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Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems?
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How many are we solving? http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALS.html
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$$ → photons photons → data data → models models → results results → $$ Breaking it down
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SecondsDescriptionPercent 104490 Assigned and available91% 42093 Shutter open40% 52684 Collecting (3026 images)50% 51806 Something else50% Operational Efficiency “representative” 8.3.1 user
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SecondsDescriptionPercent 51806 Something else100% 247s 45 Mounting22% 229s 37 Centering16% 179s 109 Strategizing38% 309s 37 Prepping24% Operational Efficiency “representative” 8.3.1 user
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NumberDescriptionPercent 446028 Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% 48 Published2% 8.3.1 in 2003 Turning data into models
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Top producing beamlines of the world http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALL.html Structures credited
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28 operating US beamlines ~10 11 ph/μm 2 exposure limit ÷ 2x10 9 ph/μm 2 /s ~ 100,000 datasets/year ÷ 1324 str in 2003 ~ 2% efficient What is the limit?
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DVD data archive
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Elven Automation Elves examine images and set-up data processing Elves run… mosflm scala solve mlphare dm arp/warp
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Apr 6 – 24 at ALS 8.3.1 Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators 56unique cells 5 KDa – 23 MDaasymmetric unit 0.94 – 32 Åresolution (3.2 Å)
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Apr 6 – 24 at ALS 8.3.1 Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures
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Overlaps Signal to noise Radiation Damage Why do structures fail?
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avoidable overlaps mosaicity phi detector c* b c a Ewald sphere
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unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere
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Overlaps Signal to noise Radiation Damage Why do structures fail?
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MAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results
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SAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results
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Minimum required signal (MAD/SAD)
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Is it real, or is it MLFSOM ?
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“We really need those high-resolution spots”
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Incremental strategy incremental_strategy.com merged.mtz auto.mat
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Overlaps Signal to noise Radiation Damage Why do structures fail?
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Distention of cryo with dose before
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Distention of cryo with dose after
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Water ring shift saturated sucrose in 250mM WO4 0 MGy
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Water ring shift saturated sucrose in 250mM WO4 184 MGy
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Water ring shift Resolution (Ǻ) Photons/s/pixel 7.5 3.8 2.5 1.9 1.5 saturated sucrose in 250mM WO4
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Protein crystal background
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Water ring shift Absorbed dose (MGy) Water ring position (Ǻ) GCN4-p1-N16A trigonal crystal crystal background saturated sucrose
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Water ring shift http://www.lsbu.ac.uk/water/amorph.html
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Water ring shift bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
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Water ring shift Hydrogen bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)
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Water ring shift Hydrogen bubbles? http://www.rcdc.nd.edu/compilations/Rxn.pdf “The hydrogen atom reacts with organic compounds by abstracting H from saturated molecules and by adding to centers of unsaturation, for example,
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Damage model system
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Data quality vs phasing quality Exposure time (min) Correlation coefficient
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Individual atoms decay at different rates Exposure time (min) Correlation coefficient to observed data
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Damage changes fluorescence spectrum Photon energy (eV) counts
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Damage changes fluorescence spectrum fluence (10 3 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 120 Exposing at 12680 eV Se cross-section at 12680 eV
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fluorescence probe for damage Absorbed Dose (MGy) Fraction unconverted Wide range of decay rates seen 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 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%
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“Can we do more with what we’ve got?”
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Interleaved Scheduling experiment queuebeamline Minor 30s Choe 120s Alberta 60s Choe 30s Minor 30s
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SuperTong
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“infinite capacity” sample carousel
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Carousel open
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CHL idlepos
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