Approximately 80 percent of information reaches our brains through our eyes. Evidently, the visualization of the object under consideration is often a means to greatly increase knowledge on it. In particular, such approaches are applied in structural biology. The elucidation of the structures of biomacromolecules and cellular elements at all levels of cell organization, as the subject-matter of this science, is the driving force for the development of modern biology as a whole. The rapid progress achieved in this field in the past years is to a large extent due to advances in X-ray diffraction analysis and new technologies based on the use of synchrotron radiation. The National Research Center "Kurchatov Institute" has developed the infrastructure necessary to implement ambitious projects in the field of structural biology. Nevertheless, the state-of-the-art research requires unique experimental mega facilities, and their creation often calls for the combined efforts of several countries. For this purpose, the Kurchatov Institute actively participates in the International (Germany, Russia, and others) project on the construction of a free-electron laser. Scientists working in the field of structural biology pin great hope on its successful implementation.

by Mikhail KOVALCHUK, Corresponding Member of the Russian Academy of Sciences, Vladimir POPOV, Corresponding Member of the Russian Academy of Sciences, Center of Nano, Bio, Info, Cognitive and Social Sciences and Technologies (NBICS Center) at the National Research Center "Kurchatov Institute" (Moscow)

TASKS AND METHODS OF VISUALIZATION

The human genome project completed in 2003 has opened a new era in the development of life sciences, and the 21st century is considered to be the Age of Biology. High-throughput methods for investigations of genomes, transcriptomes*, proteomes**, and metabolomes*** are vigorously developed and improved. Systems biology originated from bioinformatics and synthetic biology aimed at designing and producing new living systems, including those non-existing in nature, are rapidly growing and evolving. Biology has changed from being a largely descriptive discipline to being a quantitative science, thus approaching such exact sciences as physics and chemistry. This progress is to a large extent associated with advances in structural biology-the interdisciplinary field concerned with the structures of proteins, nucleic acids, their intricate multisubunit complexes, cellular organelles, membranes, cytoskeletal elements, and so on at all levels of cell organization.

It is said that to see is to realize. The visualization and the solution of the three-dimensional structures of bio-macromolecules provided an insight into the principles of operation of complex molecular bionanomachines and protein complexes, such as ribosomes responsible for assembling the proteins of the cell, ATPases providing

*A transcript is an RNA molecule that is formed as a result of the transcription (expression of the corresponding gene or the DNA region). The set of all RNA molecules produced in one cell or their population is called the transcriptome. Unlike the genome, which is generally fixed for a cell line, the transcriptome can substantially vary depending on the environmental conditions.-Ed.

**A proteome is the entire set of proteins of the body expressed by a cell, tissue, or organism at a certain time.-Ed.

***A metabolome refers to the complete set of small-molecule metabolites (metabolic intermediates and final metabolic products), which can be found both within biological samples and a single organism.-Ed.

the synthesis of the universal fuel of the cells (adenosine triphosphate), and photosynthetic reaction centers playing a key role in the photosynthesis. The elucidation of the structural features of biotarget molecules is an actual necessary stage in the design of new drugs. Without the knowledge of the three-dimensional structures of natural catalysts (enzymes), it is impossible to understand the molecular mechanisms of action of the latter and to control their properties.

At present there exist numerous physicochemical methods, which are used to study particular features of the organization of macromolecules, e.g., the nearest environment of the metal atoms in protein molecules or the specially introduced fluorescent or paramagnetic labels. However, only some of these approaches are suitable for the elucidation of the overall structures of the objects and details of their atomic and molecular structures. These are X-ray diffraction analysis (XRD), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and cryo-electron microscopy (cryo-EM).

Each of the above-mentioned methods has not only its own advantages but also limitations. Thus, X-ray diffraction analysis requires crystals of macromolecules, which are often difficult to grow. Despite considerable advances in NMR techniques providing information on the structures of macromolecules directly in solution, investigations of large biomolecules by NMR spectroscopy is still a difficult problem. Nevertheless, the combined use of the above-mentioned approaches provides researchers with rather detailed information even on complex biological systems. For example, the general information on the shape and structure of the biological system under consideration can be obtained by SAXS or cryo-EM. Then the system can be disassembled by molecular biol-

Main methods of investigating the three-dimensional structures of macromolecules and the growth rate in the number of structures in the RCSB Protein Data Bank (www.rcsb.org).

ogy techniques, the arrangement of individual fragments (domains, individual proteins) can be studied at higher level by ΝMR or X-ray diffraction, and finally the details of the structural organization can be reconstructed.

All structural data for macromolecules obtained by researchers from different countries are deposited in the Protein Data Bank (www.rcsb.org), which was established at the Brookhaven National Laboratory (USA) in 1971. The Protein Data Bank statistics shows that, beginning from the mid-1990s, there has been a spectacular increase in the number of structures due primarily to the rapid progress in methodology. By October 2012, the Protein Data Bank contained more than 85,000 entries, most of the data (88 percent) being obtained by X-ray diffraction crystallography. Therefore, notwithstanding all limitations and difficulties, this method serves as the basis for the solution of problems of structural biology.

X-RAY DIFFRACTION CRYSTALLOGRAPHY

Recall that crystallography, as a science concerned with the properties of crystals, arose as a part of geology and initially used mainly descriptive methods (angles, habitus, etc.) for the characterization of minerals. Later on, due to advances in chemistry, crystallography turned to investigations of the chemical compositions of minerals. However, it was only in the 20th century that crystallography became an independent field of physics due mainly to the discovery of the nature of X-rays. Since that time crystallography has played and is still playing an important role in the advances in modern structural biology.

X-ray diffraction analysis is based on the phenomenon of X-ray diffraction (X-rays were discovered by a German physicist Wilhelm Conrad Röntgen in 1895, the Nobel Prize in Physics in 1901) on a three-dimensional crystal lattice. This phenomenon was discovered by his compatriot Max von Laue in 1912 (the Nobel Prize in Physics in 1914). In 1913, British physicists William Henry Bragg and William Lawrence Bragg provided the theoretical basis for this phenomenon (the Nobel Prize in Physics in 1915). A Russian scientist Georg Wulff (the Corresponding Member of the Russian Academy of Sciences from 1921) made a substantial contribution to this field of science.

X-ray diffraction crystallography began to be actively used for investigations of macromolecules in 1930-1940s. The first X-ray diffraction pattern of a protein crystal (the proteolytic enzyme pepsin, which was crystallized by an American biochemist John Northrop in 1929) was obtained by British scientists John Bernal and Dorothy Crowfoot Hodgkin in 1934. In 1941, their compatriot William Astbury obtained the first X-ray diffraction pattern of DNA. Based on the X-ray diffraction images taken by British biophysists Rosalind Franklin and Maurice Wilkins, an American biologist James Watson and his British colleague Francis Crick (the latter three are Nobel Prize winners in 1962) proposed a double helix model of the DNA molecules in 1953. The first protein

Comparison of the intensities of X-ray radiation sources of different generations. Locations of the synchrotron sources: NRC Kl, Russia (National Research Center "Kurchatov Institute"); DESY, Germany (Deutsche Elektronen-Synchrotron); Spring-8, Japan (RIKEN); ESRF, France (European Synchrotron Radiation Facility, European Molecular Biology Laboratory); LCLS, USA (SLAC National Accelerator Laboratory); XFEL, Germany (Deutsche Elektronen-Synchrotron, International project).

structures to be solved were hemoglobin and myoglobin, under the supervision of British biochemists Max Perutz and John Kendrew, respectively, in 1958. Since that time, X-ray diffraction crystallography has become increasingly popular in biology.

In the Soviet Union, an enormous contribution to crystallography as a whole and specifically to X-ray diffraction crystallography was made by Academician Boris Vainshtein, who headed the A.V. Shubnikov Institute of Crystallography of the USSR Academy of Sciences (since 1991, the Institute of the Russian Academy of Sciences) for a long period of time. At his laboratory, crystallograph-ic studies of biomacromolecules were initiated as early as in 1950s, crystals of a number of important proteins were grown for the first time, and their atomic structures were determined. Under the supervision of Boris Vainshtein, the structures of the oxygen-binding plant protein leghe-moglobin (1975), the enzyme aspartate aminotransferase (1978), which is widely used in medicine for the laboratory diagnostics, and the protein catalase (1981) with a molecular weight of higher than 200,000 Da (a record for that time period) were solved and refined. These results were of world-class quality. This laboratory became one of the pioneers in the application of X-ray diffraction to studies of such large biological objects as viruses.

Vainshtein's school laid the foundation for the development of structural biology and had a strong impact on the establishment of X-ray crystallography of biomacromolecules in Russia. The Department of Protein Crystallography at the A.V. Shubnikov Institute of Crystallography of the Russian Academy of Sciences remains one of the leaders in this field. Vainshtein's disciples and followers are now actively working at the National Research Center (NRC) "Kurchatov Institute" and scientific institutions of the Russian Academy of Sciences-the Engelhardt Institute of Molecular Biology, the Institute of Protein Research (Pushchino), the M.M. Shemyakin-Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, and the A.N. Bach Institute of Biochemistry.

The Russian scientific school made also a considerable contribution to the development of another technique for the structural characterization of macromolecules with the use of X-ray radiation-small-angle X-ray scattering. The research performed by Lev Feigin (Institute of Crystallography of RAS) and his disciple Dmitry Svergum has laid the theoretical groundwork for this technique and substantially extended its application.

X-RAY RADIATION SOURCES

In experimental apparatus designed for X-ray diffraction studies, the X-ray source emits a photon beam. The beam is focused and monochromatized by passing through a number of devices and then falls on a crystal. The X-ray diffraction pattern is obtained using a detector that is located behind the crystal.

In early experiments on the determination of the three-dimensional structures of macromolecules, fully vacuum-sealed X-ray tubes were used as the X-ray source. Later on, so-called rotating-anode X-ray tubes were constructed to avoid overheating. Different modifications of these tubes are widely used in in-house X-ray sources. The drawback of these facilities is the low intensity of the

Place and tasks of the Protein Factory in the structure of the NBICS Center at the NRC "Kurchatov Institute".

incident beam, which imposes serious limitations on the size of the crystals used in experiments (the lower the intensity of the beam the larger the crystal size is required).

The design and construction of synchrotrons as a new type of resonance accelerators of charged particles contributed to marked advances in X-ray diffraction techniques. The main purpose of a synchrotron is to accelerate electrons, positrons, or protons up to high energies. As such, these particles are unsuitable for X-ray diffraction studies. However, as charged particles are forced to travel in a curved path (which takes place in synchrotrons), they emit photons-light quanta of different energies, and, under particular conditions, the latter can be used for X-ray diffraction studies. X-ray synchrotron radiation produced by modern accelerators is characterized by very high brightness. Thus, the photon flux from any synchrotron source exceeds the value achieved with the use of rotating anodes by tens of orders of magnitude. An increase in the brightness of the radiation has the following two advantages: the very fast data collection and the possibility of using smaller crystals, which is of great importance taking into account the problems with the crystallization of biological objects.

Several generations of synchrotron facilities are in operation worldwide. The main difference between them is in the type of special devices used to deflect the beam of charged particles from its original straight course, thus generating the electromagnetic radiation flux. These devices are called undulators* or wigglers and consist of periodic systems of magnets designed to deflect particles by electric or magnetic fields. Synchrotron radiation produced in large research centers by third-generation facilities allows researchers to collect X-ray diffraction data from very small crystals (20 μm or even less) and to measure complete X-ray data sets from complex objects in a few minutes.

X-ray free-electron lasers (XFEL) can provide a new breakthrough in X-ray diffraction technologies. These lasers generate radiation by a relativistic electron beam in an undulator. The undulator forces the electrons in the beam to follow a sinusoidal path, resulting in the emission of photons, whose energy depends on the energy of the electrons and the parameters of the device. The laser beam is collimated and amplified using a mirror system. The characteristic features of XFEL are that the X-ray parameters can vary over a wide range and very high X-ray intensities can be achieved, the latter significantly exceeding those produced by third-generation synchrotrons. X-ray free-electron lasers can be used to study micro- and even nanocrystals and, in the ideal case, individual macromolecules, which opens up tempting oppor-

* An idea of the magnetic undulator was proposed by a British physicist H. Motz in 1951, and it was realized for the first time in the USA in 1953.-Ed.

tunities, for example, for investigations of non-crystalline samples. It should be emphasized that Russian scientists made a significant contribution to the elaboration of the theory of free-electron lasers.

The world's first XFEL source has been launched in Stanford (USA), where pilot experiments were performed and first X-ray diffraction patters of nanocrystals and noncrystalline samples were obtained. These results confirmed the unique potential of the method. The world's most powerful X-ray free-electron laser is currently under construction at the German Particle Physics Center (DESY) in Hamburg. This ambitious project (with an investment volume of more than a billion euro) called for concentrated efforts of several countries. Russia is the full member of the project, provides approximately 25 percent of the total investment volume, and participates in the design and construction of unique facilities. It is expected that XFEL at the DESY will start to operate in 2015.

FROM GENE TO PROTEIN STRUCTURE

In any structural biology project, a number of stages should be passed, from the problem statement-the selection of objects for investigation (for example, a gene encoding a particular protein)-to the determination of the structure of the target protein or the protein complex as the final result: the cloning of the target gene and the expression (synthesis), isolation, and purification of the functional protein, preparation of crystals suitable for X-ray diffraction, collection and processing of X-ray diffraction data, and finally solution and refinement of the structure of the macromolecule. Each of these stages is, as a rule, a specific hard-to-solve research problem, where the 100 percent success is not guaranteed.

Recent advances in molecular and structural biology techniques have considerably enhanced chances for success. Nevertheless, the probability of failure does exist in each stage. In particular, the expression of the protein in the correct functional state and in amounts sufficient for its structural and physicochemical characterization is one of the most unpredictable stages, especially for proteins with a complex structural organization and proteins containing prosthetic groups (i.e., groups of the non-protein origin involved in macromolecules: metals, iron-sulfur sites, porphyrin derivatives, and so on).

The crystallization of proteins is the next equally difficult stage. As mentioned above, the necessity of obtaining crystals is the main limitation of protein X-ray crystallography. However, the crystallization of macro-molecules is the most problematic and least predictable stage of such investigations due to the lack of an adequate theory, which would allow one to relate a particular object to certain crystallization conditions. The problem of crystallization is most often solved by screening numerous crystallization conditions (from hundreds to several thousand for one protein), which is a time-consuming and laborious process.

This is why only a relatively small number of initiative structural projects culminate in obtaining information on the structure of the object of interest within a reasonable period of time. In the case of "simple" proteins, e.g., hydrolases, oxidoreductases, which do not contain co-valently bound prosthetic groups, etc., the percentage of success varies, on average, from 10 to 30 percent. As was mentioned above, the expression of the protein and the preparation of X-ray quality protein crystals are the most problematic stages (up to 60 and 90 percent of failures, respectively). It is also necessary to take into account the time consumption. Thus, several man-years may be required to obtain membrane protein crystals with desired parameters.

NBICS CENTER AT THE NRC "KURCHATOV INSTITUTE"

The National Research Center "Kurchatov Institute" founded in 1943 is one of the world-leading research institutions. Nowadays, the NRC "Kurchatov Institute" is a multi-disciplinary research center with an advanced experimental base, including nuclear research reactors and critical reactor assemblies, facilities for thermonuclear fusion and ion-plasma technologies, dedicated accelerating-storage complexes, a cluster technological line for microelectronics, and so on. The main research activities are atomic power engineering, thermonuclear fusion, nuclear medicine, and some others. Recently, the fields of research have been expanded to nanobiotechnology, nanomaterials, and nanosystems.

The Center of Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Center) was launched at the NRC "Kurchatov Institute" in 2009. The purpose of creating the NBICS Center was in forming the infrastructure basis for the convergent development of different fields of science and technologies and achieving breakthrough results due to their "cross-linking". The NBICS Center possesses several complexes, including a computer complex (a supercomputer cluster), a synchrotron-neutron complex (a synchrotron radiation source and an IR-8 neutron reactor), complexes for nuclear medicine, microelectronics, and superconductivity, as well as the Institute of NBICS research and technologies, which includes departments of crystallography and materials science, molecular biology, neurophysiology and cognitive sciences, mathematical modeling, robotic technology and microsystems, social sciences, and applied problems.

The NBICS Center incorporates world-class mega facilities (the above-mentioned synchrotron source and the neutron reactor), as well as high-class X-ray equipment, atomic force and electron microscopes, various technology devices for nanobiotechnologies and microelectronics, so-called "clean" areas, and other unique facilities.

Since the NBICS Center has a synchrotron radiation source at its disposal, which serves as a base for the implementation of projects associated with the application of X-ray diffraction and small-angle X-ray scattering methods, it provides unique opportunities for investigations in the field of structural biology. Besides, the resource base for the application of cryo-electron microscopy and nuclear magnetic resonance in studies of the structures of macromolecules is currently under development, which will greatly expand the range of tasks that researchers can accomplish.

PROTEIN FACTORY

The key goal of this specialized department, which started its work at the NBICS Center in the spring 2010, is to provide the implementation of structural biology projects at the NRC "Kurchatov Institute". The Protein Factory provides the production and purification of objects of investigation and their structure-function characterization using synchrotron radiation. These can be the whole genome sequencing data obtained at the Division of Genome Research of the NBICS Center, ge-

Three-dimensional structures of selected enzymes produced at the Protein Factory: A-thermostable alcohol dehydrogenase from T. SIBIRICUS (1.7 Å resolution); B-hexameric uridine phosphorylase from S. ONEIDENSIS (0.95 Å resolution), whose crystals were grown in microgravity; C-48-heme cytochrome C nitrite reductase from T. NITRATIREDUCENS (1.4 Å resolution) and the active site of this enzyme (D)

netic constructs for the expression of particular proteins, which are of interest, e.g., forbiomedicine, proteins with different degrees of purity, or even their crystals.

The Protein Factory has unique facilities for screening and optimization of protein crystallization conditions. To solve these problems, the Protein Factory has at its disposal, in addition to conventional methods, the following two powerful tools: a robotic crystallization system and accessories for experiments in microgravity (space experiments). The robotic crystallization system enables users to search for the starting crystallization conditions for proteins. All operations (except for the transfer of samples from one component of the robotic system to another, which is performed by an operator), from the preparation of the initial crystallization solutions to the photomonitoring and documentation of the crystallization process, are carried out in an automated mode. The crystallization in microgravity offers additional opportunities to improve the quality of crystals compared to those obtained on earth by conventional methods. In microgravity, the absence of the convective-flow transport and sedimentation provides the uniform mass transport to all growing faces of the crystal and, in some case, improves its quality. The corresponding experiments in microgravity are performed on the International Space Station in collaboration between the Russian Federal Space Agency (Roscosmos) and the Japan Aerospace Exploration Agency (JAXA).

PROJECTS UNDER IMPLEMENTATION

Several projects in the field of biotechnology and bio-medicine using a synchrotron radiation source are currently underway at the Protein Factory. Among these projects are the structure-function investigation of enzymes from extremophiles**, structural studies of intri-

*The sedimentation is the settling of particles of a dispersed phase in a liquid or gas in the gravitational field or in the field of centrifugal forces.-Ed.

** Extremophiles are organisms (including bacteria and microorganisms) that live in extreme environmental conditions (extremely high/low temperatures, extremely high pressure, and so).-Ed.

cate protein complexes and molecular nanomachines, and the design of new therapeutic agents.

The goal of the first of these projects is to find enzymes with polyextremophilic properties, such as thermal stability, halotolerance (salt resistance), and resistance to organic solvents. The genomes of thermophilic ar-chaea (the collection owned by the Doctor of Biological Science Yelizaveta Bonch-Osmolovskaya*, S.N. Wino-gradsky Institute of Microbiology of the Russian Academy of Sciences), which were sequenced at the Genome Research Center at the NBICS Center (headed by K.G. Skryabin) and the Bioengineering Center of the Russian Academy of Sciences**, serve as the starting data. Enzymes, which are of interest for biotechnology and medicine, are selected based on the respective analysis, and the relationship between the structural features of these enzymes and their ability to retain physiological activity in extreme environmental conditions is elucidated.

In particular, alcohol dehydrogenase from Thermococ-cus sibiricus was studied in detail. This enzyme exhibits a complex of unique polyextremophilic properties: stability up to 100 °C, which is an absolute record for NAD(P)-dependent enzymes***, and the ability to retain activity in the presence of high concentrations of salts (up to 4 M) and organic solvents (up to 50 percent) at high temperatures (up to 55 °C). The determination of the three-dimensional structure of the enzyme at 1.7 Å resolution revealed the structural features responsible for its extremely high resistance to external factors.

The project "Structure of Complex Molecular Nanomachines" is aimed at characterizing multisubunit proteins, including those containing different prosthetic groups and metal sites. Among these proteins are, e.g., hexameric uridine phosphorylase and hexameric cytochrome c nitrite reductase (NR), which catalyzes the six-electron reduction of nitrite or sulfite to ammonia or hydrogen sulfide, respectively, as one of the most complex reactions in nature. It should be noted that crystals of uridine phosphorylase, which were obtained in microgravity on the International Space Station, made it possible to determine the structure of this protein at very high, so-called atomic, resolution (0.95 A) and reveal the positions of even hydrogen atoms, while the quality of crystals obtained on earth was substantially lower.

* See: Ye. Bonch-Osmolovskaya, "Thermophiles: Past of the Planet, Future of Biotechnology", Science in Russia, No. 4, 2010.-Ed.

**See: N. Ravin, "Genomic Analyses and the Ecology of Microorganisms", Science in Russia, No. 5, 2011.-Ed.

***NAD(P) is an abundant natural coenzyme of the enzymes dehydrogenases, which catalyze important redox reactions in living cells.-Ed.

The physicochemical, kinetic, and spectroscopic properties of NR from different sources in solution were analyzed in detail, and more than ten structures were determined at high resolution (1.4-2.0 Å), including the structures of the apo enzyme (the active site does not contain bound molecules) and its complexes with low-molecular-weight compounds (substrates and inhibitors). The enzyme exists in solution and in the crystal as a stable symmetric hexamer and contains 48 covalently bound hemes*, which is an absolute record in the number of the latter per protein molecule. Eight hemes of each monomer of ΝR form a canonical three-dimensional arrangement typical of other multiheme cytochromes as well. The determination of the structures of NR and its complexes at high resolution made it possible to reveal a number of unique structural features of the active site of the enzyme responsible for the high efficiency of the catalysis.

Let us briefly mention the project "Design of New Therapeutic Agents". In particular, the structure and function of the protein parkin are investigated. This protein possibly plays an important role in the pathogenesis of Parkinson's disease. The investigation of the protein named mechano-growth factor (MGF) is aimed at finding ways of enhancing the repair and regeneration of muscle tissues. More than 20 tyrosine kinases were cloned and expressed at the Protein Factory. This enzyme is one of the main target proteins for the design of a new generation of anticancer agents.

Since the launch of the Protein Factory two years ago, more than 40 proteins were expressed, isolated, and purified, and crystals of 29 of them (70 percent) were obtained. More than 80 X-ray diffraction datasets were collected (including the data sets for complexes with li-gands) for 22 individual proteins. Twenty seven structures were deposited in the Protein Data Bank, more than 70 percent of the structures being solved at resolution higher than 2 A. These facts clearly show that the Protein Factory is a powerful tool promoting in-depth structure-function studies of biomacromolecules at the modern methodological level.

*The heme is a non-protein part (the so-called prosthetic group) of many proteins, e.g., of hemoglobin, in which the heme is responsible for its color-Ed.