Building a user-friendly beamline Aina Cohen and Paul Ellis

PDB structures May-July ’03 HOME SOURCES SYNCHROTRONS

MR v. PHASE MEASUREMENT MOLECULAR REPLACEMENT EXPERIMENTAL PHASES

EXPERIMENTAL PHASING MIR SIR MAD SAD AB INITIO

ANOMALOUS SCATTERERS

Optimised sulfur anomalous

Xenon anomalous

ANOMALOUS SCATTERERS

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

ANOMALOUS SCATTERERS

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

ANOMALOUS SCATTERERS

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

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

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

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

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

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

BL9-2 Oversubscribed

What Else Do We Have?

9-1 & : eV 11-1: eV (9-2: eV) + Good flux + Access to useful energy ranges minutes to 1/2 hour at best to change energy

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

Energy Tracking Requirements: To change energy from eV to 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

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)

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 = – MonochromatorTheta x – MonochromatorTheta 2 x Write a Tcl/Tk script

Typical Se Edge Scans BL9-1 BL9-2

The Results

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

The Scan Tab

Automated MAD scans

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

SSRL Crystal Mounting System

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

The Dispensing Dewar

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

Robot Demonstration

Crystal screening tab in BLU-ICE

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

Cassette Tool Kit Demonstration

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

Automated Calibration

View of the Robot System on 1-5, 9-1, 9-2 and

+ 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

Beam Line Relative Intensity SPEAR40X15X20X15X7XX Relative Intensity SPEAR3200X75X200X75X35X100X Wavelength Range (Å) Energy Range (keV) Detector Readout (sec) Detector Size (mm) 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).

Unreliable Hardware

New Final Beam Conditioning System

150 mm 75 mm

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

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

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

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

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