Designing an X-ray experiment Data collection strategies

Transcription

1 Designing an X-ray experiment Data collection strategies Macromolecular Crystallography School Madrid. May, José Miguel Mancheño. Insto. Rocasolano. CSIC.

2 The context Macromolecular Crystallography (MX) (from) the crystal... (through the) three dimensional structure... last experimental step (to) hypothesis about the biological role of a protein the smoothness of the subsequent steps critically depends on the data quality phasing, Fourier maps, refinement rotation method (X-ray diffraction experiments)

3 Contents of the talk The main objective of data collection determining factors what is a good crystal? Cryoprotection and radiation damage introduction efects of RD and cryoprotectants strategies when cryoprotection fails Geometry of diffraction: rotation method Ewald sphere mosaicity and divergence background contribution thick vs thin datasets Completeness: total rotation range and blind region consequences of symmetry in the diffraction pattern incomplete datasets and splitting the rotational range Anomalous diffraction data introduction strategies of data collection additional considerations Initial inspection of the diffraction image and decisions Some final notes on choices on data collection

4 The main objective High quality X-ray diffraction data set that fulfills your expectatives Dataset must be complete Maximum resolution achievable With no systematic errors hkl I hkl σ(i hkl ) crystal quality % solvent huge unit cells proper estimation : counting statistics I hkl /σ(i hkl ): significant values in the higher resolution shell overlapping overloads radiation damage diffraction power of the crystal experimental parameters: detector type, exposure time symmetry of crystal lattice reciprocal lattice geometry of the experimental setup What exactly constitutes a useful data set largely depends on the purpose of the data collection- phasing and refinement have different requirements. The low resolution part of the diffraction data is important for phasing, and high resolution data help model building and refinement. B. Rupp Chapter 8 In Biomolecular Crystallography Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F Dauter (1999) Acta Cryst. D55,

5 Determining factors Qualitative Quantitative x-ray source detector type time available cryoprotection crystal symmetry cell parameters resolution crystal reproducibility crystal quality: intrinsic low diffracting power B. Rupp Chapter 8 In Biomolecular Crystallography Protein crystals low relative intensity large unit cells large number of reflections significant intrinsic disorder: solvent content low relative intensity radiation damage sensitivity Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

6 What is a good protein crystal? Single: only one lattice Diffracts to high angle Low mosaicity check by indexing pattern and looking for unpredicted spots check completeness high resolution shell Large better signal/noise; fewer overlaps diffracted intensity proportional to illuminated volume; not much gain when larger than beam ( µm) smaller crystals may freeze better (lower mosaicity) Good freeze The best that you have no ice, minimum amount of liquid (low background) the best worst

7 Cryoprotection and Radiation Damage A brief introduction > 90% diffraction experiments in MX are done at 100 K (flashcooled crystals) N 2 gas (100 K); N 2 liquid (<77 K); ethane, propane The benefits of cooling the crystals are multiple: it prolongs the lifetime of the crystal by a factor of aprox. 70; a complete dataset can be collected with one crystal: no merging and nonisomorphism; elimination of radiation damage due to radicals 3rd generation synchrotron radiation (SR) sources (late 1990s): intense radiation damage (RD). Symptoms of RD include: loss of high resolution data and decrease in diffraction intensities increase in unit cell volume increase in mosaicity increase in values of B-Wilson factors increase in B factors in the refined model ongoing systematic studies on different aspects of RD are increasing our understanding of the underlying physics and chemistry of mechanisms breakage of covalent bonds: Cys, Glu, Asp, Tyr, Met radiation damage phasing (RIP, RIPAS) Ramagopal et al., (2005) Acta Cryst D61, Holton (2009) J. Synchrotron Rad. 16(2), exploits the difference between datasets obtained from native and severely radiation-damaged crystals. Usually, the presence of heavy atoms can dramatically increase the damage to crystals. Garman & Schneider (1997) J. Appl. Cryst 30, Garman & Owen (2006) Acta Cryst D62, 32-47

8 Cryoprotection and Radiation Damage A brief introduction > 90% diffraction experiments in MX are done at 100 K (flashcooled crystals) N 2 gas (100 K); N 2 liquid (<77 K); ethane, propane The benefits of cooling the crystals are multiple: it prolongs the lifetime of the crystal by a factor of aprox. 70; a complete dataset can be collected with one crystal: no merging and nonisomorphism; elimination of radiation damage due to radicals 3rd generation synchrotron radiation (SR) sources (late 1990s): intense radiation damage (RD). Symptoms of RD include: loss of high resolution data and decrease in diffraction intensities increase in unit cell volume increase in mosaicity increase in values of B-Wilson factors B. Rupp Chapter 8 In Biomolecular Crystallography increase in B factors in the refined model ongoing systematic studies on different aspects of RD are increasing our understanding of the underlying physics and chemistry of mechanisms breakage of covalent bonds: Cys, Glu, Asp, Tyr, Met radiation damage phasing (RIP, RIPAS) exploits the difference between datasets obtained from native and severely radiation-damaged crystals. Usually, the presence of heavy atoms can dramatically increase the damage to crystals. Ramagopal et al., (2005) Acta Cryst D61, Holton (2009) J. Synchrotron Rad. 16(2), Garman & Schneider (1997) J. Appl. Cryst 30, Garman & Owen (2006) Acta Cryst D62, 32-47

9 Cryoprotection and Radiation Damage A brief introduction > 90% diffraction experiments in MX are done at 100 K (flashcooled crystals) N 2 gas (100 K); N 2 liquid (<77 K); ethane, propane The benefits of cooling the crystals are multiple: it prolongs the lifetime of the crystal by a factor of aprox. 70; a complete dataset can be collected with one crystal: no merging and nonisomorphism; elimination of radiation damage due to radicals 3rd generation synchrotron radiation (SR) sources (late 1990s): intense radiation damage (RD). Symptoms of RD include: loss of high resolution data and decrease in diffraction intensities increase in unit cell volume increase in mosaicity increase in values of B-Wilson factors increase in B factors in the refined model ongoing systematic studies on different aspects of RD are increasing our understanding of the underlying physics and chemistry of mechanisms breakage of covalent bonds: Cys, Glu, Asp, Tyr, Met radiation damage phasing (RIP, RIPAS) exploits the difference between datasets obtained from native and severely radiation-damaged crystals. Usually, the presence of heavy atoms can dramatically increase the damage to crystals. Ramagopal et al., (2005) Acta Cryst D61, Holton (2009) J. Synchrotron Rad. 16(2), Garman & Schneider (1997) J. Appl. Cryst 30, Garman & Owen (2006) Acta Cryst D62, 32-47

10 Cryoprotection and Radiation Damage Objective of cryoprotection Avoid the formation of ice crystals: glass-like (vitreous) water ice crystals: loss of internal order of the protein crystal ice crystals: interference with protein diffraction pattern Vitrification of pure water in micrometric drops: 10-5 s ( 10 6 K s -1 ) Johari et al., (1987) Nature 330, the cooling rate for pure water to remain vitreous is too high 10 6 K s -1. In the presence of a cryoprotectant agent: 1-2 s The cooling rate to remain vitreous dramatically decreases which is why cryoprotectants are used glycerol-like cryoprotectants oil-like cryoprotectants: Paratone N, Silicone D-200 Riboldi-Tunnicliffe & Hilgenfeld, (1999) J. Appl. Cryst. 32, B. Rupp Chapter 8 In Biomolecular Crystallography Garman & Schneider (1997) J. Appl. Cryst 30, Garman & Owen (2006) Acta Cryst D62, 32-47

11 Cryoprotection and Radiation Damage Objective of cryoprotection Possible strategy for finding an appropiate cryoprotectant buffer Garman & Schneider (1997) J. Appl. Cryst 30, Garman & Owen (2006) Acta Cryst D62, 32-47

12 Cryoprotection and Radiation Damage Nevertheless, sometimes cryoprotection fails Annealing (cryoshutter; re-mounting) Annealing refers to a cyclic thermal treatment of cryocooled crystals, which are (sometimes repeatedly) warmed up and cooled again. Not all crystals survive such a treatment. If it works it leads to a drastic reduction in mosaicity with a substantially increased resolution. NCP NCP Harp et al., (1998) Acta Cryst. D54, _our_beamlines/id29/annealing

13 Cryoprotection and Radiation Damage Nevertheless, sometimes cryoprotection fails Annealing (cryoshutter; re-mounting) Annealing refers to a cyclic thermal treatment of cryocooled crystals, which are (sometimes repeatedly) warmed up and cooled again. Not all crystals survive such a treatment. If it works it leads to a drastic reduction in mosaicity with a substantially increased resolution. NCP NCP Harp et al., (1998) Acta Cryst. D54, _our_beamlines/id29/annealing

14 Cryoprotection and Radiation Damage Nevertheless, sometimes cryoprotection fails Annealing (cryoshutter; re-mounting) Annealing refers to a cyclic thermal treatment of cryocooled crystals, which are (sometimes repeatedly) warmed up and cooled again. Not all crystals survive such a treatment. If it works it leads to a drastic reduction in mosaicity with a substantially increased resolution. Dehydration (soaking; evaporation) In some cases, it has been reported a spectacular improvement of diffraction upon dehydration. One method consists in transferring the crystal to a dehydration solution containing a higher precipitant concentration. Another method involves the transfer of the crystal from the drop to a new crystallization drop which is equilibrated against dehydrating solution. Heras et al., (2003) Structure 11, NCP NCP Harp et al., (1998) Acta Cryst. D54, _our_beamlines/id29/annealing

15 Cryoprotection and Radiation Damage Nevertheless, sometimes cryoprotection fails Hyperquenching The cooling rate for pure water to remain vitreous is too high 10 6 K s - 1. At any rate, dipping crystals directly into liquid nitrogen would seem to be the optimal and fastest procedure. Nevertheless, most cooling occurs in the cold gas layer above the liquid. By removing this cold layer, cooling rates are increased to K s -1 (100-fold over best current practice). Glycerol concentrations (in protein-free aqueous samples) drop from 28% to as low as 6%. Warkentin et al., (2006) J. Appl. Crystallog. 39, Slow cooling: The complete removal of water from the surface of the crystal by swiping through oils (Paratone-N; perfluoroethers) in certain cases allows slow cooling of the crystals without harm. It is presently unknown whether slow cooling is a generally applicable cryocooling method. B. Rupp Chapter 8 In Biomolecular Crystallography

16 Determining factors Qualitative Quantitative x-ray source detector type time available cryoprotection crystal symmetry cell parameters resolution crystal reproducibility crystal quality: intrinsic low diffracting power B. Rupp Chapter 8 In Biomolecular Crystallography Protein crystals low relative intensity large unit cells large number of reflections significant intrinsic disorder: solvent content low relative intensity radiation damage sensitivity Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

17 Geometry of diffraction the rotation method Basic concepts Phenomenological interpretation of diffraction For geometric considerations, the diffraction from a crystal can be treated as a reflection of x-rays from planes in the crystal. All geometric considerations of diffraction can be rationalized using the concept of the Ewald sphere, which illustrates the Bragg s law in three dimensions. The radiation of wavelength λ is represented by a sphere of radius 1/λ centered on the x-ray beam. The crystal is represented by the reciprocal lattice, with its origin at the point on the ES where the direct beam leaves it. Each reciprocal-lattice point lies at the end of a vector perpendicular to the corresponding family of crystal planes and with a length inversely proportional to the interplanar spacing d. If the reciprocal-lattice point lies on the surface of the ES, the following trigonometric condition is fulfilled: λ = 2 d sinθ, which is precisely the Bragg s law. Therefore, when a RLP with indices hkl lies at the surface of the Ewald sphere, the interference condition for that particular reflection is fulfilled and it gives to a diffraction beam directed along the line joining the sphere centre with the RLP at the surface. x-ray beam Ewald sphere [stationary radiation (λ)] crystal reciprocal lattice (RL) RL origin Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

18 Geometry of diffraction the rotation method Basic concepts Phenomenological interpretation of diffraction Since crystals of macromolecules have unit-cell dimensions much larger than the wavelength λ of the radiation used the RL is densely populated in relation to the size of the ES. For any particular crystal orientation, only a few reflections can be in the diffracting position, but most of them will not lie on the surface of the ES. To observe the diffraction from a number of reflections, the RLPs have to be moved to the surface of the ES and this requires that the crystal be rotated to bring successive reflections into diffraction. Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

19 Geometry of diffraction the rotation method Basic concepts Phenomenological interpretation of diffraction Since crystals of macromolecules have unit-cell dimensions much larger than the wavelength λ of the radiation used the RL is densely populated in relation to the size of the ES. For any particular crystal orientation, only a few reflections can be in the diffracting position, but most of them will not lie on the surface of the ES. To observe the diffraction from a number of reflections, the RLPs have to be moved to the surface of the ES and this requires that the crystal be rotated to bring successive reflections into diffraction. Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

20 Geometry of diffraction the rotation method Basic concepts Phenomenological interpretation of diffraction Since crystals of macromolecules have unit-cell dimensions much larger than the wavelength λ of the radiation used the RL is densely populated in relation to the size of the ES. For any particular crystal orientation, only a few reflections can be in the diffracting position, but most of them will not lie on the surface of the ES. To observe the diffraction from a number of reflections, the RLPs have to be moved to the surface of the ES and this requires that the crystal be rotated to bring successive reflections into diffraction. The RLPs are arranged in densely populated planes. A family of planes perpendicular to the x-ray beam project onto a flat detector as a family of concentric circles. In the general case, the planes will be offset from the beam direction, and the intersection is a family of ellipses. B. Rupp Chapter 8 In Biomolecular Crystallography Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

21 Geometry of diffraction the rotation method Basic concepts Phenomenological interpretation of diffraction Since crystals of macromolecules have unit-cell dimensions much larger than the wavelength λ of the radiation used the RL is densely populated in relation to the size of the ES. For any particular crystal orientation, only a few reflections can be in the diffracting position, but most of them will not lie on the surface of the ES. To observe the diffraction from a number of reflections, the RLPs have to be moved to the surface of the ES and this requires that the crystal be rotated to bring successive reflections into diffraction. The RLPs are arrange in densely populated planes. A family of planes perpendicular to the x-ray beam project onto a flat detector as a family of concentric circles. In the general case, the planes will be offset from the beam direction, and the intersection is a family of ellipses. If the crystal is rotated, the start and end orientations of the plane form two intersecting ellipses with all reflections recorded between them in the form of a lune. The width of each lune varies around its circunference. They are widest in the direction perpendicular to the rotation axis, when the width is proportional to the rotation range per frame. Along the rotation axis, the width is very small. The larger the cell constants, the closer the lunes and the smaller the rotation increment needs to be.

22 Geometry of diffraction the rotation method Mosaicity and divergence Experimental/intrinsic broadening: crystal mosaicity (η) and beam divergence (δ) If practice, the ES itself has a finite width which is due to two factors: first, the x-ray beam has a finite divergence ( º on rotating anodes; µrad in synchrotron sources), and second x- ray is monochromated to a defined narrow wavelength window with a bandpass (δλ/λ) of the order 10-4 or smaller at synchrotron beamlines. δ η δ + η η δ Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

23 Geometry of diffraction the rotation method Mosaicity and divergence Experimental/intrinsic broadening: crystal mosaicity (η) and beam divergence (δ) If practice, the ES itself has a finite width which is due to two factors: first, the x-ray beam has a finite divergence ( º on rotating anodes; µrad in synchrotron sources), and second x- ray is monochromated to a defined narrow wavelength window with a bandpass (δλ/λ) of the order 10-4 or smaller at synchrotron beamlines. Real crystals are formed by a large number of individual growth domains which are slightly misaligned against each other: this is the origin of mosaicity. The spread in orientation causes the corresponding RLPs to remain in reflection condition over a longer rotation increment, and appear elliptically elongated perpendicular to the ES. ((δλ/λ) + δ) B. Rupp Chapter 8 In Biomolecular Crystallography

24 Geometry of diffraction the rotation method Partially and fully recorded reflections Mosaicity and divergence φ > (η+δ) frame n The total effect of divergence and mosaicity (rocking curve) has important consequences for the data collection strategy. In the rotation method, if the rotation range per frame is small compared with the rocking curve, individual reflections can be spread over several images: partial reflections. Conversely, reflections having all their intensity present on a single image are termed full reflections. partials (n+1) full (n) partials (n-1) Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

25 Geometry of diffraction the rotation method Partially and fully recorded reflections Mosaicity and divergence φ > (η+δ) frame n+1 The total effect of divergence and mosaicity (rocking curve) has important consequences for the data collection strategy. In the rotation method, if the rotation range per frame is small compared with the rocking curve, individual reflections can be spread over several images: partial reflections. Conversely, reflections having all their intensity present on a single image are termed full reflections. partials (n+2) full (n+1) partials (n) Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

26 Partially and fully recorded reflections Geometry of diffraction the rotation method Mosaicity and divergence The total effect of divergence and mosaicity (rocking curve) has important consequences for the data collection strategy. In the rotation method, if the rotation range per frame is small compared with the rocking curve, individual reflections can be spread over several images: partial reflections. Conversely, reflections having all their intensity present on a single image are termed full reflections. Two approaches can be considered: a wide-slicing approach based on collecting images wider than the rocking curve (usually of the order of 0.5º or more), and fine slicing when images are much narrower than the reflection width (<0.5º).The first approach renders thick datasets, and the latter thin datasets. φ = 0.75º φ = 0.25º º º º º φ > (η+δ) full spots overlaps high background contribution lower I/σ(I) φ < (η+δ) partials no overlaps low background contribution higher I/σ(I) Pflugrath (1999) Acta Cryst. D55,

27 Overlapping Geometry of diffraction the rotation method Mosaicity and divergence º φ = 0.75º For the wide slicing approach, a few factors must be taken into account for selection of the maximum rotation range per frame. In principle, it should be small enough to avoid overlap of neighboring lune. A simple formula has been derived to estimate φ max. φmax = (180d/πa) - η d: interplanar spacing (resolution) a: unit cell dimension along the beam direction η: mosaicity φ > (η+δ) It is very difficult to collect data from crystals which have one very large unit-cell dimension if the latter lies along the beam direction. Always try to orientate long unit cell axis along the rotation axis. Pflugrath (1999) Acta Cryst. D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

28 Geometry of diffraction the rotation method Background Background º φ = 0.75º One major disadvantage of the wide slicing relates to the fact that the rotation range is greater than the rocking curve. As a consequence, each reflection profile is overlapped on the background which accumulates during the whole exposure even when reflections do not diffract. The higher the contribution of the background the lower the Ι/σ(Ι) considering identical experimental conditions. φ > (η+δ) Bourenkov & Popov (2006) Acta Cryst. D62, Pflugrath (1999) Acta Cryst. D55,

29 Geometry of diffraction the rotation method Background Background One major disadvantage of the wide slicing relates to the fact that the rotation range is greater than the rocking curve. As a consequence, each reflection profile is overlapped on the background which accumulates during the whole exposure even when reflections do not diffract. The longer the crystal-to-detector distance, the better the Ι/σ(Ι) since the contribution of the background decreases with the square of the distance, whereas the peak intensity decreases less. Nevertheless, the distance should be adjusted to match the maximum resolution of the diffraction. The higher the contribution of the background the lower the Ι/σ(Ι) considering identical experimental conditions. Bourenkov & Popov (2006) Acta Cryst. D62, Bourenkov & Popov (2006) Acta Cryst. D62, Pflugrath (1999) Acta Cryst. D55,

30 Geometry of diffraction the rotation method Background Background One major disadvantage of the wide slicing relates to the fact that the rotation range is greater than the rocking curve. As a consequence, each reflection profile is overlapped on the background which accumulates during the whole exposure even when reflections do not diffract. The higher the contribution of the background the lower the Ι/σ(Ι) considering identical experimental conditions. The longer the crystal-to-detector distance, the better the Ι/σ(Ι) since the contribution of the background decreases with the square of the distance, whereas the peak intensity decreases less. Nevertheless, the distance should be adjusted to match the maximum resolution of the diffraction. Nevertheless, it is also possible that the physical size of a reflection increases with the crystal-to-detector distance. In some cases there may be a decrease in the Ι/σ(Ι) because of noise introduced by the detector. The beam size should not be much larger than the size of the crystal. Setting the beam cross-section to significantly smaller than the sample size should be justified. This is so because the diffraction intensity and the size of the spot profiles are affected when the beam is smaller than the crystal. The volume of the cryosolution should be minimized. Bourenkov & Popov (2006) Acta Cryst. D62, Pflugrath (1999) Acta Cryst. D55,

31 Geometry of diffraction the rotation method A brief summary thick vs thin datasets Thick datasets More fully recorded reflections Fewer partials More spatial overlaps Higher x-ray background More saturated pixels Lower total number of images. Thin datasets No fully recorded reflections Fewer spatial overlaps Lower x-ray background Few saturated pixels Higher total number of images. More time consumed (reading out the detector). In general, integration of reflections in each image is performed with software such as MOSFLM or HKL. Each reflection has a two-dimensional profile. Intensity estimates of reflections recorded partially in adjacent images are summed after integration. Each frame has an individual scale factor. Leslie (1999) Acta Cryst. D55, Otwinowski & Minor (1997) Methods Enzymol. 276, A three dimensional integration of reflections is performed with software such as XDS or d*trek. In these cases, individual scale factors are not applied to each portion of a reflection on separate images. Instead pixels from adjacent images are integrated to create full reflections which are then divided in scaling batches based on consecutive rotation ranges of a few images each. Kabsch (1988) J. Appl. Cryst. 21, Messerschmidt & Pflugrath (1999) J. Appl. Cryst. 20, Howart et al., (1987) J. Appl. Cryst. 20, Blum et al., (1987) J. Appl. Cryst. 20, Pflugrath (1999) Acta Cryst. D55,

32 Completeness total rotation range and blind region Total rotation range To use the Ewald sphere construction to understand which parts of reciprocal space are measured, it is easier to fix the resolution sphere of all the RLPs within a maximum resolution, and rotate the Ewald sphere. The region collected is the volume swept out by the leading and trailing surfaces of the sphere. In a rotation of 180º above, the lower boundary of the initial sphere sweeps out the volume coloured green & the upper boundary the light brown part. The dark brown part is measured twice, and the blue part not at all. Because of Friedel s law: F(-h,-k,-l) = F(h,k,l) this dataset is complete (apart from the blind region), but if complete anomalous differences are required (breaking the Friedel s law), then 180º + 2θ is required (unless there is symmetry). Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

33 Completeness total rotation range and blind region The blind region The blind region is smaller for short wavelenghts, as the Ewald sphere is flatter. Diffraction vectors close to the rotation axis will never pass through the sphere, even in a 360º rotation. The blind region may be filled in by collecting a second set of data, offsetting the crystal by at least 2θ max. or by symmetry (no in P1). Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

34 Completeness total rotation range and blind region Consequences of symmetry in the diffraction pattern If rotational symmetry is present, we can get a complete dataset with less than 180º rotation. By examination of the rotational symmetry of the intensities, the point group may be determined. The point group determines the total rotational range. An orthorhombic example Rotation of an orthorhombic crystal around one axis by 90º starting from an axis gives a complete dataset (except for the blind region). A 90º rotation starting at a diagonal collects the same 45º twice, and gives incomplete data. Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

35 Completeness total rotation range and blind region Consequences of symmetry in the diffraction pattern If rotational symmetry is present, we can get a complete dataset with less than 180º rotation. By examination of the rotational symmetry of the intensities, the point group may be determined. The point group determines the total rotational range. An orthorhombic example In general, the required rotation range depends on the crystal orientation. For example, in 3 symmetry the AU is a wedge 60º wide but spanning the space between the three fold axis along c and the plane ab. If rotated around the c axis, the 3 crystal requires only 60º of data, but if rotated around a vector in the ab plane 90º are necessary. A 90º rotation starting at a diagonal collects the same 45º twice, and gives imcomplete data. Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

36 Completeness total rotation range and blind region Incomplete datasets and splitting the rotational range φ = 90º ( φ mín. 180º) In general, a given fraction of the rotation range yields a larger fraction of data. For example, after 90º rotation when 180º is required, the completeness may reach about 65%. Characteristically, the high resolution data are completed first and the missing region at lowest resolution is only filled when the rotation approaches 180º. It is possible to obtain higher completeness without increasing the total rotation range covered by splitting the whole range into smaller parts. 45º of data colected twice but separated by a 45º gap will give much higher completenes than a single 90º pass. Use strategy programs (eg MOSFLM) to give you the smallest required range (eg 90º for 222, or 2 X 30º) and the start point. Simplest: collect 180º (or 360º in P1 to get full anomalous data) Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

37 Completeness total rotation range and blind region Incomplete datasets and splitting the rotational range φ = 90º ( φ mín. 180º) Systematic, correlated losses in reciprocal space has dramatic consequences in the electron density maps. Conversely, randomly missing parts are generally not a problem. In general, a given fraction of the rotation range yields a larger fraction of data. For example, after 90º rotation when 180º is required, the completeness may reach about 65%. Characteristically, the high resolution data are completed first and the missing region at lowest resolution is only filled when the rotation approaches 180º. Use strategy programs (eg MOSFLM) to give you the smallest required range (eg 90º for 222, or 2 X 30º) and the start point. Simplest: collect 180º (or 360º in P1 to get full anomalous data) B. Rupp Chapter 8 In Biomolecular Crystallography Dauter (1999) Acta Cryst D55, Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F

38 Anomalous diffraction data Brief introduction The presence of anomalous scatterers breaks the centrosymmetry of the reciprocal space and centrically opposed Friedel mates of each reflection, F(-h,-k,-l) and F(h,k,l), must be recorded. Of those, only the non-centric pairs can show intensity differences in the presence of anomalous scattering: anomalous differences. These anomalous differences, as well as dispersive differences between data recorded at different wavelengths, form the basis of the powerfull phasing technique MAD. Both Friedel mates must be collected: F(-h,-k,-l) and F(h,k,l) B. Rupp Chapter 8 In Biomolecular Crystallography

39 Anomalous diffraction data Brief introduction The presence of anomalous scatterers breaks the centrosymmetry of the reciprocal space and centrically opposed Friedel mates of each reflection, F(-h,-k,-l) and F(h,k,l), must be recorded. Of those, only the non-centric pairs can show intensity differences in the presence of anomalous scattering: anomalous differences. These anomalous differences, as well as dispersive differences between data recorded at different wavelengths, form the basis of the powerfull phasing technique MAD. Both Friedel mates must be collected: F(-h,-k,-l) and F(h,k,l) In the MAD case: datasets must be recorded at different wavelengths (2 to 4). Appropriate selection of these wavelengths is critical. Briefly, anomalous differences are largest at the absorption edge maximum (maximum of f ), and the dispersive differences are largest between the remote dataset and the dataset recorded at the inflection point of the edge jump (minimum in f ). In the SAD case: strictly, it does not require measurement of an X-ray absorption scan. The experiment must simply be conducted at or somewhat above the absorption edge of the scatterer. B. Rupp Chapter 8 In Biomolecular Crystallography

40 Anomalous diffraction data Brief introduction The presence of anomalous scatterers breaks the centrosymmetry of the reciprocal space and centrically opposed Friedel mates of each reflection, F(-h,-k,-l) and F(h,k,l), must be recorded. Of those, only the non-centric pairs can show intensity differences in the presence of anomalous scattering: anomalous differences. These anomalous differences, as well as dispersive differences between data recorded at different wavelengths, form the basis of the powerfull phasing technique MAD. Both Friedel mates must be collected: F(-h,-k,-l) and F(h,k,l) B. Rupp Chapter 8 In Biomolecular Crystallography

41 Anomalous diffraction data Very important considerations As in many cases the anomalous signal is very low (2-5%) special care must be taken to collect the best dataset. The sensitivity of the crystals to radiation damage suggests the the strategy of collecting anomalous data in so-called inverse beam geometry. After each rotation increment, the φ-axis is rotated 180º, and the corresponding inverse image containing the Friedel or Bijvoet mates is collected. This reduces potential difficulty in the later scaling stage. In addition, this strategy has the benefit that the anomalous difference can be determined rapidly which permits adjusting measurement parameters. Setting the crystal so that Bijvoet-related reflections are collected in the same frame is not always possible (low symmetry point groups). Care is needed to ensure exact centering of the crystal and that the total diffracting volume of the crystal is fully illuminated by the X-ray beam. High redundancy is better than long exposures. Reduce time & resolution if necessary to avoid radiation damage. High-resolution experimental phases are not necessary to obtain high resolution maps: is more efficient to collect medium- or low-resolution data for phasing and then use phase extension with high resolution data. Friedel completeness usually must be close to 100% for SAD phasing; for 2-3 λ MAD phasing lower completeness is acceptable. Recollect first part of data at end to assess radiation damage. B. Rupp Chapter 8 In Biomolecular Crystallography

42 Initial inspection of the diffraction image Once the crystal has been centered on the goniostat, a first diffraction image is recorded, generally with φ 1º rot. range. The crystal is exposed for a few seconds (synchrotron) or up to several minutes on in-house sources. Does the crystal diffract sufficiently or not? As a general rule, low resolution data (>4.0 Å) can be usefull for phasing, but often insufficient to refine a quality model. Both manual and automated model building, as well as refinement, become considerably more reliable once the resolution reaches or exceeds Å. Exposure time Too short exposure times means noisy images with poor resolution, and too long exposure times lead to saturated spots. Overexposing the crystal in an attempt to record highresolution data at the expense of saturating low-resolution data invariably leads to problems with phasing and to poor electron density maps. B. Rupp Chapter 8 In Biomolecular Crystallography

43 Initial inspection of the diffraction image Once the crystal has been centered on the goniostat, a first diffraction image is recorded, generally with φ 1º rot. range. The crystal is exposed for a few seconds (synchrotron) or up to several minutes on in-house sources. Does the crystal diffract sufficiently or not? As a general rule, low resolution data (>4.0 Å) can be usefull for phasing, but often insufficient to refine a quality model. Both manual and automated model building, as well as refinement, become considerably more reliable once the resolution reaches or exceeds Å. Anisotropy Diffraction can be highly anisotropic, that is, the resolution in certain reciprocal lattice directions is sometimes significantly different. A good practice is to record at least a second, 45º or 90º offset image in the initial inspection. Exposure time Too short exposure times means noisy images with poor resolution, and too long exposure times lead to saturated spots. Overexposing the crystal in an attempt to record highresolution data at the expense of saturating low-resolution data invariably leads to problems with phasing and to poor electron density maps. B. Rupp Chapter 8 In Biomolecular Crystallography

44 Initial inspection of the diffraction image Once the crystal has been centered on the goniostat, a first diffraction image is recorded, generally with φ 1º rot. range. The crystal is exposed for a few seconds (synchrotron) or up to several minutes on in-house sources. Does the crystal diffract sufficiently or not? As a general rule, low resolution data (>4.0 Å) can be usefull for phasing, but often insufficient to refine a quality model. Both manual and automated model building, as well as refinement, become considerably more reliable once the resolution reaches or exceeds Å. Anisotropy Diffraction can be highly anisotropic, that is, the resolution in certain reciprocal lattice directions is sometimes significantly different. A good practice is to record at least a second, 45º or 90º offset image in the initial inspection. Large unit cells When the cell constants are very large ( Å), the RLPs lie very densely and the reflections begin to overlap on the detector. In this case, distance can be increased (or a longer λ can be selected) to resolve the diffraction spots. Exposure time Too short exposure times means noisy images with poor resolution, and too long exposure times lead to saturated spots. Overexposing the crystal in an attempt to record highresolution data at the expense of saturating low-resolution data invariably leads to problems with phasing and to poor electron density maps. B. Rupp Chapter 8 In Biomolecular Crystallography

45 Initial inspection of the diffraction image Split reflections and multiple diffraction patterns. When spots visibly split into distinct diffraction patterns or streak, it is commonly an indication of multiple domains orientations in the crystal. If one of them dominates and can be indexed separately, it may be possible to obtain useful data. Epitaxial, non-merohedral twinning can also lead to separate but interpenetrating lattices. In certain cases, if even one of these patterns can be indexed independently the data may be usable. B. Rupp Chapter 8 In Biomolecular Crystallography

46 Initial inspection of the diffraction image Split reflections and multiple diffraction patterns. In the case of merohedral twinning reflections from the distinctly oriented domains will perfecly superimpose and the diffraction pattern will look unsuspicious and normal. When only two types of domains grow in a specific, perfect orientation to each other they are related by a unique symmetry operation (twin operator) and the twinning is defined as hemihedral. Mosaicity By far the largest source of finite spot width. As long as it is smaller than the rotation angle (1º or so) generally causes no problem. Larger values may be tractable with a thin slicing strategy (rotation angle º). B. Rupp Chapter 8 In Biomolecular Crystallography

47 Initial inspection of the diffraction image Ice rings? Ice rings may affect nearby reflections; nevertheless, frames with not too excessive ice rings can be processed if the crystal still diffracts. B. Rupp Chapter 8 In Biomolecular Crystallography

48 Some final notes on choices in data collection Which source? The most important properties of a source are: intensity, divergence, beam size and spectral distribution. The ideal source matches the properties of your crystal. Intensity A strongly diffracting crystal does not need SR. A good combination is a high-resolution native collected at a synchrotron source with derivatives collected at home. Divergence The lower the better; nonetheless, it does not help with a crystal with high mosaicity. Beam size The beam should not be significantly bigger than the crystal. If the beam is smaller than the crystal, different volumes of crystal may be in the beam depending on the orientation. This will adversely affect the relative intensities of the reflections. Wavelength and dispersion Ideally, tune the wavelength to optimize the anomalous signal, but do not neglect the use of anomalous phasing at nonoptimum wavelengths. Which crystal? Single, diffracts at high resolution, low mosaicity, large, good freeze the best worst!! Background Low background improves signal-to-noise ratio. Minimize the solvent around the crystal. For large, robust crystals, the loop can be smaller than the crystal. Thin, fragile crystals, need a large loop. Redundancy High redundancy produces more accurate data and allows reliable rejection of outliers. With CCDs detectors collection of 180º or even 360º of data is reasonably fast and simplifies the strategy. Completeness Is critical. Systematic omission of data will distort all maps. Use strategy programs when collecting data. Resolution Check completenes and other statistics in the high resolution shell. Sometimes you can position the detector a little closer the the apparent maximum resolution, provided there is no overlap. Width Thin slicing may improve data quality. Evans (1999) Acta Cryst. D55,

49 Bibliography Biomolecular Crystallography. Principles, Practice, and Application to Structural Biology Bernhard Rupp; Garland Science (2010) CCP4 Study Weekends Dauter & Wilson, (2001) Chapter 9 in Int. Tables for Crystallography vol F Acta Cryst. D55, part 10, Acta Cryst. D62, part 1, 1-123