Macromolecular crystallography Lecture 1 Pavel Plevka •Lecture 1 – Introduction to X-ray crystallography, basic diffraction •Lecture 2 – Solution of phase problem, model building, and structure validation • •Development of crystallography •Waves, radiation, and diffraction WILHELM CONRAD RÖNTGEN (1845-1923) •1901 Nobel Laureate in Physics • discovery of the remarkable rays subsequently named after him. MAX VON LAUE (1879-1960) •1914 Nobel Laureate in Physics • for his discovery of the diffraction of X-rays by crystals laue img59 Friedrich and Knipping Waves Coherent beam Addition of waves Particles & waves Diffraction of light Diffraction of light MAX VON LAUE (1879-1960) •1914 Nobel Laureate in Physics • for his discovery of the diffraction of X-rays by crystals laue img59 Wavelength and diffraction Wavelength comparison of X-rays and visible light 38l 70μm SIR WILLIAM HENRY BRAGG (1862-1942) SIR WILLIAM LAWRENCE BRAGG (1890-1971) •1915 Nobel Laureates in Physics • for the analysis of crystal structure by means of X-rays wh-bragg wl-bragg nl = 2d sinq James Batcheller Sumner (1879-1960) •1946 Nobel Laureate in Chemistry • for his discovery that enzymes can be crystallized FRANCIS HARRY COMPTON CRICK (1916~2004) JAMES DEWEY WATSON (1928~) MAURICE HUGH FREDERICK WILKINS (1916~2004) •1962 Nobel Laureates in Physiology and Medicine • for their discoveries concerning the molecular structure of nuclear acids and its significance for information transfer in living material. wcwf03 wcwf02 Rosalind Franklin Maurice Wilkins James Watson and Francis Crick Max Ferdinand Perutz (1914 – 2002) John Cowdery Kendrew (1917 – 1997) •1962 Nobel Laureates in Physics • for their studies of the structures of globular proteins Information from X-ray diffraction experiment [0;0;0] x y z aadensity Representative electron density for amino acid side chains Electron density maps calculated at 1.5 Angstrom resolution. Comparison of microscope and diffraction Waves and Radiation Wave as a vector •F=Acosa+iAsina •F=Aexp(ia) a A Real axis A- wave amplitude a- wave phase F A X-rays scatter from electrons in all directions Primary beam Secondary beams X-ray scattering from several electrons Primary beam When do electrons scatter “in phase” – waves add constructively? Molecule is composed of many electrons Each electron will scatter secondary radiation uppon exposure to x-rays The scattered secondary beams will interact and cause interference The scattering from a molecule is dependent on number of and distances between electrons In other words, scattering from molecule is dependent on its structure If we would know the amplitudes and phases of scattered molecule, we could calculate the structure of molecule... •Scattering from a single molecule is not detectable •If molecules are all oriented in the same way, the scattering from individual molecules will add in certain directions –Which directions? • There is no path and PHASE DIFFERENCE when rays reflect from a plane nl = 2d sinq Bragg’s law: There is NO PHASE DIFFERENCE if the path differences are equal to whole number multiplies of wavelength (l) w sinq = w/d 2w = nl nl = 2d sinq Bragg’s law: sinq = w/d 2w = nl There is NO PHASE DIFFERENCE if the path differences are equal to prime number multiplies of wavelength (l) w (h, k, l) Diffraction pattern from a protein crystal (h, k, l) nl = 2d sinq aadensity piccat piccatfftm picmanxfft Observed amplitudes Fourier amplitudes and phases Real space cat Fourier transform Circular rainbow scale of phases Linear intensity scale of amplitude size Electron density equation + PHASE PROBLEM Summary: 1. X-rays have suitable wavelength for study of molecular structures 2. Crystals allow measurement of useful diffraction data because they diffract strongly in certain directions 3. Our goal is to obtain three-dimensional distribution of electron density, because it shows the shape of a molecule 4. Diffraction experiments provide only amplitudes of structure factors => Phase problem Human cardiovirus Saffold virus 3 (2.5Å resolution) 1. Rentgenové paprsky se používají ke studiu makromolekulárních struktur protože: A.) Mají vlnovou délku podobnou meziatomovým vzdálenostem. B.) Jako jediné elektromagnetické záření interagují s biologickým materiálem. C.) Byly objeveny v době intenzivního zájmu o strukturu makromolekul a z historických důvodů se používají dodnes. 2. To, že makromolekuly tvoří krystaly znamená že: A.) Mají enzymatickou aktivitu B.) Jsou součástí kostry buňky (cytoskeletu) C.) Mají stabilní strukturu. 3. Mapa elektronové hustoty, která je výsledkem rentgenové analýzy krystalů: A.) Ukazuje tvar molekul, které tvoří krystal B.) Má vždy bílou barvu C.) Ukazuje tvar molekuly po denaturaci Macromolecular crystallography Lecture 2 Pavel Plevka •Phase problem and its solution •Building macromolecular structures based on X-ray diffraction data •Validation of macromolecular structures nl = 2d sinq Bragg’s law Bragg’s law Adition of waves F=Acosa+iAsina Electron density equation & PHASE PROBLEM piccat picmanxfft Observed amplitudes Fourier amplitudes and phases Real space cat Fourier transform Circular rainbow scale of phases Linear intensity scale of amplitude size Fourier cat •Molecular replacement •1. source of initial phases is a model •2. the model is oriented and positioned to obtain the best agreement with the x-ray data •3. phases are calculated from the model •4. The calculated phases are combined with the experimental data • Solving the phase problem by: Molecular Replacement was invented by Michael Rossmann piccat picmanx Observed amplitudes Phases unknown! Unknown structure, unknown orientation Known structure Fourier cat Cat Fourier transform Diffraction experiment picmanx Manx cat Wrong orientation! Calculated amplitudes and phases FT of Manx cat picmanx Observed amplitudes Phases unknown! Known structure Fourier cat Fourier transform, try different orientations picmanx Manx cat Wrong orientation! Calculated amplitudes and phases FT of Manx cat picmanx picmanx picmanx picmanx picmanx picmanx picmanx piccatmanx3 piccatmanx3fft Observed amplitudes (tailed cat), calculated phases (Manx cat) Even the tail becomes visible! Inverted Fourier transform picduck picduckfft piccatduckfft piccatduck Duck amplitudes + cat phases Duck Fourier transform of duck Looks like a cat!! Model Bias Fourier transform Inverted Fourier transform •Multiple/Single Isomorphous Replacement (MIR/SIR) •source of phases – intensity differences between data from native and derivative (heavy atom containing) crystals •Positions of heavy atoms identified from isomorphous difference Patterson maps Solving the phase problem by: •Multiple/Single-wavelength anomalous diffraction (MAD/SAD) •source of phases – intensity differences between structure factors due to the presence of atom that specifically interacts with X-rays of a given wavelength •Positions of heavy atoms identified from anomalous difference Patterson maps Solving the phase problem 3 Model building & resolution Fitting of protein sequence in the electron density Easy in molecular replacement More difficult if no initial model is available Unambiquous if resolution is high enough (better than 3.0 Å) Can be automated, if resolution is close to 2Å or better Model building & refinement Model building & refinement Validation •Assesment of model quality: •Is the model in agreement with experimwntal data? •How the geometry of amino acids look like? •Are atoms far / close enough from each other? •Are residues “happy” in their environment? •Are the hydrogen donors/acceptors satisfied? R-factor, Rfreefactor R-factor Rfree factor Ramachandran plot Ψ φ φ Ψ ω Geometry and stereochemistry Bond lengths Dihedral angles Real-space fit Data deposition •Protein Data Bank (PDB) •Some structures are wrong! Summary 1. X-rays have suitable wavelength for study of molecular structures 2. Crystals allow measurement of useful diffraction data because they diffract strongly in certain directions 3. Our goal is to obtain three dimensional distribution of electron density, because it shows the shape of a molecule 4. Diffraction experiments provide only amplitudes of structure factors => Phase problem 5. Solution of the phase problem: Molecular replacement Isomorphous replacement Anomalous diffraction 6. Model building, refinement, validation, deposition 1. Virus purification 2. Crystallization 3. Diffraction data 4. Solve structure Rhinoviruses – 40% of common cold cases – economic losses $16bn/year in USA McMinn et al. Clin Infect Dis 2001. Image by Heng Soy, KI Media 2012. Structural studies of human picornaviruses Enteroviruses (EV71) – hand-foot-and-mouth-disease – encephalitis I would like to start my presentation by defining biological significance of my project. My research is focused on structural studies of picornaviruses that include some of the most common human pathogens. For example, rhinoviruses are responsible for about 40$ of common cold cases. The economic damage caused by rhinoviruses was estimated to be 16bn dollars per year in the united states alone. Similar expenses are expected for European union. Enterovirus 71 induces yearly outbreaks of hand-foot-and-mouth disease mostly in south-east Asia. About 5% of the infected children develop encephalitis that can be fatal or result in permanent brain damage. This magnetic resonance image shows inflammation in left section of cerebellum caused by EV71 infection. The three years old patient lost ability to speak. Currently no antiviral drugs are available against rhinoviruses or enteroviruses. More basic research is needed to improve our understanding of picornavirus biology to allow identification of new targets for antiviral drugs. My research will address these requirements by -> next slide Picornavirus replication cycle … aspiring to answer basic biological questions about picornavirus life cycle. It is well established that picornaviruses enter cells by receptor mediated endocytosis. However, it is not known how is the picornavirus ssRNA genome delivered from virions across the membrane of the endosome into cell cytoplasm. That is our first research question. When in the cytoplasm the genome is translated into a polyprotein that is then cleaved into functional subunits. The viral proteins induce formation of so called virus-replication factories that contain lipid vesicles derived from intra-cellular membranes, viral and cellular proteins, ribosomes and viral RNAs. We will use modern cryo-electron tomography approaches to characterize the factories on molecular level. In addition we will structurally characterize picornavirus genome replication and recombination – processes that happen in the vicinity of the replication factories. Finally, we will determine picornavirus virion assembly mechanism. Human cardiovirus Saffold virus 3 (2.5Å resolution) Human Parechovirus 1 @ 3.1Å 1.) Jakou část strukturního faktoru můžeme změřit v difrakčním experimentu: a) amplitudu (ve formě intensity) b) fázi 2.) Nejčastější metoda pro získání fází je: a) molekulární nahrazení (molecular replacement) b) isomorfní nahrazení c) anomální diffrakce 3.) Ramachandran plot ukazuje: a.) distribuci úhlů v hlavním řetězci proteinu b.) vzdálenosti mezi atomy c.) konformace postranních řetězců aminokyselin Honeybee viruses … aspiring to answer basic biological questions about picornavirus life cycle. It is well established that picornaviruses enter cells by receptor mediated endocytosis. However, it is not known how is the picornavirus ssRNA genome delivered from virions across the membrane of the endosome into cell cytoplasm. That is our first research question. When in the cytoplasm the genome is translated into a polyprotein that is then cleaved into functional subunits. The viral proteins induce formation of so called virus-replication factories that contain lipid vesicles derived from intra-cellular membranes, viral and cellular proteins, ribosomes and viral RNAs. We will use modern cryo-electron tomography approaches to characterize the factories on molecular level. In addition we will structurally characterize picornavirus genome replication and recombination – processes that happen in the vicinity of the replication factories. Finally, we will determine picornavirus virion assembly mechanism. cutaneous leishmaniasis metastasis to nasopharyngeal tissues Ives et al. Science, 2011 Olivier. Nature, 2011 Free Dictionary Leishmania RNA virus 1 1. Virus purification 3. cryo-EM 4. Reconstruction 2. Grid preparation Johann Deisenhofer (1943) Robert Huber (1937) Hartmut Michel (1948) •1988 Nobel Laureates in Chemistry • for the determination of the structure of a photosynthetic reaction centre Venkatraman Ramakrishnan (1952) Thomas A. Steitz (1940) Ada E. Yonath (1939) •2009 Nobel Laureates in Chemistry • for studies of the structure and function of the ribosome X-ray crystallography •First method to determine structure of molecules with atomic resolution •As of September 17, 2013 there were more than 70,000 structures determined by protein crystallography in Protein Data Bank •Macromolecular structures are crucial for our understanding of life at the molecular level •28 Nobel prizes 14 Bravais Lattices Description of lectromagnetic waves •E- electric field strength •A- amplitude •l - wavelenght • E = A cos 2pz/l E = A cos (a+2pz/l) •z - position along beam path •a - phase • l l nl = 2d sinq aadensity Diffraction pattern from a protein crystal Phase problem α α α Phase problem α α α Building macromolecular structures Building macromolecular structures Building macromolecular structures Phase problem Electron density equation Wave as a vector •F=Acosa+iAsina •F=Aexp(ia) a A Real axis A - wave amplitude α - wave phase F A Patterson function, Patterson space