1. Understanding biology through structures Course work 2006 Structure Determination and Analysis : X-ray Crystallography

2. Understanding biology through structures Course work 2006 X-rays Intensity of diffracted beam Diffraction SourceSampleDetector

3. Understanding biology through structures Course work 2006 Energy = hc λ

4. Understanding biology through structures Course work 2006 X-rays It is possible to translate information in the diffraction pattern into atomic structure using Bragg’s law, which predicts the angle of reflection of any diffracted beam from specific atomic planes Unlike using a light microscope, there is no way of re-focusing diffracted x-rays. Instead we must collect a diffraction pattern (spots).

5. Understanding biology through structures Course work 2006 A typical crystallography experiment Pure protein Grow crystal Characterize crystals Collect diffraction data Solve phase problem Calculate electron density map Build/rebuild model Refine model Analyze structure

6. Understanding biology through structures Course work 2006 The Beginning

7. Understanding biology through structures Course work 2006 Principles of X-ray diffraction What is a crystal? The unit cell is the basic building block of the crystal The unit cell can contain multiple copies of the same molecule whose positions are governed by symmetry rules

8. Understanding biology through structures Course work 2006 Proteins and crystallisation Proteins must be homogenous & monodispersed. Need large amount (mg quantities) Is it stable ( salt, pH, temp) Will modifications have to be made? What type of protein is it? Has anything similar been crystallized before? Proteins must be pure (> 99%) & fully folded Check the activity of your protein if you have an assay Check folding by other spectroscopic methods

9. Understanding biology through structures Course work 2006 Crystallisation of proteins ‘controlled’ precipitation of the protein. Protein aggregates associate & form intermolecular contacts that resemble those found in the final crystal. Aggregates reach the critical nuclear size, growth proceeds by addition of molecules to the crystalline lattice. The processes of nucleation and crystal growth both occur in supersaturated solutions. Precipitant Cover-slip sealed with vacuum grease Protein in “Hanging drop” Process controlled by: Temp pH Salt conc Precipitants (PEG, ethanol)

10. Understanding biology through structures Course work 2006 Diffraction Apparatus

11. Understanding biology through structures Course work 2006 Synchrotron radiation More intense X-rays at shorter wavelengths mean higher resolution & much quicker data collection

12. Understanding biology through structures Course work 2006 Experimental setup

13. Understanding biology through structures Course work 2006 Remove cover slip and fish out crystal with a small nylon loop Mount loop on goniostat in a stream of nitrogen gas Surface tension of the liquid in the loop holds crystal in place Mounting crystals

14. Understanding biology through structures Course work 2006 Diffraction Each image represents the rotation of the crystal 1 degree in the X-ray beam. Each images gives us the position of each spot relative to all the others & there intensity. Intensity = square of amplitude.

15. Understanding biology through structures Course work 2006 Diffraction Principles n  = 2dsin 

16. Understanding biology through structures Course work 2006 Diffraction Principles A string of atoms Corresponding Diffraction Pattern

17. Understanding biology through structures Course work 2006 The reciprocal lattice and the geometry of diffraction X-ray source X-ray detector

18. Understanding biology through structures Course work 2006 Spacing between diffraction spots defines unit cell 1/ a 1/ b

19. Understanding biology through structures Course work 2006 Waves & the phase problem The amplitudes of the diffracted X-rays can be experimentally measured, but the phases cannot = phase problem. i.e. we don’t know the phase of each diffracted ray relative to the others! X ? A Z X Y

20. Understanding biology through structures Course work 2006 The Phase Problem Diffraction data only records intensity, not phase information (half the information is missing) To reconstruct the image properly you need to have the phases (even approx.) –molecular replacement –direct methods –isomorphous replacement –anomolous dispersion

21. Understanding biology through structures Course work 2006 Structure factors & Fourier transforms unit cell F (h,k,l) = V  x=0  y=0  z=0  (x,y,z).exp[2  I(hx + ky + lz)].dxdydz A reflectionelectron density All reflections phase  ( x,y,z ) = 1/V  h  k  l  F ( h,k,l )  exp[2  I (h x + k y + l z ) + i  ( h,k,l ) Electron density amplitude At a point The vector (amplitude and phase) representing the overall scattering from a particular set of Bragg planes is termed the structure factor ( F ). Structure factors for various points on the crystal lattice correspond to the Fourier transform of electron density within the unit cell and vice-versa.

22. Understanding biology through structures Course work 2006 F T Fourier Transform of a molecule

23. Understanding biology through structures Course work 2006 Fourier Transform of a crystal

24. Understanding biology through structures Course work 2006 The Phase Problem Diffraction data only records intensity, not phase information (half the information is missing) To reconstruct the image properly you need to have the phases (even approx.) –molecular replacement –direct methods –isomorphous replacement –anomolous dispersion

25. Understanding biology through structures Course work 2006 Molecular replacement Requires a starting model for structure Can calculate back from structure to electron density to structure factors Works if model is 30 to 40 % identical to correct answer

26. Understanding biology through structures Course work 2006 Molecular Replacement By determining the correct orientation and position of a molecule in the unit cell using a previously solved structure as a ‘search model’. This model can then be used to calculate phases

27. Understanding biology through structures Course work 2006 Isomorphous replacement (IR) Provides indirect estimates of the protein phase angles by observing the interference effects of the intensities on scattered beams by a heavy atom marker. All the electrons in the heavy atom will scatter essentially in the same phase. We can solve the positions of these heavy atoms because they are few in number and strong in signal. Using this estimate we can deduce the positions of the protein atoms and their phases

28. Understanding biology through structures Course work 2006 Anomalous scattering Scattering information of an atom whose absorption frequency is close to the wavelength of the source beam produces phase information Resolved anomalous scattering requires intensity measurements at one wavelength Multi-wavelength anomalous dispersion, requires intensity measurements at several wavelengths

29. Understanding biology through structures Course work 2006 Using the structure factor calculation we can produce electron density maps for the whole protein. We then fit our protein model (co- ordinates X,Y,Z) inside the map.

30. Understanding biology through structures Course work 2006 Resolution 1.2 Å 2 Å 3 Å

31. Understanding biology through structures Course work 2006 Resolution 6Å : Outline of the model, feature such as helices can be identified. 3Å : Can trace polypeptide chain using sequence data, establish folding topology. Assign side chains. 2Å : Accurately establish mainchain conformation, assign sidechains without sequence data, I.d water molecules. 1.5Å : Individual atoms are almost resolved, detailed discription of water structure. 1.2Å : Hydrogen atoms may become visible.

32. Understanding biology through structures Course work 2006 Final Structure But the work is not over yet!

33. Understanding biology through structures Course work 2006 Refinement The process of building and rebuilding a model can cause many errors in the structure. 1.Bond length, 2.Bond angle 3.Atomic clashes etc It is necessary to subject the structure to refinement in order to remove these errors and produce a better structure. Minimization Thermal parameters In order to further improve the model, it is refined using a simulated annealing protocol Refinement progress is monitored by following the agreement between the the observed data ( data collected) and the calculated data (data calculated from current model) = R factor

34. Understanding biology through structures Course work 2006 R-factor The agreement between the the observed data (data collected) and the calculated data (data calculated from current model) the lower the number the better; typically around 20% Resolution The higher the resolution the more detail that can be seen 3.0Å is fairly low whilst 1.1Å is approaching atomic resolution B-factor Measure of thermal motion. i.e. how much energy each atom contains. Gives us information on mobility & stability Rms deviation Deviation of bond lengths & angles from ideal Quality of the structure?

35. Understanding biology through structures Course work 2006 Deviation of bond lengths & angles from ideal. All based on the geometry of small molecules. Rms deviation for bond lengths should be less than 0.02Å and less than 4º for bond angles Determined using a Ramachandran plot. Rms deviation of bond length & bond angle

36. Understanding biology through structures Course work 2006 Absorption of Light

37. Understanding biology through structures Course work 2006 Protein chromophores: Peptide bond Amino acid side chains Prosthetic groups Peptide bond absorbance: 210 nm due to n   transition 190 nm due to    transition Amino acid side chain absorbance: Asp, Glu, Asn, Gln, His and Arg have transitions at the same wavelength where peptide absorbs Protein concentration can be measured by measuring absorbance at 280 nm and by assuming that 1 mg ml -1 solution of protein has absorbance of 1.0 Absorption in the UV and visible range

38. Understanding biology through structures Course work 2006 Absorption and emission spectra of individual tryptophan residues, in the absence of energy transfer

39. Understanding biology through structures Course work 2006 Fourth derivative absorption spectrum Fourth derivatives of the absorption spectra have been documented as a valuable tool for studying structural changes in proteins. Protein fourth derivative spectra have been shown to be very sensitive to changes in the microenvironment (polarity, hydration, hydrophobic interactions, packing density) of tyrosine and tryptophan residues Chauhan and Mande, Biochem J, 2001

40. Understanding biology through structures Course work 2006 Measurements of conformational properties using optical activity

41. Understanding biology through structures Course work 2006 Linearly polarised light Right circular polarisation Left circular polarisation

42. Understanding biology through structures Course work 2006 Nearly all molecules of life are optically active There are four ways that an optically active sample can alter the properties of transmitted light: optical rotation, ellipticity, circular dichroism, circular birefringence Linear Circular Elliptical

43. Understanding biology through structures Course work 2006 After passing through an optically active absorbing sample, the light is changed in two aspects: 1.The maximal amplitude E is no longer confined to a place, instead it traces an ellipse Ellipticity = tan -1 (minor/major axis) 2.The orientation of the ellipse is an indication of optical activity. If the sample did not absorb any light, the ellipse would such small axial ratio that it would be equivalent to a plane-polarised light. In this case we will say that the plane polarised light has been rotated. 3.Orientation of the ellipse is the optical rotation. Optical rotation as a function of wavelength is called the optical rotatory dispersion (ORD).

44. Understanding biology through structures Course work 2006 Circular Dichroism

45. Understanding biology through structures Course work 2006 CD spectrum of a protein

46. Understanding biology through structures Course work 2006

47. Where can Circular Dichroism be used?

48. Understanding biology through structures Course work 2006 Measurements of conformational properties using fluorescence

49. Understanding biology through structures Course work 2006 Fluorescence Chromophores are components of molecules which absorb light They are generally aromatic rings

50. Understanding biology through structures Course work 2006 Fluorescence ENERGY S0S0 S1S1 S2S2 T2T2 T1T1 ABS FL I.C. ABS - AbsorbanceS 0.1.2 - Singlet Electronic Energy Levels FL - FluorescenceT 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal ConversionIsC - Intersystem CrossingPH - Phosphorescence IsC PH [Vibrational sublevels] Jablonski Diagram Vibrational energy levels Rotational energy levels Electronic energy levels Singlet StatesTriplet States

51. Understanding biology through structures Course work 2006 Simplified Jablonski Diagram S0S0 S’ 1 Energy S1S1 hv ex hv em

52. Understanding biology through structures Course work 2006 Fluorescence The longer the wavelength the lower the energy The shorter the wavelength the higher the energy eg. UV light from sun causes the sunburn not the red visible light

53. Understanding biology through structures Course work 2006 Fluorescence Excitation Spectra Intensity related to the probability of the event Wavelength the energy of the light absorbed or emitted

54. Understanding biology through structures Course work 2006 Corrected excitation spectra (corrected for source output and monochromator throughput) can be obtained by using a reference channel equipped with a "quantum counter". This is a concentrated dye solution (typically 3 mg/mL rhodamine B in ethylene glycol). A tiny fraction of the excitation beam is diverted to the reference detector. The quantum counter absorbs all of this light, and converts it (with 100% efficiency to fluorescence), the intensity of which is independent of wavelength between 220 and 580 nm. Any changes in lamp output or monochromator throughput will cause corresponding alterations in the output of the reference channel. By dividing the fluorescence signal by the reference signal, these wavelength-dependent variations are cancelled out. Unfortunately, the quantum counter will not entirely correct the emission spectrum. However, instrument manufacturers supply correction factors for their monochromators. Application of these will give an approximately correct spectrum. If more accuracy is needed, the spectrum of a known standard compound (fluorescing in the region of interest) can be compared to published standards. j. Biological fluorophores 1) Intrinsic fluorophores a) Proteins Tryptophan dominates protein fluorescence spectra - high molar absorptivity - moderate quantum yield - ability to quench tyrosine and phenylalanine emission by energy transfer. Free tyrosine has a relatively high fluorescent output, but is strongly quenched by trptophan in native proteins. Unless tyrosine and tryptophan are absent, emission from phenylalanine is not observed in protein fluorescent spectra.

55. Understanding biology through structures Course work 2006 Tryptophan is a good fluorophore note that this fluorescence expt used an excitation of 270nm we can consider solvent effects on its emission wavelength in the same way we did for absorption... note that the fluorescence looks like a mirror image of the 280nm absorption peak (and not the 220nm peak)

56. Understanding biology through structures Course work 2006 Absorption vs Emission for Trp comparing our diagrams for absorption and emission – and assuming that protein interiors behave like organic solvents(!) – we predict: Abs. in water buried in protein  E abs protein waterabsorption  E em emissionprotein water Em.

57. Understanding biology through structures Course work 2006 Effect of Ca 2+ on Intrinsic Trp-fluorescence and on Fluorescence Anisotropy ▼ Wild type Dome loop mutant Blue shift and intensity enhancement upon addition of Ca 2+ Change in anisotropy upon titration in the wild type, but not in the mutant

58. Understanding biology through structures Course work 2006 Raman Scatter A molecule may undergo a vibrational transition (not an electronic shift) at exactly the same time as scattering occurs This results in a photon emission of a photon differing in energy from the energy of the incident photon by the amount of the above energy - this is Raman scattering. 488 nm excitation 575-595 nmThe dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation this would give emission at 575-595 nm

59. Understanding biology through structures Course work 2006 Rayleigh Scatter Molecules and very small particles do not absorb, but scatter light in the visible region (same freq as excitation) Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)

60. Understanding biology through structures Course work 2006 Probes for Proteins FITC488525 PE488575 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 ProbeExcitationEmission

61. Understanding biology through structures Course work 2006 Hoechst 33342 (AT rich) (uv)346460 DAPI (uv)359461 POPO-1 434456 YOYO-1 491509 Acridine Orange (RNA)460 650 Acridine Orange (DNA)502536 Thiazole Orange (vis)509525 TOTO-1514533 Ethidium Bromide526604 PI (uv/vis)536620 7-Aminoactinomycin D (7AAD)555655 Probes for Nucleic Acids

62. Understanding biology through structures Course work 2006 DNA Probes AO –Metachromatic dye concentration dependent emission double stranded NA - Green single stranded NA - Red AT/GC binding dyes –AT rich: DAPI, Hoechst, quinacrine –GC rich: antibiotics bleomycin, chromamycin A 3, mithramycin, olivomycin, rhodamine 800

63. Understanding biology through structures Course work 2006 Probes for Ions INDO-1 E x 350E m 405/480 QUIN-2E x 350E m 490 Fluo-3 E x 488E m 525 Fura -2E x 330/360E m 510

64. Understanding biology through structures Course work 2006 pH Sensitive Indicators SNARF-1488575 BCECF488525/620 440/488525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] ProbeExcitationEmission

65. Understanding biology through structures Course work 2006 Probes for Oxidation States DCFH-DA(H 2 O 2 )488525 HE(O 2 - )488590 DHR 123(H 2 O 2 )488525 Probe Oxidant ExcitationEmission DCFH-DA- dichlorofluorescin diacetate HE- hydroethidine DHR-123- dihydrorhodamine 123

66. Understanding biology through structures Course work 2006 Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium

67. Understanding biology through structures Course work 2006 Other Probes of Interest GFP - Green Fluorescent Protein –GFP is from the chemiluminescent jellyfish Aequorea victoria –excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm –contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence –Major application is as a reporter gene for assay of promoter activity –requires no added substrates

68. Understanding biology through structures Course work 2006 Energy transfer excitation emission transfer A B phycoerythrin-Texas RedECD phycoerythrin-cyanine5PC5

69. Understanding biology through structures Course work 2006 Energy Transfer Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

70. Understanding biology through structures Course work 2006 non-radiative(quenching) excited states ground state AB Energy transfer Molecule A absorbs light and is excitedexcitation transfer A passes the energy onto molecule B emission Molecule B emits light