docRIP and RIPAS phasing with the help of SHELXC/D/E
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RIP and RIPAS phasing with the help of SHELXC/D/E
Radiation Damage Induced Phasing Max Nanao ACA 2016 RADIATION DAMAGE INDUCED PHASING REVIEW X-rays induce changes to macromolecular crystals via radiation damage Global changes/damage Molecular rotations Unit cell changes Specific changes/damage Lowered occupancy of heavy atoms (Se, Br, Hg…) Breakage of S-S bonds Decarboxylation Dehydroxylation RNAse, +/- 8 σ Trypsin, +/- 8 σ SPECIFIC DAMAGE CAN BE USED TO PHASE MACROMOLECULES: PHASES FOR FREE! 01/08/16 3 RIP PHASING PREMISE + Hg Hg SIR RIP Page 4 l Title of Presentation l Date of Presentation l Author SYNCHROTRON RIP – A ONE MINUTE REVIEW Specific radiation damage used in an “inverted SIR” experiment Specific damage àSignal Disulfides, metals, carboxylates General damageàNoise Which is the native? Un-damaged = “Derivative” aka “Before damage” Damaged = “Native” aka “After damage” Substructure and phases can be determined experimentally août 1, 2016 5 SOME RIP POSITIVES No modification of the protein necessary Works for disulfides and/or HAs (ok, a little modification) Can be performed on a fixed energy beamlines High degree of isomorphism if dose is managed (same xtal, same position) RIP PHASING SCHEME “burn” “after” “before” Calculate Differences, and statistics (SHELXC) Determine structure of damage (SHELXD) Calculate phases of whole protein, build model (SHELXE) 01/08/1 6 7 RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 8 RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 9 RIP substructure can be improved iteratively by difference Fourier based substructure revision: “recycling” (SHELXE) RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 10 RIP substructure can be improved iteratively by difference Fourier based substructure revision: “recycling” (SHELXE) RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 11 Early RIP maps revealed many small negative peaks in unexpected places à Fafter overscaled! RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 12 Early RIP maps revealed many small negative peaks in unexpected places à Fafter overscaled! overscaled!à Many substructures not solvable! RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 13 For substructure determination: down scale “After” dataset by K For Phasing: scale can be refined (e.g. in SHARP) RIP ISSUES AND DIFFICULTIES No downscaling Radiation damage substructures are more complex Large Negatively occupied sites Scaling between before and after datasets can be difficult SHELXD CC K 01/08/16 14 RIP ISSUES AND DIFFICULTIES Radiation damage substructures are more complex Phase error vs. scale Large Negatively occupied sites Scaling between before and after datasets can be difficult 01/08/16 15 RIP substructure can be improved by adjusting the before/after scale (SHELXC DSCA) IMPORTANT SHELX KEYWORDS FOR RIP SHELXC RIP RIPA DSUL DSCA SHELXE -b Revised heavy atom sites -h Heavy atoms in native (-z) still untested (-a) may reduce need for recycling? 01/08/1 6 16 THE MANY FACES OF RIP Classic RIP A-burn-B Classic UV RIP A-UV-B (Laser or LED) Segmented RIP Classic RIP Classic UV RIP Segmented RIP Serial X-ray RIP Collect large dataset, split up into sub datasets Serial X-ray RIP Serial UV-RIP Collect sub datasets from many crystals High intensity RIP Serial UV-RIP Collect sub datasets from many crystals High intensity RIP At high fluence (i.e. XFEL) , heavier atom occupancy preferentially reduced 01/08/16 17 THE MANY FACES OF RIP Classic RIP A-burn-B +Easy -Difficult to estimate burn dose -Diffractive life of crystal is wasted Classic UV RIP A-UV-B (Laser or LED) Segmented RIP Collect large dataset, split up into sub datasets Serial X-ray RIP Collect sub datasets from many crystals 01/08/16 18 Burn: With accurate estimates of absorbed dose (calibrated diodes, RADDOSE) and/or online microspectrophotometers, this process can be simplified THE MANY FACES OF RIP Classic RIP A-burn-B Classic UV RIP A-UV-B (Laser or LED) +Improved signal Less noise from global damage -Fewer groups affected (no carboxylates) Different wavelength of laser? -Difficult to estimate UV burn Interleave X-ray datasets with UV burns -Penetration depth is small Smaller xtals (see serial approach) -Alignment of light source can be difficult UVLEDs Acta Crystallogr D Struct Biol. 2016 Mar;72(Pt 3):395-402. Radiation-damage-induced phasing: a case study using UV irradiation with light-emitting diodes. de Sanctis D, Zubieta C, Felisaz F, Caserotto H, Nanao MH. 01/08/16 19 THE MANY FACES OF RIP Classic RIP A-burn-B Large oscillation dataset Classic UV RIP A-UV-B (Laser or LED) “before” dataset “after” dataset Segmented RIP Collect large dataset, split up into sub datasets +No burn +Signal:Noise can be optimised computationally +Diffractive life not wasted -Computationally intensive 01/08/16 20 RIP THE MANY FACES OF RIP Classic RIP A-burn-B Classic UV RIP A-UV-B (Laser or LED) Segmented RIP Collect large dataset, split up into sub datasets Serial X-ray RIP Collect sub datasets from many crystals +Dose multiplier! +Extend useful range of crystal by RIP (needed single xtals big enough for multiple datasets before) -Non isomorphism, merging difficult -Long collection times (diffractive map, sub-dataset collection) 01/08/16 21 MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines. Zander U, Bourenkov G, Popov AN, de Sanctis D, Svensson O, McCarthy AA, Round E, Gordeliy V, Mueller-Dieckmann C, Leonard GA. Acta Crystallogr D Biol Crystallogr. 2015 Nov;71(Pt 11):2328-43 Repeat until specific damage is accumulated (normally at 2 MGy) THE MANY FACES OF RIP Classic RIP A-burn-B Classic UV RIP A-UV-B (Laser or LED) Segmented RIP Collect large dataset, split up into sub datasets Serial X-ray RIP Collect sub datasets from many crystals Serial UV-RIP Collect sub datasets from many crystals High intensity RIP At high fluence (i.e. XFEL) , heavier atom occupancy preferentially reduced 01/08/16 22 “before”/low fluence “after”/high fluence Towards RIP using free-electron laser SFX data. Galli L, Son SK, White TA, Santra R, Chapman HN, Nanao MH. J Synchrotron Radiat. 2015 Multiwavelength anomalous diffraction at high x-ray intensity. Son SK, Chapman HN, Santra R. Phys Rev Lett. 2015 Towards phasing using high X-ray intensity. Galli L, Son SK, Barends TR, White TA, Barty A, Botha S, Boutet S, Caleman C, Doak RB, Nanao MH, Nass K, Shoeman RL, Timneanu N, Santra R, Schlichting I, Chapman HN. IUCrJ. 2015 THE MANY FACES OF RIP Classic RIP A-burn-B Classic UV RIP A-UV-B (Laser or LED) Segmented RIP Collect large dataset, split up into sub datasets Serial X-ray RIP Collect sub datasets from many crystals Serial UV-RIP Collect sub datasets from many crystals High intensity RIP At high fluence (i.e. XFEL) , heavier atom occupancy preferentially reduced 01/08/16 23 Towards RIP using free-electron laser SFX data. Galli L, Son SK, White TA, Santra R, Chapman HN, Nanao MH. J Synchrotron Radiat. 2015 Multiwavelength anomalous diffraction at high x-ray intensity. Son SK, Chapman HN, Santra R. Phys Rev Lett. 2015 Towards phasing using high X-ray intensity. Galli L, Son SK, Barends TR, White TA, Barty A, Botha S, Boutet S, Caleman C, Doak RB, Nanao MH, Nass K, Shoeman RL, Timneanu N, Santra R, Schlichting I, Chapman HN. IUCrJ. 2015 CONCLUSIONS Comclusions Specific radiation damage can be used in various types of MX experiments to determine phases de novo RIP is similar but not identical to SIR and requires that we take specific measures SHELX has been specifically modified to work with RIP Page 24 l Title of Presentation l Date of Presentation l Author THE MANY FACES OF RIP ACKNOWLEDGEMENTS Classic RIP A-burn-B Daniele de Sanctis Raimond Ravelli Classic UV RIP A-UV-B (Laser or LED) George Sheldrick Segmented RIP Elspeth Garman Collect large dataset, split up into sub datasets Andrew McCarthy Serial X-ray RIP Lorenzo Galli Collect sub datasets from many crystals Serial UV-RIP Sang-Kil Son Collect sub datasets from many crystals HenryRIP Chapman High intensity Nicolas Foos At high fluence (i.e. XFEL) , heavier atom occupancy preferentially reduced +Novel phasing method for FELs -Still theoretical 01/08/16 25