1. Building a user-friendly beamline Aina Cohen and Paul Ellis

2. PDB structures May-July ’03 HOME SOURCES SYNCHROTRONS

3. MR v. PHASE MEASUREMENT MOLECULAR REPLACEMENT EXPERIMENTAL PHASES

4. EXPERIMENTAL PHASING MIR SIR MAD SAD AB INITIO

5. ANOMALOUS SCATTERERS

7. Optimised sulfur anomalous

8. Xenon anomalous

9. ANOMALOUS SCATTERERS

13. Krypton & Xenon ► Underutilized ► Tend to be relatively isomorphous ► Must be stable in cryoprotectant ► Good chance of useful derivative ► Quillin: large-to-small mutations KrXe “on” ratefastslow “off” ratefastslow bindingweakerstronger MAD?yes (K)no

14. ANOMALOUS SCATTERERS

15. Derivatizing with quick soaks ► Quick soaks can be much less time consuming than traditional long soaks or cocrystallizing ► High concentrations can be destructive of crystal order ► Ions used include: – Br -, I - – Cs +, Rb + – Sr 2+ – Gd 3+, Ho 3+, Sm 3+, Eu 3+ “traditional”“quick” heavy atom concentrationmMM soaking time≥ hours≤ minutes

16. ANOMALOUS SCATTERERS

18. Beamline parameters To cover the great majority of samples: »?

19. Beamline parameters To cover the great majority of samples: »Energy range: <6-17 keV

20. Beamline parameters To cover the great majority of samples: »Energy range: <6-17 keV »Fast energy moves

21. Beamline parameters To cover the great majority of samples: »Energy range: <6-17 keV »Fast energy moves »Resolution: ~1 eV

22. Beamline parameters To cover the great majority of samples: »Energy range: <6-17 keV »Fast energy moves »Resolution: ~1 eV »Spot size: 250 µm - <50 µm

23. SSRL BL9-2 + Good Flux + Useful Energy Range (6-16 keV) + Rapid Energy Changes

24. BL9-2 Oversubscribed

25. What Else Do We Have?

27. 9-1 & 11-1 9-1: 12500-16500 eV 11-1: 10500-15000 eV (9-2: 6000-16000 eV) + Good flux + Access to useful energy ranges -- 15 minutes to 1/2 hour at best to change energy

28. Energy Moves at Side Stations To change energy at BL9-1 or BL11-1 the following must be repositioned: monochromator theta table slide (theta) monochromator bend table vertical table pitch table horizontal table yaw Weight (kg) Q315 detector: 140 Positioners: ~340 Goniometer: ~70 Robotic Mounting System: ~90 Counter Weight: 72 Other Devices: ~45 Tabletop: 225 Total - ~ 1000 kg

29. Energy Tracking Requirements: To change energy from 12500 eV to 16500 eV, the experimental table at BL9-1 must move almost a meter (as measured from the end of the table). The mechanical components must be highly reproducible (better than 50 µm). Most of the effort to implement this system was in trouble-shooting and replacing components that were not to spec. Reliable Computer Controlled Positioners

30. Energy Tracking Requirements: Advanced Hardware Control System (DCSS) Beam Line Optics Experimental Hardware Detector System Fl. Detector Sensor A/D DHS NT DHS VMS GalilGalil GalilGalil Distributed Control System Server (DCSS) Central Database / Scripting Engine BLU-ICE GUI SGI BLU-ICE GUI SGI BLU-ICE GUI linux (remote) GalilGalil GalilGalil GalilGalil DHS linux DHS SGI (fileserver)

31. Fit these values to a polynomial function of monochromator theta. Creating the DCS script Optimize the beam line at different energies and record the motor positions Table Slide Position (mm) verses Monochromator Theta Difference Between Measured and Calculated Table Slide Positions (microns) TableSlide = – 2052.82 + MonochromatorTheta x 165.354 – MonochromatorTheta 2 x 0.219763 Write a Tcl/Tk script

32. Typical Se Edge Scans BL9-1 BL9-2

33. The Results

34. Further Automation of MAD Data Collection Reliable Computer Controlled HardwareAdvanced Control System (DCS) +

35. The Scan Tab

36. Automated MAD scans

37. What bottlenecks remain? Sample Mounting - Hutch access is time consuming - Crystals commonly lost due to human error - Data often not collected from the best crystal Data Collection - Detector Readout Time - Exposure Times of 10 seconds or more Unreliable Hardware - Difficult to maintain and trouble-shoot - Increases alignment time - Frequent break downs

38. SSRL Crystal Mounting System

39. Cassette Stores 96 Samples Mount 3 cassettes at the beam line Ship 2 cassettes inside a Taylor Wharton or MVE dry shipper Store 20 cassettes inside a Taylor Wharton HC35 storage device NdFeB ring magnet Standard Hampton pins

40. The Dispensing Dewar

41. Vertically opening gripper arms The Robot and Gripper Arms Z U θ1θ1 θ2θ2 Fingers to Hold Dumbell Magnet Tool Cryo-tong Cavity Epson ES553 Robot

42. Robot Demonstration

43. Crystal screening tab in BLU-ICE

44. Cassette Tool Kit Supplied (A) Sample Cassette and Hampton pins (B) Alignment Jig – to aid mounting pins into cassettes (C) Transfer Handle – for handling cold cassettes (D) Magnetic Tool – to mount pins in cassette & to test pin size (E) Dewar Canister – replaces stock canister in dry shipping dewars (F) Styrofoam Spacer – keeps single cassette in place when shipping (G) Teflon Ring – to support the canister in the shipping dewar Styrofoam box holds liquid nitrogen for loading cassettes

45. Cassette Tool Kit Demonstration

46. Vertically opening gripper arms Force Sensor Fingers to Hold Dumbell Magnet Tool Cryo-tong Cavity Force Sensor

47. Automated Calibration

48. View of the Robot System on 1-5, 9-1, 9-2 and 11-1 9-1 1-5 9-2 9-1 11-1 11-3

49. + 3x3 array of CCD modules + Active area of 315 x 315 mm + 51 micron pixel size One Second Readout ADSC Quantum-315 at BL9-2, BL9-1, BL11-1 & coming to BL11-3 This readout speed is 10 times faster than the Quantum-4

50. Beam Line11-111-39-29-17-11-5 Relative Intensity SPEAR40X15X20X15X7XX Relative Intensity SPEAR3200X75X200X75X35X100X Wavelength Range (Å)0.82-1.20.97-0.980.62-2.10.73-0.991.080.77-2.1 Energy Range (keV)10.5-1512.6-12.85.9-2012.5-16.511.55.9-16 Detector Readout (sec)111140-9010 Detector Size (mm)315 180-345188 SPEAR3 The relative intensities of the SMB crystallography beamlines (~1 Å and 0.2 mm collimation) for the current SPEAR at 100 mA (measured) and for SPEAR3 at 500 mA (estimated).

51. Unreliable Hardware

52. New Final Beam Conditioning System

53. 150 mm 75 mm

54. Solutions Sample Mounting with SSRL Robotic System + Screen up to 288 crystals without entering the experimental hutch + Feedback systems and calibration checks ensure reliable operation + Many crystals are quickly screened and data collected from only the best Data Collection Times Reduced + 1 second readout + higher intensities + better focus Upgraded Final Beam Conditioning System + Modular design enables rapid replacement of broken components + easy to maintain - compact, few cables, He tight + increased functionality, and feed back

55. Where do we go from here? Automated data collection from the best crystals Automatic structure solution Sample tracking database More feedback Automated beam line alignment and calibration Remote Access

56. The Macromolecular Crystallography Group Department of Energy, Office of Basic Energy Sciences The Structural Molecular Biology Program is supported by: National Institutes of Health, National Center for Research Resources,Biomedical Technology Program NIH, National Institute of General Medical Sciences and by the Department of Energy, Office of Biological and Environmental Research. SSRL Director Keith Hodgson SMB Leader Britt Hedman MC Leader Mike Soltis Günter Wolf, Scott McPhillips, Paul Ellis, Aina Cohen, Jinhu Song, Zepu Zhang, Henry Van dem Bedem, Ashley Deacon, Amanda Prado, Jessica Chiu, John Kovarik, Ana Gonzalez, John Mitchell, Panjat Kanjanarat, Mike Soltis, Hillary Yu, Ron Reyes, Lisa Dunn, Tim McPhillips, Dan Harrington, Mike Hollenbeck, Irimpan Mathews, Joseph Chang, Irina Tsyba, Ken Sharp, Paul Phizackerley

58. Ideal Hampton Pin Lengths for Cassette Hampton Mounted CryoLoop in a MicroTube Hampton CrystalCap Magnetic Hampton 18mm CrystalCap Copper Magnetic

59. Carrier for Two Modified ALS “Pucks” Carrier that Mounts in place of Cassette In Dispensing Dewar ALS Puck with SSRL-style Ring Magnets Inside ALS Tapered Pin