Structural Bioinformatics (C3210) Experimental Methods for Macromolecular Structure Determination

Transcription

1 Structural Bioinformatics (C3210) Experimental Methods for Macromolecular Structure Determination

2 Introduction Knowing the exact 3D-structure of bio-molecules is essential for any attempt to understand how they work and how they interact with each other. Unfortunately, getting to the structure of such macromolecules in the lab is not easy. X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, cryo-electron microscopy and neutron diffraction are the four main techniques that provide protein structures experimentally. The distribution by source of the structures available in the protein data bank is indicated in the diagram below (neutron diffraction not shown - it was approx. 66 structures in PDB in 2013). PDB distribution in 2013: X-ray: (88%) NMR: (11%) EM: 650 (0.7%) ND: 66 (0.01%) 2

3 X-ray Crystallography X-ray crystallography is a technique in which the pattern generated by the diffraction of X-rays passing through the single crystal lattice of a given material is recorded and then analyzed to reveal the 3D structure of the molecules composing the lattice. X-ray crystallography involves the following steps: protein production growing of crystal measurement of diffraction data electron density calculation structure determination and refinement 3

4 Protein Production and Purification The first step is to get large amounts of protein we want to crystallize. This is generally done by cloning the protein in various expression systems such as microorganism, bacteria, plant etc... The protein is then purified, since only highly purified solutions can give rise to crystals and not every protein can be purified to a sufficient degree. 4

5 Molecular Cloning Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. It can also be used to produce protein codded by the DNA sequence. The procedure consists of the following steps: preparation of protein coding DNA (isolation from fragmented cell DNA) inclusion of the DNA fragment into plasmid (cyclic DNA) transformation of DNA fragment into bacteria cultivation of bacteria cells that include required gene isolation of protein from bacteria cells and its purification (cell disruption, precipitation, ultracentrifugation, chromatography...) 5

6 Growing of Single Crystal Many approaches are used to produce crystals but the general method is to start with a solution of the protein and to slowly change the conditions so that saturation is exceeded. This can be done by changing parameters such as ionic strength (by adding sallts), ph, temperature of the solution. Supersaturation has to be reached slowly so that only a few nucleation sites are created which, with time, will grow larger. Thousands of solution conditions may be tried before finding one that succeeds in crystallizing the molecules, and ultimate success is never guaranteed. 6

7 Methods of Growing Protein Crystal The most common method is as follows. A protein is dissolved in a buffer solution and precipitant (salt or polyethyleneglycol) is added very slowly to reduce the solubility close to spontaneous precipitation. Then water is slowly removed by controlled evaporation resulting in precipitation. If left undisturbed for a few days ideally a few large crystals will grow. There are various methods implementing this approach. The most common is hanging drop - vapor diffusion method - a drop of protein solution is suspended over a reservoir containing buffer and precipitant (of higher concentration than in the drop). Water diffuses from the drop to the solution leaving the drop with optimal crystal growth conditions. Sitting drop - vapor diffusion method is similar only different placement of a drop is used. hanging drop sitting drop lysozyme crystal 7

8 Crystalization Screening Different proteins crystallize under different conditions (concentrations of protein and precipitant, ph, temperature...) which is difficult to find Crystallization screening is the search for conditions that allow crystal growth in a multi-component solution Automated systems are used to prepare solutions with different concentrations of protein, precipitant and other components Mosquito Crystal robot automates high density 96-well hanging drop plate preparation; see video: 8

9 Properties of Protein Crystals The protein crystals are not as dense packed as crystals of small molecules. They are loosely packed, with large solvent-filled holes and channels that may occupy up to 50% of the crystal volume. Protein crystal are very fragile. The protein crystals must always be kept in contact with their mother liquor or saturated vapour of the liquor even during exposition to X-ray beams, so crystals are usually closed in glass capillary during measurement (see figure). Alternatively, a crystal can be cooled to very low temperature (approx 100 K) which preserve the crystal in the X-ray beam <- Packing of protein molecules in crystal. Black areas are filled with water. -> Protein crystal in sealed capillary prepared for X-ray measurement. 9

10 The Single Crystal The crystal for X-rays measurement must be a crystalline solid in which the crystal lattice of the sample is pure, continuous and unbroken. The size of a protein crystal must be approximately mm in all three dimensions to diffract strongly enough to achieve the resolution needed for a successful 3D structure determination. The basic unit of a crystal is called the unit cell, which is defined by three principal axes (a, b and c) and three angles (alpha, beta and gamma). 10

11 Collecting the Diffraction Data The crystal is bombarded by X-rays, and the interference between rays scattered by different parts of the crystal give rise to diffraction. This diffraction pattern is dependent on the 3D location of the atoms. 11

12 X-ray Difractiometer When X-rays encounters a crystal lattice, electrons diffract the X-rays, which results in a diffraction pattern. In four-cycle diffractometer the crystal is mounted in a goniometer head located at the centre of the diffractometer and irradiated with X-ray beam. Intensity of diffracted X-ray radiation is measured by detector for different crystal rotations. See video:

13 X-ray Generation Evacuated cathode-ray tube: Electrons traverse the evacuated tube from cathode to anode and generate X-ray by high energy collision with anode A high-energy electron collided with and displace an electron from a low-lying ortbital in a target metal atom of anode. Then an electron from a higher orbital drops into the resulting vacancy, emitting its excess energy as an X-ray photon 13 Cathode-ray tube Scheme of synchrotron Synchrotron in Grenoble

14 X-ray Generation Synchrotron radiation: Synchrotron are particles accelerators that circulate electron beams at speeds close to the speed of light Whenever electron beam is forced to change direction, electromagnetic radiation is released Produced radiation covers broad range of wavelengths about 1 A X-ray radiation is two orders of magnitude more intense, so measurements are much faster than with evacuated cathoderay tube radiation Synchrotrons are big and expensive devices Web links: Video: ESFR, Grenoble, France 14 Cathode-ray tube Scheme of synchrotron Synchrotron in Grenoble

15 Electron Density Calculation The diffraction pattern is analyzed by mathematical and computation methods (Fourier transform analysis) to produce an electron density map. These maps show contour lines of electron density. Since electrons more or less surround atoms uniformly, it is possible to determine where atoms are located Unfortunately, since hydrogen has only one electron, it is difficult to map hydrogens 15

16 Electron Density Maps at Different Resolution Quality of density maps is described by its resolution Very low resolution 8 to 6 Å: solvent/protein boundary visible, may see helices or beta sheet (unlikely), no sign of ligands Low resolution 6 to 3.5Å: secondary structure visible Medium resolution 3.5 to 2.4Å: side chains visible, some still blurred, unambiguous secondary structure, ligands clearly visible, some water (primary solvation shell) visible Medium high resolution 2.4 to 2.0Å: most side chains unambiguously defined High resolution 2.0 to 1.6Å: very accurate ligand-protein interactions, multiple side chain conformations, secondary solvation shell visible Very high resolution 1.6 to 0.9Å: individual atoms visible 16

17 Structure Determination and Refinement Structure determination: Electron maps are interpreted and fitted to a 3D structure model For low and medium resolution electron maps, information abut residues sequence is necessary, because some amino acids have very similar side-chain electron density Structure model can be build manually using interactive graphics system or automatically using software tools Structure refinement: The atomic model must be further refined. Original model is used to generate modified models using molecular dynamicsbased method or other methods. For these models, theoretical diffraction patterns are calculated and compared to experimental data. This process continues until good agreement with experimental data is achieved. 17

18 Structure Validation The quality of the structure is validated by comparing various parameters with typical values in proteins Distribution of backbone dihedral angles (Ramachandran plot) should correspond to values observed in majority of proteins Distribution of hydrophobic and hydrophilic amino acids is also useful indicator. Hydrophobic acids dominates in inferior of the protein while hydrophobic proteins are more common on protein surface. 18

19 Structure Quality Estimation At the end of the process, the 3D structure of the macromolecule is obtained as a list of coordinates (x, y and z). The overall quality of the coordinates is usually measured by two quantities: The resolution - this is the ability to resolve two points separated by some distance from each other. The higher the resolution, the "sharper" our view of the structure. To obtain structures of reasonable quality measurement should have resolution have resolution less than 2.0 A The crystallographic R factor - it measures the agreement between observed diffraction patterns and those calculated from determined structure. The value 0.0 corresponds to perfect agreement of calculated and observed intensities. A well-refined structure of a macromolecule has an R factor not larger 0.2. Fobs is derived from the measured intensity of a reflection in the diffraction pattern, and Fcalc is the intensity of the same reflection calculated from the current model. The sum is over points in space. 19

20 The Advantages of X-ray Crystallography X-ray crystallography has major advantages, making it the most practical method for bio-molecule structure determination. It provides accurate atomic resolution models and allows scientists to resolve relatively large structures and complexes. Recent examples are structures of viral capsides and the ribosome, each composed of tens of thousands of atoms. 20

21 The Limitations of X-ray Crystallography X-ray crystallography also has some major limitations: The information provides only one snapshot of the protein that does not reflect its dynamic behavior Contacts between molecules in the crystal (crystal packing) and the dense packing might affect the structures The procedure is very slow There are many macromolecules which are difficult to crystalize, such as proteins with significant hydrophobic portions (membrane proteins) or highly flexible proteins Positions of hydrogens are difficult to obtain (can be determined at very high resolution, but with limited reliability only) 21

22 NMR Spectroscopy Nuclear magnetic resonance (NMR) is a physical phenomenon based upon the magnetic properties of the nuclei of some atoms. NMR spectroscopy of macromolecules was developed in the mid1980s enabling the elucidation of 3D structures of macromolecules in solution. The first NMR structures deposited at the PDB started to appear in the early 1990s. 22

23 Nuclear Magnetic Resonance Some atomic nuclei, such as 1H, 13C, 15N and 31P have magnetic moment cause by their spin When the nuclei are placed in a strong external static magnetic field they align with or against an applied field. Exposition of the nuclei to a second oscillating magnetic field of specific (resonance) frequency can cause the spin to reverse its orientation. The resonance frequency depends on strength of static magnetic field. This external magnetic field is partially shielded by electron cloud around nuclei. Because local electronic environment differs for different atoms, effective field acting on the nuclei is also different that causes different resonant frequencies Difference between resonant frequency of specific nuclei and reference frequency is called chemical shift Chemical shift depends on electronic environment of an atom; higher values of the shift correspond to higher electronic shielding 23

24 NMR Spectroscopy NMR spectroscopy is experimental method based on measurement of resonant frequencies (chemical shift respectively) of atomic nuclei in molecules Because atoms in specific atomic groups have characteristic chemical shifts, NMR spectra can be used to identify atom groups of molecule Measurement of 1H is the most common Measurement of 13C is also common but much less sensitive mainly because 13C isotope forms of about 1% only of C isotopes (the most common isotope is 12C). Small molecules and biological macromolecules (proteins, DNA) are usually measured in liquid state (solution) although solid state measurement is also possible (e.g. in material research) 24

25 NMR Instrumentation Current NMR spectrometers utilize high-field superconducting magnets producing magnetic field up to 24 T and working with frequency of RF field up to 1000 MHz The sample (approx microlitres with mm of protein) is positioned in the magnetic field and excited via pulsations in the radio frequency input circuit. The realigned magnetic fields induce a radio signal in the output circuit which is used to generate the output signal. 25

26 NMR in CEITEC NMR spectrometers in CEITEC: 950/850/700/700/600/500 MHz 26

27 Spin-Spin Coupling Effective field acting on nuclei is influenced by magnetic field due to spin of neighbouring nuclei. This effect is called spin-spin coupling. Spin-spin coupling give rise to the peak splitting in NMR spectra This effect is observable only between protons on covalently bonded atoms and only if the distance between nuclei is less than or equal to three bond lengths. This is because in spin-spin coupling the magnetic field of neighbouring nuclei does not act directly but through electron cloud surrounding the nuclei. Spin-spin coupling is never caused by neighbouring atoms that are bonded to the same atom and have identical electronic environment (e.g. H in CH2 group does not split peak of the second H) Number of new peaks and their distance bring information about atoms connectivity because they occur only between protons on covalently bonded atoms. Information about torsional angles can also be obtained from spin-spin coupling measurements. 27

28 Spin-Spin Coupling 28

29 Nuclear Overhauser Effect Nuclear Overhauser Effect (NOE) is another interaction between spins of neighbouring nuclei that can occur between noncovalently bonded nuclei which are close in space NOE differs from spin-spin coupling in the respect that NOE is observed through space, not through bonds This interaction is distance dependent, usually only protons separated by less than 5 or 6 Å can be observed Because the nuclei distance can be derived from the observed NOEs, precise three-dimensional structure of the molecule can be reconstructed. NOE measurements play important role in determination of 3D structure of biological macromolecules because many of atoms in these big structures are covalently distant while they can be close in a 3D space 29

30 2D NMR Classical NMR spectra is useless for analyzing the 3D structure of macromolecules due to the spectral overlap of hundreds of protons. To solve this problem, special technique called 2D NMR was developed which produces 2D spectra with better separation of individual peaks Measurement of 2D NMR spectra is based on application of two radiofrequency pulses separated in time. The time is changing during series of measurement. For each time different classical spectrum is measured. 30

31 2D NMR 3D NMR is improvement which is based on the same principles as 2D spectra, but two time differences between RF pulses are varying. Various methods of 2D/3D NMR use specific series of RF pulses to obtain spectra with desired information. Correlated spectroscopy (COSY) gives rise to cross peaks corresponding to the covalent structure (spin-spin couplings) while in NOESY cross peaks are obtained from the NOE effect. 31

32 Interpretation of Protein NMR Spectra Peaks in the spectra must be assigned to hydrogen atoms in specific residues in protein Spin-spin coupling measurements (e.g. COSY 2D NMR) enable to identify to which amino acid type individual H atom belong, its position within side-chain and give information about dihedral angles of side chain atoms. But it does not provide information about sequence order of amino acids. The sequence information can be obtained from NOE measurements (e.g. NOESY 2D NMR) Information obtained from NMR is not sufficient for direct assessment of 3D structure. First, 3D models consistent with the set of constraints (dihedral angles, distances) are generated. These models (usually several tens) can be refined by calculating NOEs from the model and fitting against the measured NOE intensities, as well as by optimizing for more favorable structural properties. 32

33 NMR Spectra and Dynamics of Molecules NMR spectrum of biological macromolecules is measured in liquid phase (solution of protein) Molecule in solution has very dynamic behaviour thus the structure of molecule is not rigid (compare with rigid structure in X-ray crystallography) Measured data correspond to weighted average of all structures in solution. This complicates assessment of 3D structure form 3D data but brings information about dynamic behaviour of the molecule Reconstruction of 3D structure from NMR data usually provides few tens of structures with different conformations (called ensemble) 33

34 The Advantages and Limitations of NMR Advantages: The main advantage of NMR spectroscopy over X-ray crystallography is that there is no need to have the sample in a crystalline state (which restricts the applicability of X-ray crystallography). Further information about the dynamics of the molecule in solution can be deduced. Motions that occur on a time-scale ranging from about 10 picoseconds to about 10 nanoseconds can be detected. Limitations: The precision of the technique is still not as high as in X-ray crystallography. Only relatively small bio-molecules can be solved, for proteins usually smaller than 300 residues Rapid developments in equipment and software has led however, to continual improvement in these areas 34

35 The Advantages and Limitations of NMR Advantages: The main advantage of NMR spectroscopy over X-ray crystallography is that there is no need to have the sample in a crystalline state (which restricts the applicability of X-ray crystallography). Further information about the dynamics of the molecule in solution can be deduced. Motions that occur on a time-scale ranging from about 10 picoseconds to about 10 nanoseconds can be detected. Limitations: The precision of the technique is still not as high as in X-ray crystallography. Only relatively small bio-molecules can be solved, for proteins usually smaller than 300 residues Rapid developments in equipment and software has led however, to continual improvement in these areas 35

36 Electron Microscopy Electron microscopy (EM) is another approach that provides information about the 3D structure of macromolecules. It has recently become popular in structural biology as it can provide molecular structural data about large molecular complexes. 36

37 Principles of Electron Microscope Electron microscope is based on similar principles as light microscopes but electron beam is used instead of light beam It has much higher magnification or resolving power than a regular light microscope, allowing it to see very small objects such as cells and large molecules Source of illumination is a cathode that emits electrons at the top of the column Since electrons are scattered by collisions with air molecules, column must be under a vacuum 37

38 Cryo-electron Microscopy Important breakthrough in solving bio-molecule structures by EM, is cryo-electron microscopy ("cryo-em") The images are created with an electron microscope equipped with a "cold stage". This means that the samples are scanned in a frozen state, usually liquid nitrogen temperatures (77 K = -196 C). This procedure protects samples from radiation damage and keeps them in a native state A version of cryo-electron microscopy is cryo-electron tomography (CET) where a 3D reconstruction of a sample is created from tilted 2D images (also measured at cryogenic temperatures, either liquid nitrogen or helium). 38

39 The Advantages of Electron Microscopy EM is able to resolve very large biological complexes not accessible or X-ray crystallography Structures determined by EM can serve as an initial reference for interpretation of X-ray diffraction patterns Structures from EM can be combined with X-ray data to obtain structural information about large molecules 39

40 The Limitations of Electron Microscopy The limitation of EM is the structural resolution that can be achieved. The current resolution (about 3.5 Å) is not sufficient to determine the location of the amino-acid side chains. 40

41 Neutron Diffraction Neutron diffraction is based on scattering of neutrons on atoms nuclei. Nuclear reactors are used as a source of neutrons. The technique is similar to X-ray diffraction but due to the different scattering properties of neutrons versus x-rays complementary information can be obtained Because hydrogen nuclei are strong scatterers for neutrons, the position of hydrogen (or deuterium) atoms in a crystal structure can be determined far more precisely with neutron diffraction than with X-ray diffraction Deuterium atoms is easier to detect then 1H atoms The studies with deuterium can be used to identify the presence of H/D exchange sites (which usually corresponds to acidic hydrogens) Typical resolution of neutron diffraction is between 1.5 and 4.0 A (i.e. individual atoms are visible including hydrogens) Disadvantage of the method is necessity to have access to neutron source (nuclear reactor) 41