Center for Structures of Membrane Proteins 2006 Optimizing x-ray structure determination James Holton LBNL/UCSF April 6, 2006 Beamline staff Acknowledgments George Meigs Jane Tanamachi UCSF UC Berkeley Plexxikon MD Anderson Alberta Synchrotron Institute PRT Members Funding Optimizing structure determination How many are we solving? Optimizing structure determination How many are we solving? What is the limit? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? How many are we solving? How many are we solving?Jiang & R.M. Sweet (2004) How many are we solving? How many are we solving? How many are we solving? Breaking it down $$ photons Breaking it down $$ photons photons data Breaking it down $$ photons photons data data models Breaking it down $$ photons photons data data models models results Breaking it down $$ photons photons data data models models results results $$ Breaking it down $$ photons photons data data models models results results $$ Breaking it down $$ photons 2x10 11 photons/s $600,000/year 6x10 12 photons/dollar Breaking it down $$ photons photons data data models models results results $$ Breaking it down $$ photons photons data data models models results results $$ Breaking it down Operational Efficiency representative user SecondsDescriptionPercent Assigned to user- Operational Efficiency representative user SecondsDescriptionPercent Assigned to user Light available Operational Efficiency representative user SecondsDescriptionPercent Assigned to user Light available91% Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open40% Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open40% Collecting (3026 images) Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open40% Collecting (3026 images)50% Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open40% Collecting (3026 images)50% Something else Operational Efficiency representative user SecondsDescriptionPercent Assigned and available91% Shutter open40% Collecting (3026 images)50% Something else50% Operational Efficiency representative user SecondsDescriptionPercent Something else50% Operational Efficiency representative user SecondsDescriptionPercent Something else100% 45 Mounting Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 37 Centering Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% 109 Strategizing Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% 179s 109 Strategizing38% Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% 179s 109 Strategizing38% 37 Prepping Operational Efficiency representative user SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% 179s 109 Strategizing38% 309s 37 Prepping24% Operational Efficiency representative user SecondsDescriptionPercent Something else32% 10s 45 Mounting1% 30s 37 Centering2% 140s 109 Strategizing29% 0s 37 Prepping0% Operational Efficiency expert user SecondsDescriptionPercent Something else100% 10s 45 Mounting3% 30s 37 Centering7% 140s 109 Strategizing90% 0s 37 Prepping0% Operational Efficiency expert user $$ photons photons data data models models results results $$ Breaking it down $$ photons photons data data models models results results $$ Breaking it down Turning data into models NumberDescriptionPercent Images in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% Data sets in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% MAD/SAD in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% Published in 2003 Turning data into models NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% 48 Published2% in 2003 Turning data into models Top producing beamlines of the worldStructures credited Top producing beamlines of the worldx10 6 unique HKLs Top producing beamlines of the world Structures/10 20 photons Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? What is the limit? 28 operating US beamlines What is the limit? 28 operating US beamlines 2x10 13 ph/sWhat is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 13 ph/s Henderson et al (1990) What is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /sWhat is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /s = 400,000 datasets/year What is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /s ~ 200,000 datasets/year What is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /s ~ 100,000 datasets/year What is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /s ~ 100,000 datasets/year 1324 str in 2003 Jiang & R.M. Sweet (2004) What is the limit? 28 operating US beamlines ~10 11 ph/m 2 exposure limit 2x10 9 ph/m 2 /s ~ 100,000 datasets/year 1324 str in 2003 ~ 2% efficient What is the limit? NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% 48 Published2% in 2003 Turning data into models Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? DVD data archive Breaking it down $$ photons photons data data models models results results $$ Elves examine images and set-up data processing Elves run mosflm scala solve mlphare dm arp/warp Elven Automation Elves examine images and set-up data processing Elves run mosflm scala solve mlphare dm arp/warp Elven Automation Elves examine images and set-up data processing Elves run mosflm scala solve mlphare dm arp/warp How often does it really work? Elven Automation Apr 6 24 at ALS Elven Automation How often does it really work? Apr 6 24 at ALS Elven Automation 27,686images collected Apr 6 24 at ALS Elven Automation 27,686images collected 148datasets (15 MAD) Apr 6 24 at ALS Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators Apr 6 24 at ALS Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators 56unique cells Apr 6 24 at ALS Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators 56unique cells 5 KDa 23 MDaasymmetric unit Apr 6 24 at ALS Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators 56unique cells 5 KDa 23 MDaasymmetric unit 0.94 32 resolution (3.2 ) Apr 6 24 at ALS Elven Automation 148datasets Apr 6 24 at ALS Elven Automation 148datasets 117succeded Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures NumberDescriptionPercent Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% 48 Published2% in 2003 Turning data into models Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems? Why do structures fail? Overlaps Why do structures fail? Overlaps Signal to noise Why do structures fail? Overlaps Signal to noise Radiation Damage Why do structures fail? Overlaps Signal to noise Radiation Damage Why do structures fail? Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31 failed ~61 (0-231)hours 2 / 15MAD structures unavoidable overlaps detector unavoidable overlaps phi detector unavoidable overlaps mosaicity phi detector unavoidable overlaps mosaicity phi detector c* unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* Ewald sphere unavoidable overlaps mosaicity phi detector c* b c a unavoidable overlaps mosaicity phi detector c* b c a unavoidable overlaps mosaicity phi detector c* b c a unavoidable overlaps mosaicity phi detector c* b c a unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere Overlaps Signal to noise Radiation Damage Why do structures fail? Overlaps Signal to noise Radiation Damage Why do structures fail? Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures Apr 6 24 at ALS Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures What is a good exposure time? How much signal do I need? MAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results SAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results Minimum required signal (MAD/SAD) SAD phasing experiment Anomalous signal to noise ratio Correlation coefficient to published model MR simulation Signal to noise ratio Correlation coefficient to correct density corrupted data MR simulation Signal to noise ratio Correlation coefficient to correct density corrupted data MR simulation Rmsd from perfect search model ( ) Correlation coefficient to correct density corrupted model MR simulation Fraction of full search model Correlation coefficient to correct density trimmed model Is it real, or is it MLFSOM ? Background scattering Resolution () Electron equivalents The form-factor of the cryostream measured theoretical Background scattering Resolution () Photons/s/pixel Se edge with detector at 100 mm We really need those high-resolution spots Incremental strategy incremental_strategy.com merged.mtz auto.mat Incremental strategy incremental_strategy.com merged.mtz auto.mat We have a problem with non-isomorphism Proteins move Overlaps Signal to noise Radiation Damage Why do structures fail? Overlaps Signal to noise Radiation Damage Why do structures fail? thaw Radiation Damage Distention of cryo with dose before Distention of cryo with dose after Water ring shift saturated sucrose in 250mM WO4 0 MGy Water ring shift saturated sucrose in 250mM WO4 37 MGy Water ring shift saturated sucrose in 250mM WO4 80 MGy Water ring shift saturated sucrose in 250mM WO4 184 MGy Water ring shift Resolution () Photons/s/pixel saturated sucrose in 250mM WO4 Water ring shift Resolution () Photons/s/pixel saturated sucrose in 250mM WO4 Water ring shift Resolution () Photons/s/pixel saturated sucrose in 250mM WO4 Water ring shift Resolution () Photons/s/pixel saturated sucrose in 250mM WO4 Water ring shift Resolution () Photons/s/pixel saturated sucrose in 250mM WO4 Water ring shift Absorbed dose (MGy) Water ring position () saturated sucrose in 250mM WO4 Protein crystal background Water ring shift Absorbed dose (MGy) Water ring position () GCN4-p1-N16A trigonal crystal Water ring shift Absorbed dose (MGy) Water ring position () GCN4-p1-N16A trigonal crystal crystal background saturated sucrose Water ring shift Water ring shift Water ring shift bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995) Water ring shift Hydrogen bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995) Water ring shift Hydrogen bubbles?The hydrogen atom reacts with organic compounds by abstracting H from saturated molecules and by adding to centers of unsaturation, for example, Water ring shift Hydrogen bubbles?The hydrogen atom reacts with organic compounds by abstracting H from saturated molecules and by adding to centers of unsaturation, for example, Damage model system 67 consecutive data sets Data quality vs exposure Exposure time (min) Correlation coefficient Data quality vs exposure Exposure time (min) Data quality vs exposure Exposure time (min) Data quality vs exposure Exposure time (min) Resolution limit Data quality vs exposure Exposure time (min) R sym Experimentally-phased map Data quality vs phasing quality Exposure time (min) Correlation coefficient Specific Radiolysis of Selenomethionine 67 consecutive data sets Individual atoms decay at different rates Exposure time (min) Correlation coefficient to observed data Damage changes fluorescence spectrum Photon energy (eV) counts Damage changes fluorescence spectrum Photon energy (eV) counts Damage changes fluorescence spectrum Photon energy (eV) counts Damage changes fluorescence spectrum fluence (10 3 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol Exposing at eV Damage changes fluorescence spectrum fluence (10 3 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol Exposing at eV Se cross-section at eV Damage changes fluorescence spectrum Absorbed dose (MGy) Fraction unconverted 25mM SeMet in 25% glycerol Half-dose = 10.6 MGy Exposing at eV fluorescence probe for damage Absorbed Dose (MGy) Fraction unconverted Wide range of decay rates seen Half-dose = 41.7 4 MGy GCN4 in crystal Half-dose = 5.5 0.6 MGy 8 mM SeMet in NaOH Protection factor: 660% 94% Can we do more with what weve got? SecondsDescriptionPercent Something else100% 247s 45 Mounting22% 229s 37 Centering16% 179s 109 Strategizing38% 309s 37 Prepping24% Beamline Efficiency representative user SecondsDescriptionPercent Something else32% 10s 45 Mounting1% 30s 37 Centering2% 140s 109 Strategizing29% 0s 37 Prepping0% Beamline Efficiency expert user SecondsDescriptionPercent Something else100% 10s 45 Mounting3% 30s 37 Centering7% 140s 109 Strategizing90% 0s 37 Prepping0% Beamline Efficiency expert user Interleaved Scheduling experiment queuebeamline Minor 30s Choe 120s Alberta 60s Choe 30s Minor 30s cool hand luke Hampton Pin Syrrx Pin plastic Pin Yale Pin what we have here is failure to communicate SuperPin SuperTong Hampton PinSuper Tong Syrrx PinSuper Tong plastic PinSuper Tong Yale PinSuper Tong infinite capacity sample carousel 6-foot conveyor Carousel open Carousel cold CHL idlepos Beamline staff Acknowledgments George Meigs Jane Tanamachi Is it real, or is it MLFSOM ? Download Elves from: