basic tutorials