1. Automation of Macromolecular Crystallography at SSRL
The Australian Synchrotron
New Zealand Users Workshop
September 2003
Automation of Macromolecular Crystallography at SSRL
Robot still
Aina Cohen, Stanford Synchrotron Radiation Laboratory,
SSRL is funded by the US Dept. of Energy and the National Institutes of Health

2. PDB structures May-July ’03
Before you can think about meeting users needs, you have to know what their needs are…
Stats from the taken from three recent month of PDB releases to give you and idea of what sort of resources crystallographers are using.
This shows that about ¾ of all pdb depositions come from synchrotron data - so the first you can conclude is that synchrotrons are the preferred method collecting crystallographic data in most cases.
About 1020 total deposited structures – most don’t have complete information – 910 specified what x-ray source they used, and only 921 specified what method they used
87% of structures were from bacterial sources
85 se met mads – 72% of structures – in those structures the average ratio of protein / selium is 5.7 kDaltons /Se not counting n-terminal methionine (100 daltons / amino acid)
10/day world
HOME SOURCES
SYNCHROTRONS

3. MR v. PHASE MEASUREMENT MOLECULAR EXPERIMENTAL REPLACEMENT PHASES
You can break down synchrotron structures to two cases – those solved by molecular replacement and those solved by experimentally determined phases. About 71% of structures were solved using molecular replacement.
Generally molecular replacement does not put a lot of demands on a beamline but synchrotrons have great advantages over a home source for small or weakly diffracting crystals. Molecular replacement data can be collected at any reasonable wavelength (typically around 1 angstrom) and it is not strongly effected by energy resolution.
I am not going to talk much about molecular replacement because you can collect good data on almost any beamline.
MOLECULAR
REPLACEMENT
EXPERIMENTAL
PHASES

4. EXPERIMENTAL PHASING MIR MAD SAD AB INITIO SIR
This is not the case for the 29% of structures that were solved using experimental phases. Experimental phasing generally relies on tunability or benefits from it.
This is obviously the case for MAD which accounts for 64% of experimental phasing and SAD structures often optimize the energy to enhance of anomalous.
And I would should also point out that even even the MIR and SIR were generally MIRAS and SIRAS – almost every experimentally phased structure used some sort of anomalous phasing.
So since you can collect M. replacement data almost anywhere, the parameters of the beamline are generally set by anomalous phasing.
SIR

5. ANOMALOUS SCATTERERS
What anomalous scatterers did people use – and what energy range would give the best data.
This is a breakdown of the anomalous scatterers used in the experimentally phased structures – the blocks represents the energy range you would use to collect MAD data for those scatterers.
There is a critical block around Se – where you have the first, second and fifth most used anomalous scatterer. So whatever you do, you want to cover this range ( ).
But there are some other important ones such as Zn and Fe which cover a more substantial range (down to 7.1)
2 structures with Cd – Ta from Tantallium Bromide clusters,

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

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

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

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

10. 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
Resolution to get the most signal out of sharp edges
Normal cases – not interested in collecting data from crystals that will not survive long enough to give a complete dataset – so that generally limits you to crystals of about 50 microns or larger.

11. SSRL BL9-2 + Good Flux + Useful Energy Range (6-16 keV)
+ Rapid Energy Changes
Example of a real beamline that meets most of the needs is BL9-2
End station on a 2 Tesla wiggler – good flux
Collimating primary mirror - See that the mirror cuts the energy at about 16 keV
Double crystal monochromator and a toroidal focusing mirror
Double crystal monochromator with an encoder – self correcting – easy to change energy
So it is accurate and stable
Well optimized for MAD data collection
(all of our other wiggler beamlines were fixed wavelength)
As soon as it was commissioned - over subscribed
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Source 1.9 tesla wiggler on the 3 GeV spear ring – producing a spectrum extending well over 20 keV
Left see brightness of source
Right intensity after the optics
(mono fully tuned – cannot have any x-rays greater 5.9 x 3 = 17.7 – third harmonic
Losing intensity at low energy because of absorption from Be windows and Carbon filters

12. BL9-2 Oversubscribed

13. What Else Do We Have?
We saw how much our users liked 9-2, so we decided to try to give them some of same capabilities on our other beamlines.
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Other beamlines we had at that time were …
BL1-5 was also a focused double crystal monochromator station – however it had very limited flux
BL7-1 physically incapable of reaching the Se edge

14. What Else Do We Have? Chose to do this first on BL9-1 and BL11-1
Talking how we automated energy moves at 9-1
as a basic example of beamline automation at SSRL
Later on I will talk about some more sophisticated examples

15. 9-1 & 11-1 + Good flux + Access to useful energy ranges
-- 15 minutes to 1/2 hour
at best to change energy
9-1: eV
11-1: eV
(9-2: eV)
Both these beamlines are wiggler side stations
Read ranges
Both cover Se and mercury (the two top anomalous scatterers)
These lines use a single crystal side scattering monochromator
Advantage simple and cheap
And reflects the beam sideways which gives us space away from the BL9-2 beam and allowed us tto install an additional experimental hutch.
However, for MAD datacollection, is a disadvantage because as you change energy the beam moves across the experimental hutch
And this has to be tracked by the experimental table

16. 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: ~80
Robotic Mounting System: ~90
Counter Weight: 72
Other Devices: ~55
Tabletop: 225
Total - ~ 1000 kg
This slide Illustrates a bit more on why it took so long to change energy at BL9-1
the beamsize is small
And you have to move a long way as you change energy – over a meter across the experimental hutch
To track energy requires moving the table in horizontal, vertical, pitch and yaw – to keep the beam on the sample and parallel to the experimental table
Not easy to do with 2000 lbs of equipment on the table

17. Energy Tracking Requirements:
Reliable Computer Controlled Positioners
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.
This project had two parts
The idea was to write software to use the table motions to automatically track the beam as you change energy.
Obviously for that to work – the table motions have to be reproducible
Given the size of the beam – 250 microns Full Witch Half Max vert
2mm in horizontal
To have any hope of being able to track the beam with energy
Reproducible to 50 micron in vertical
400 in the horizontal
And this was not at all the case – which was one of the reasons we had trouble even moving the table manually.
First had to identify the problems with the table mechanisms and repair them –
Eventually, we go to a repeatability of about 20 microns in the vertical horizontal motors and 250 microns in slide
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).

18. Energy Tracking Requirements:
Advanced Hardware Control System (DCSS)
BLU-ICE GUI
SGI
BLU-ICE GUI
SGI
BLU-ICE GUI
linux (remote)
Distributed Control System Server (DCSS)
Central Database / Scripting Engine G a l i G a l i G a l i
DHS
SGI (fileserver)
DHS
VMS
DHS
linux
DHS NT G a l i G a l i
Now that we had the physical infrastructure worked out, how did we go about writing the software
Fortunately we had a very sophisticated control system on the SSRL beamlines – DCS
Developed by Tim and Scott McPhillips
+ has access to all the real motors and low level resources of each beamline
+ Passes requests from the BLU-ICE gui, for data collection and staff operations
+ incorporates a scripting engine (tcl/tk) for building complex devices
Detector
System
Beam Line
Optics
Experimental
Hardware
Fl. Detector
Sensor A/D

19. Creating the DCS script
Table Slide Position (mm) verses Monochromator Theta
Optimize the beam line at different energies and record the motor positions
Difference Between Measured and Calculated
Table Slide Positions (microns)
Fit these values to a polynomial function of monochromator theta.
How did we use the DCS scripting engine to automate the table motions at BL9-1
First we optimized the beamline optics and table positions at different energies and recorded the motor positions
Then we fit these values as a polynomial function of Mono theta
And created a script for a new motor called energy that uses these equations to calculate where to move each of the table positions for any given energy
Here is an example of how this worked for table slide.
Plot of the measured and best fit values of table slide verses mono theta
This shows the errors between the measured and calculated positions – the worst case is a little over 200 microns (which is about a 10th of the beam width)
Here is the best fit equation
This shows it incorporated into a tcl script
TableSlide = –
+ MonochromatorTheta x
– MonochromatorTheta2 x
Write a Tcl/Tk script

20. Typical Se Edge Scans BL9-1 BL9-2
We were now able move quickly to anywhere within the energy range of the beamline, but we still didn’t really know how useful the beamline would be for MAD.
We were actually quite surprised how well it worked. Here’s a comparison of a typical Se-edge scan at 9-1 on the top compared to 9-2 on the bottom. As you can see, the energy resolution of the single crystal monochromator at 9-1 isn’t quite as good as the double-crystal monochromator at 9-2, but there is still plenty of signal for phasing.
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For Se, Br, Kr but there is little practical difference for most samples - and several structures were solved on BL9-1 by MAD or SAD
11-1 resolution was very poor(c. 20 eV) however several structures were solved there using SAD
We have changed the monochromater crystal at BL11-1 to a different asymmetric cut which dramatically increased the energy resolution at Se.

21. The Results And it does actually work…
Our users have been routinely using this capability for over a year and here are some of the structures solved with SAD or MAD at beamlines 9-1 and 11-1 by our structural genomics group.
Mention Ashley Deacon

22. Further Automation of MAD Data Collection
Reliable Computer Controlled Hardware
Advanced Control System (DCS) +
Energy tracking is an important example of how the combination of reliable hardware and an advanced control system can give users access to new capabilities. What are some of the other things we have done?
Another simple example is enabling users to control the beam size. This can be very important to reduce background or to illuminate only part of a crystal. This is the setup tab on the BLU-ICE GUI. You can simply type in values for the horizontal and vertical beam size and hit “start” and the 8 beam definition slits will be moved accordingly.

23. The Scan Tab
Somewhat more complicated is energy selection for MAD data collection. Before automation, this step was a real problem for many users as it involved:
Entering the experimental hutch
Manually moving a fluorescence detector up to the sample
Closing the hutch and resetting the interlocks
Moving the beam energy to just above the absorption edge
Opening the experimental shutter
Manually inserting filters until the detector wasn’t saturating
Collecting the edge scan
Analyzing the edge scan using “chooch”
Reentering the hutch
Manually moving the detector away from the sample
Reclosing the hutch and resetting the interlocks again
REMEMBERING TO REMOVE THE FILTERS!
Copying the chooch results to the collection parameters
Users had problems at almost every stage…so we automated the whole process
The user only has to select the absorption edge and the system will do all the steps to collect and analyze the scan.

24. Automated MAD scans Here’s a short video illustrating this
Periodic Table – choose edge of the element that they are interested in, and click start – a fl. Scan will be automatically collected.
Table at BL9-1 getting in postion. The fl. Detector also moves into position and if necessary the beam is attenuated to prevent overloading the detector.
Once the edge-scan is complete, the program chooch will automatically run and calculate f prime and f double prime curves and the system will suggest the peak, infection and remote energies to use for data collection.
You can look at the curves and change the energies if you like, and then go to the collection menu and click update to copy the energies into the collection parameters.

25. 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
OK. Now we have fast energy moves and automated MAD scans, but there were still obvious areas for improvement. We identified three major bottlenecks.
Sample mounting. This isn’t just the time it takes to get a crystal onto the beamline. It also includes crystals lost by human error and collecting lower quality data because experimenters didn’t have the patience to screen many crystals or were afraid to take a crystal off the beamline once it was on.
Exposure and readout times. Ie. How long it takes to actually collect your data once things are set up
Unreliable hardware. Which wastes beamtime on repair and realignment.

26. 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
First going to discuss sample mounting

27. SSRL Crystal Mounting System
The obvious way to address the first bottleneck is crystal mounting robots. And many of these have been constructed around the world. This is a schematic representation of the SSRL system.
The samples are stored in a dewar filled with liquid nitrogen and are transferred to and from the goniometer by the commercial pick and place robot.
I am going to introduce you to the basic components of the system but don’t worry about trying to remember all the details because I will also show you a video that will make things very clear.

28. Cassette Stores 96 Samples
Standard Hampton pins
Mount 3 cassettes at the beam line
The system is based on a sample storage cassette which use ring magnets to hold 96 crystals mounted on Hampton pins.
We chose Hampton pins because this is what most experimenters that collect data at SSRL were already using
Three of the cassettes mount within the dewar at the beamline – making almost 300 samples available without reentering the hutch.
The cassettes are easy to ship as two cassettes fit into most standard dry shippers such as the MVE SC4 or The taylor wharton CX100
They are also easy to store – for example 20 cassettes fit inside a Taylor Wharton HC35 storage device
In these two pictures you can see that a transfer handle clips onto the top of the cassette making it easy to move.
NdFeB
ring magnet •
Ship 2 cassettes inside a Taylor Wharton or MVE dry shipper •
Store 20 cassettes inside a Taylor Wharton HC35 storage device

29. The Dispensing Dewar
This is cutaway view of the LN2 dewar showing the three cassette locations and the magnet tool used by the robot to manipulate the samples.
Using the transfer handle - it easy to load the cassettes into this dewar as these rods poke up to just beneath the surface of the liquid nitrogen.
Which goes into an conical hole in the bottom of the cassette – then the cassettes rotates easily until it locks into place on the indexing pin.

30. The Robot and Gripper Arms Vertically opening gripper arms
Epson ES553 Robot Z U
Vertically opening gripper arms θ1 θ2
Here is a detail of the robot. It is an off-the-shelf Epson model which makes the design relatively inexpensive and also very reliable.
This model was picked out by Dr. Paul Phiz our former group leader who did some of the initial work on this project.
We outfitted this robot with a pneumatic actuator and custom gripper arms.
Ends similar to traditional cryo-tongs – pair of fingers used to grip a magnet tool as shown in the previous slide.
Animation shows how the grippers open and close.
Cryo-tong Cavity
Fingers to Hold
Dumbell Magnet Tool

31. Robot Demonstration
Once at the beamline, you simply use the transfer handle to remove the cassettes from the shipping dewar and place them inside the beamline dewar. For most users this will be the last time an they need to enter the hutch before their beamtime is over.
This demonstrates how the robot mounts crystals – for the video – left out LN2 so you can see what is going on. The grippers pick up the magnet tool. This tool actually has a strong magnet at one end and a weaker magnet at the other. It used the strong magnet to pull the pin out of the cassette.
Crystal transported from under liquid nitrogen to the goniometer in under 4 seconds
Rise in temperature less than 5K and it arrived at a temperature below the cold stream (100K)
Entire mounting operation about 40 seconds.
Between operations the tongs reside inside the deicer. The deicer uses heated and dried compressed air to dry the tongs because they can pickup some condensation.
Immediately after the crystal is mounted and the tongs are in the deicer – an autocentering routine is used to center the loop. When the crystal is as large as the loop, this works for the large majority of cases.
When you are done, the robot will dismount the crystal. It starts by pre-cooling the tongs.
The tongs are out of LN2 for less than 7 seconds – sample returns to the dewar at a temperature of less than 110 K.
Now is puts the pin on the weaker side of the magnet tool.
Dismounting 45 seconds

32. Crystal screening tab in BLU-ICE
So we have all this hardware, but obviously hardware its useless without good software.
This shows a new tab added to blu-ice for crystal mounting and screening (the screening tab was developed and added to the blu-ice gui by Guenter Wolf – one of the JCSG staff programmers)
Information about the crystals contained in the cassettes can be uploaded from an excel spread sheet.
You can set this up for screening hundreds of samples without being at the beamline - return look at snap-shots decide which are the best crystals to collect.
Or you can use it interactively – it gives you the option to pause after operations to decide what to do next
We are working on tools that will automatically evaluate the snapshots from screening and give crystal quality stats – making screening even easier.

33. Cassette Tool Kit Supplied
is the tools we provide to the users to prepare for their beamtime.
Top right hand side is a snap shot of the excel spread sheet that users can use to enter information about their samples while loading cassettes
You can download the template from our website – and after you fill it out with your sample information, you can then upload it through the web and it will be ready for use when you arrive at SSRL.
Obviously none of this will work unless users can get cassettes and know how to load samples into them.
So we are providing our users is what we call a cassette kit which includes every thing necessary to prepare samples and ship them to SSRL.
Mention Irina
The people that have been using cassettes have also found the cassettes very convenient for storage and a lot easier to use than traditional canes and cryo-vials.
Will be commercially available in October
Styrofoam box holds liquid nitrogen for loading cassettes
(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

34. Feedback
Sensor
And in regards to the robot, the last thing I want to mention are the feedback systems that we have added to make it more reliable.
This shows a force and torque sensor that is attached to the robot gripper arms. It is a 6-axis sensor – measure the forces in the x, y, and z directions plus torques along these axes.
Mitch Miller….
This system provides feedback useful both for monitoring if any collisions occur with the gripper arms and it also very useful to calibrate robot position to the external componants such as the cassette location, dumbbell magnet and even the alignment of the grippers as they are attached to the robot arm.
The last video that I will be showing today will demonstrate how we use feedback for robot alignment.
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Made by ATI – uses silicon strain gages
ATI Industrial Automation force/torque sensor

35. Automated Calibration
This is Jinhu Song. He and Henry Van Dem Bedem have been working on the automation of robot calibration using this force sensor
The first step is for a staff member to lower the tongs inside the dispensing dewar – there is a brake on the top of the robot that releases the arm so you can do this.
From that point on, the process is all automated.
The first routine locates the dumbell magnet cradle. The reason why we do this first is that this is normally done when the dewar is filled with LN2 and it was difficult for our staff to see the cradle through the LN2 to manually insert the tongs inside the cradle.
It first locates the side of the cradle and gets the tongs parallel to it. It then finds the cradle center by touching each end – this process takes about four minutes so from this point on I have sped up the footage to 3 times the actual speed. It locates the top of the cradle.
When it is done with this – it picks up the dumbell magnet tool and proceeds to fine tune the calibration by lowering the tongs inside the cradle and moves in all directions to minimize the forces. This step takes about a minute.
The next thing it does is locate the center of the dumbell magnet tool. Once this is complete it does this as a check of the calibration. It would then normally go on to calibrate the other side of the magnet tool.
Next the cassette positions are calibrated and I have now sped up the video to 4 times the actual speed.
For this we use a special alignment casssette – that is made to tighter tolerances than our normal cassettes and has a slightly different shape.
It is now calibrating the x and y position of the cassette. Now the z position of the cassette is being calibrated as well as the z-offset of the opposite sides of the magnet tool. And finally it determines the rotational offset of the cassette position. It would now go on to calibrate the other two cassette locations.
To make sure that the sample goniometer is in the correct position in x, y, and z before mounting or removing a pin,
A laser displacement sensor is used to monitor their positions. This sensor looks at the variation of the distance to an angled part of a magnet as a function of phi.
If the pin is in the zero position for x and y then the readings should remain constant as phi is rotated to 4 positions. The distance reading of the sensor gives the z position.
This most difficult part of this process was to get the sensor to work reliability in the fog from the cryo-stream.
Finally, A second sensor is used to make sure the sample pin has been properly mounted or removed from the goniometer.
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Range 60 – 140 mm mm +-40mm

36. Impacts: Accelerating Difficult Structures
Yeast RNA Polymerase II (Roger Kornberg’s group, Stanford University)
Transcription of DNA into RNA - key step in gene expression underlying all aspects of cellular metabolism
Large 450 kDa complex; 10 subunits
10 years of data collection; refinement of crystallization and cryo-cooling conditions; derivatives
Regular access to BL9-2 significantly accelerated the screening process
During the last 6 months of last run, our in-house staff as part of the Joint Center for Structural Genomics used the system to screen over 3000 crystals – and during the last 18 hours of the run they managed to screen 246 crystals from 21 different protein targets.
Also during the last month of the run, just prior to the SPEAR3 shutdown, we made the system available to our general user community on BL11-1. Six independent research groups used the system. And in particular, Roger Kornberg’s group from the department of structural biology at Stanford University reported a major success.
Took 10 years from beginning to end – K group stated that access to BL9-2 in the last two years was critical to the eventual success.
Kornberg’s group has focused on transcription and transcription regulation, working with relatively large protein complexes such as RNA polymerase II, and these often form weakly diffracting crystals. Previously, the determination of a 3.0Å resolution structure of RNA polymerase II was significantly hampered due to the intensive screening effort required to identify suitable crystallization conditions. They had worked for almost 10 years to get suitable crystals and eventually solved the structure using data collected on BL9-2. They felt that the process would not have taken nearly as long if they would have had access to our robotic screening system back then.
Using the SSRL system last run, they were able to screen 130 crystals of polymerase complexes in about seven hours of beam time. In contrast, earlier in the year they manually screened 100 crystals in a 24 hour period and lost several crystals and much sleep in the process. During their automated screening run they managed to find one crystal that diffracted to 0.6 Å resolution beyond anything observed to date. From which they collected a complete data set.
Addition to helping remote users – extremely useful to our users as well as those that are very close by
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shows all atoms of an RNA polymerase caught in the act of gene transcription.
P. Cramer, et al. Science, 288, 640 (2000)

37. View of the Robot System on 1-5, 9-1, 9-2 & 11-1
11-3
1-5
11-1
We have now installed the system on all our beamlines and it should be in routine use next run.
9-1

38. 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
The second bottle neck I mentioned was the read-out and exposure time.

39. High Speed Detector: The ADSC Quantum-315
Installed at BL9-2, BL9-1, BL11-1 & coming to BL11-3
Fast readout (1 second)
10X faster than Quantum-4
3 x 3 array of CCD modules
Large active area (315 mm x 315 mm)
50 um pixels in full readout mode
100 um pixels in binned mode
We have addressed the readout time in the same way as other synchrotrons by going to CCD detectors.
This is a photo of the ADSC quantum 315 that we have installed on BL9-1, BL9-2 and BL11-1 and we have one on order for BL The readout time in bin mode is about a second which is about 10 times faster than our older CCD detectors and about 100 times faster than image plates.
16 bits – 2to 3 bits of noise – 65536

40. 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).
Beam Line
11-1
11-3
9-2
9-1
7-1
1-5
Relative Intensity SPEAR
40X
15X
20X 7X X
Relative Intensity SPEAR3
200X
75X
35X
100X
Wavelength Range (Å)
1.08
Energy Range (keV)
5.9-20
11.5
5.9-16
Detector Readout (sec) 1
40-90 10
Detector Size (mm)
315
188
The second half of the equation is the beam flux at the sample. As you know the SPEAR ring at SSRL is currently being rebuilt.
It’s a completely new lattice – it will have much lower emittance and higher current so all of our beamlines will have greatly improved flux.
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both because of the decreased emmittance and increased current and higher bend magnet fields, this will increase the flux at our beamlines by a factor of between 5X and 100X the present values.
I will just mention at this point that all of these changes will obviously lead to a much higher rate of data collection – even now users are spending a lot of time trying to back up their datasets before they leave SSRL. Our group has been working with the SanDiego super computing center to use their petabyte storage systems. Starting next run, users will be able to easily transfer their data to and from this facility.
The magnets and vacuum chamber for the 3 GeV SPEAR storage ring will be completely replaced, reducing the natural emmittance of the machine from 130 to 18 nm-rad and raising the stored current from 100 to 500 mA. This configuration increases focused beam flux for insertion device beamlines by an order of magnitude, side station beamlines by a factor of 5 and bending magnet beamlines by nearly two orders of magnitude.
at 100 mA x and 9-2 twice

41. 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
And very quickly I just want to mention the last bottle neck which was unreliable hardware.

42. Unreliable Hardware
We have already touched on this with regard to the table and energy tracking, but there are still other older components that we are working to replace.
This is a photo of our current final beam definition system. You can see that it consists of a bunch of components bolted together with a variety of cable, pneumatic tubes, and helium lines. It is bulky, messy, has a lot of helium sealing issues, and the individual components are relatively unreliable. And what is worse, despite the fact the fact the motors on these componants are tiny, we are use standard SSRL drivers which take up a lot of rack space.

43. New Final Beam Conditioning System
So a project and Paul and I our working on currently is a new compact modular system.
It consists of interchangeable functional “modules” inserted into slots in a single small He-tight box. The planned modules for the prototype unit are beam intensity monitors, foil inserters, beam definition slits, a fast x-ray shutter and a motorized scatter guard.
This modularity will permit quick replacement of faulty modules and make it easy to add new types of componants.
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Rather than the many cables and tubes attached to our older system, the main enclosure will only have He supply and return, a power cable, ethernet and a TTL signal to syncrhonize the shutter with the phi axis, and a signal from the sensor inside our beamstop.

44. New Final Beam Conditioning System
75 mm
This is an example cross section of a module – the shutter in this case.
All the electronics are on the left side, this includes all the drivers so we completely replace all the rack electronics and cables, and on the right hand side are the mechanical componants.
Each module is about 75x150 mm by about 30 mm deep. We plan to finish construction on the prototype system in a few months. And if you would like to know more about the design, you can ask Paul or I after the talk.
150 mm

45. 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
In summary, read slide

46. Where do we go from here? Remote Access
Ok that is where we are currently – where are we going from here – our group is working on a number of projects. There are too many of them for me to mention them all but some of them are
Automatic data collection – so you can setup the robot to not only screen the crystals but to actually do the entire data collection automatically from the best crystals. Going beyond this – our structural genomics group is working towards automating all steps in structure determination.
With this automation, we need a way to track samples, where they came from, screening results, and data collected, and experimental goals – again JCSG has been using similar data bases to track their projects and samples all the way from target selection to final deposited structures. And we are working on ways to make similar technology available to our wider community.
Like I mentioned earlier – we are adding More feedback – means that we are installing encoders on all the critical motions as well as more feed-back on the beam it self.
If we were building a beamline today we would install these from scratch because the only way to keep a mechanical system reliable is to have feedback.
And this connects to the last point because this will make it much easier for us to check beamline alignment and even have the beamline calibrate itself automatically.
What does all this automation ultimately allow us to do?
Well it allows us to stay in bed and collect data – or collect data from where ever you are in the world with internet access. I expect that this will be useful for all of you when you collect data from the US but even if you planned on using the new Australian synchrotron, remote access should be easier and cheaper.
Automated data collection from the best crystals
Automatic structure solution
Sample tracking database
More feedback
Automated beam line alignment and calibration

47. The Macromolecular Crystallography Group
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
None of this work was done by just one person – so I would like to thank the entire MC group and in particular our fearless leader Mike Soltis.
And the funding agencies –
And finally, I would like to thank the organizers for the opportunity to speak and all of you for your attention.
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.

48. Further reading
For more details of the SSRL mounting robot, please refer
to the published description of the prototype system:
Cohen et al. (2002). “An automated system to mount cryo-cooled
protein crystals on a synchrotron beamline, using compact sample
cassettes and a small-scale robot” J. Appl. Cryst., 35,    

49. Cassette Tool Kit Demonstration
The cassette kit arrives in a cardboard box with styrofoam packing material.
It includes all the tools you will need to load crystals into cassettes.
The first thing Amanda removed is a canister that can replace the canister of your dry-shipper incase the cassette fits inside yours too snuggly.
It also comes with a cassette filled with 96 hampton-style pins. And a transfer handle for moving cold cassettes.
The teflon ring she just laid down goes inside the dry shipper as extra support for the canister.
When you are done unloading your cassette kit, you should be sure not to throw the styrofoam box that the kit comes in away because it is also used. We recommend putting this box in a secondary container for safety.
To load cystals into your cassette, the first thing you need to do is fill the styrofoam box with LN2 - it takes about 14 liters of LN2 to do this so she will need to make a few trips. You should not lift the box once it contains LN2. And when you are done, you should place the lid back on the box to prevent icing.
She is now going to load a few more crystals into a cold cassette. So first she uses the tranfer handle to remove the cassette from the dry shipper and she places it inside the sytrofoam box – it has a notch in the side to hold the handle.
When she is ready to load the next crystal – she removes the lid from the box and places a guide tool over the port – this tool works if the adjacent ports are filled with pins or empty.
She then puts a hampton pin on the magnet wand tool and picks up the a crystal inside the loop on the pin.
She then flash freezes the crystal while loading it into the cassette.
guide tool to make using the wand tool easier and both these tools are included in the cassette kit.
And when you are done, you simply use the transfer handle to put the cassette back in the dry shipper. The dry shipper holds two cassettes, but if you are just sending one, we provide a piece of styrofoam packaging material to keep it in place during shipping.
The cassette is now ready to be shipped to SSRL for data collection.
I should mention that initially we are loaning cassette kits to many of our user groups – but they will also be comercially available in October. At that time information on how to buy them will be available on our website.