Collecting Diffraction Data at the IMCA-CAT Beamlines

Welcome to the general instruction manual for data collection at the IMCA-CAT facilities at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 USA. We are the Industrial Macromolecular Crystallography Association Collaborative Access Team, and we provide crystallographic facilities, primarily for macromolecular diffraction experiments. We are funded through a contract between the Industrial Macromolecular Crystallography Association (IMCA) and The Center for Advanced Radiation Sources (CARS) at The University of Chicago (UofC). The contract provides for the majority of the beamtime at the IMCA facilities to be used by the macromolecular crystallographic groups in the twelve companies that comprise IMCA; the remainder is used by researchers at UofC and elsewhere. This manual is designed to help you understand what is involved in getting access to the crystallographic facilities of IMCA-CAT and to help you acquire and process your diffraction data. Comments on this manual and all other IMCA-CAT documentation is always welcome. Thanks!

Steps in getting access to the IMCA-CAT facilities

• Determine whether your institution is a candidate for use of the IMCA facilities.
  The IMCA facilities are available first to the twelve companies of the IMCA consortium. Each of the companies is invited to use roughly one-thirteenth of the user beamtime during any APS "run", and the allotment of time is determined by mutual agreement and compromise, about six weeks in advance of a run. Each company has designated one scientist to act as liaison between the other scientists in his or her group and the staff for these purposes. IMCA company beamtime can be either proprietary or non-proprietary. The APS charges a fee for proprietary time, and non-proprietary time is considered public research.
  
  Users from commercial entities other than the twelve IMCA companies will not, in general, be offered access to the IMCA-CAT facilities (except through the General User Program; see below). No proprietary experiments will be allowed on the IMCA-CAT facilities except those sponsored by the twelve IMCA companies.
  
  Academic and governmental scientists can request access through five mechanisms:
  • The most effective way for non-IMCA scientists to get beam-time at IMCA-CAT is through the General User Program. The official General User Program for IMCA-CAT's insertion-device beamline, 17-ID, is now in operation; the General User Program for IMCA-CAT's bending-magnet beamline, 17-BM, is not yet operational. On 17-ID, IMCA-CAT is performing its own reviews of general user projects, but it is using the APS's centralized proposal submission system. A user can submit a proposal to this system at http://www.aps.anl.gov/user/beamtime/prop_submission.html. Most proposals to IMCA-CAT's General User Program are submitted for a single visit, but project-level (multi-visit) proposals will also be entertained. Typically the proposal is sent at least a month before the end of an APS beam run, and is then be considered for ranking and scheduling for the following APS run.

    Currently the deadline for submission is set by the CAT itself; by late 2001, IMCA-CAT's submission deadlines will be rendered consistent with that of the APS's centralized system.

  • The IMCA companies sometimes allow academics to use a fraction of their beam-time for the academics' own use. The representatives and alternates on the IMCA Supervisory Board are the arbiters of this type of usage; the staff only plays a role in safety oversight and user training in this case.
  • UofC gets one-thirteenth of the user beamtime on the IMCA-CAT beamlines, just as each company gets one-thirteenth of the beamtime. The IMCA-CAT Director has discretionary authority over this time. The majority of it is currently being allocated to two projects, one being an in-house project funded by the National Institute of Standards and Technology and the other being a collaboration with scientists at the Center for Advanced Research in Biotechnology and The Institute for Genomic Research; this project is funded by the National Institutes of Health. A few days a year remain unallocated, and are available to qualified academics.
  • The IMCA CAT staff is allowed a few days of beamtime in each run for commissioning studies associated with new equipment or new types of experiment. Some of this time involves actual diffraction experiments. The IMCA Supervisory Board has instructed the staff to mount samples of its own during these periods, or, if the staff has no samples appropriate for these experiments, to offer the time to IMCA companies. If the companies have no appropriate samples either, then this time can be offered to academic or governmental scientists who do have the right kinds of samples. Contact the CAT Director for details.

  You may wish to be aware that other groups at the Advanced Photon Source are conducting macromolecular crystallographic experiments, including Structural Biology Center Collaborative Access Team (Sector 19, SBC-CAT) and the Biological division of the Consortium for Advanced Radiation Sources (Sector 14, BioCARS).

• Decide whether IMCA's facilities are well-suited to the experiments you wish to do.
  IMCA-CAT's facilities are designed primarily for macromolecular crystallography, but they work well for small-molecule single-crystal diffraction studies as well. We will shortly be investigating the practicality of using the experimental equipment for powder diffraction; we will report on the results of these experiments after they are complete. IMCA-CAT has an insertion-device beamline featuring a standard APS Undulator A, and a standard APS bending-magnet beamline as well.

  The insertion device line, 17-ID, is in routine operation by IMCA company scientists, UofC researchers, and general users. On 17-ID we provide monochromatic X-rays through a cryogenically cooled Si(111) monochromator arrangement, and the beam is currently focused in the vertical direction by means of a one-meter-long, cylindrically bent, Pt/Pd-coated ULE mirror. Flux on a 0.2x0.2mm sample is about 10¹² photons/sec at typical energies.

  Monochromatic and MAD experimental capabilities over a range from 6.4 to 13.5 KeV are currently available, and this range will be extended soon. Horizontal focusing will be added to the facilities within the next year. The experimental equipment available on this beamline is a single-element, 165-mm Mar Research CCD mounted on a standard Mar base with a single spindle and a computer-controlled sample-to-detector distance adjustment, an Oxford Cryosystems "Cryostream" sample cooling unit, a Bicron fluorescence detector and associated electronics, and computer and electronic support for all of the above.

  The IMCA-CAT bending-magnet beamline, 17-BM, is currently available to IMCA corporate users and UofC scientists. This line will be declared operational soon, and the General User program on this line will be activated about a year after that declaration. The bending-magnet line provides horizontal focusing through a sagittally bent second crystal in the monochromator and provide vertical focusing through another 1-m Pt/Pd-coated ULE mirror. Another Mar USA 165mm CCD system and Marbase are installed on this line, along with electronics, computer hardware, and software, a simple fluorescence detection system for setting up multi-wavelength experiments, and an Oxford Instruments "CryoJet" sample-cooling system.

• Secure institutional access to the APS.
  Any institution--corporation, university, or government agency--whose employees or students wish to collect data at any beamline at the Advanced Photon Source must secure institutional access to the APS. This is accomplished by arranging for the institution to sign a User Agreement with the APS; under this user agreement the institution agrees to abide by rules and conditions set up by the Advanced Photon Source, Argonne National Laboratory, and the U.S. Department of Energy. The APS User Office can help your institution get and sign this user agreement. No user may be trained and allowed to do hands-on work at the APS unless his or her institution has signed this agreement. Your institution may already have a user agreement in place; a list of institutions that have signed agreements is available. If not, directions for getting the agreement signed are available as part of the APS's general instructions on access to the site.
• Get scheduled.
  As mentioned above, the scheduling for IMCA company time during any given APS run (typically about nine weeks long, with three to five week gaps in between) is done several weeks before the beginning of the run. The staff acts as brokers and negotiators in this process. If you seek access during UofC time or staff commissioning time, and you feel you qualify under the conditions described above, contact the CAT Director. The scheduling of these periods is done with a similar amount of lead time.

Preparing for a Visit to the IMCA-CAT Facilities

In preparing for a visit to the IMCA-CAT facilities, it is useful to recognize what resources are available on-site and what resources you'll be expected to bring from home. The IMCA-CAT facilities are designed for macromolecular crystallography, and a large fraction of the equipment and supplies that any protein crystallographic group accumulates are available locally. But because IMCA-CAT's facilities are relatively new and the in-house research efforts are only now getting off the ground, some resources you might take for granted as being available in a crystallography lab may be absent at IMCA-CAT.

Accordingly, you may wish to examine the IMCA-CAT Equipment List so you can bring with you anything you need that does not appear on the list. The list is changing rapidly; call the staff if you are unsure about a particular item.

There are supplies available at the IMCA-CAT facilities as well; you should consult the Supply List to help you assemble your list of consumables.

Aside from assembling a list of supplies and equipment to bring along to supplement what the CAT provides, there are some steps you can take to prepare for your visit:

• Read the IMCA-CAT documentation, beginning with the IMCA-CAT Sector Orientation Guide, this document, and the CAT's Frequently Asked Questions list.
• Survey the IMCA-CAT Safety Plan.
• Think about what experiments you want to perform and how you wish to carry them out. This sounds obvious, but not everyone does it. The more carefully you've planned your trip, the more likely you are to accomplish your goals--especially in terms of experiments that you do at 2 a.m.
• Call or e-mail your CAT user representative (refer to the current beam schedule to determine which is your user rep) assigned to you to summarize your special data-collection needs. What we need to know about are the unit cell sizes (if known), the absorbing atoms present (if any), the crystal sizes, and any unusual requirements. Unit cells up to about 500 Ångstroms can be accommodated without special preparation; so can crystals with volumes down to about 10^-5 mm³.
  If you haven't done this by the week before your visit, the CAT user rep will call and ask you for the summary. You can call or e-mail any of the IMCA-CAT user support staff members, or you can target the person who is assigned to handle your beam time. They are:
  • Lisa Keefe, CAT Director; 630-252-0544, pager 630-314-0544, keefe@anl.gov
  • Rong Huang, Beamline Scientist; 630-252-0522, page 630-314-0522, rhuang@anl.gov
  • Kevin Battaile, Crystallographer; 630-252-0529, pager 630-314-0529, battaile@anl.gov
  • Anne Mulichak, Crystallographer; 630-252-0525, pager 630-314-0525, mulichak@anl.gov
  • Davita Findlay, User Support Associate, 630-252-0530, pager 630-314-0530, findlay@imca.aps.anl.gov
  • Xiaochun Yang, Software & Scientific Support Specialist, 435-A006, 630-252-7377, pager 630-218-3009, yangx@imca.aps.anl.gov
  • Ann Bertling, CAT-Coordinator; 630-252-0520, bertling@anl.gov
• Prepare your samples for transport. The IMCA-CAT Safety Plan permits you to bring samples on-site either by hand-carrying them or by shipping them via common carrier. Detailed instructions for hazardous and non-hazardous shipping are available. There are some restrictions:
  • Samples packed in liquid or solid propane must be shipped by common carrier via ground transportation; the APS will not allow propane-packed samples to be received if they have been shipped by air. Propane-packed samples must be shipped through the Central Receiving Facilities of Argonne National Laboratory in Building 5 of ANL. Generally we can get deliveries from Building 5 on working days within a half-day of their arrival. Check with Lisa Keefe for further information on propane samples.
  • Samples that have been pre-cooled in liquid nitrogen may be shipped in the so-called "dry shippers" either in your own vehicle or by common carrier. Any other samples containing liquid-nitrogen must be treated as with propane (see above).
  • Samples shipped at room temperature in sandwich boxes or other crystallization trays can be transported without restriction except as noted below. Double-container all samples; this is good laboratory practice in general, and will improve relations between IMCA-CAT and the Argonne Environmental Health and Safety officers.
  • Crystals that have been pre-soaked in heavy-atom reagents can be brought on-site if they are double-containered. Two heavy-atom reagents are absolutely forbidden: dimethyl mercury and tetraethyl lead.
  • We can now accommodate actual heavy-atom soaks on-site, provided that the compounds in question are non-radioactive and of low or no volatility; uranyl acetate is forbidden for the first reason and dimethyl mercury for the second. You must follow the IMCA-CAT Heavy-Atom Soak Standard Operating Procedure if you wish to do soaks onsite.
• If your samples are macromolecular, you should plan to collect your data at low temperature. Accordingly, your productivity at the beamline will be substantially higher if you determine the type and concentration of cryoprotectant that you will use when you mount your crystals. So some experiments at home to find the best cryo-conditions will pay off later.
• If you are shipping your samples, alert your CAT user representative to the fact that they are coming.
• Assemble a team to acquire and process data. For a visit of less than 24 hours, two trained people is enough. For longer visits three users will be much more productive than one or two. If you try to collect data on insufficient sleep you will find it harder to acquire data profitably and to process it as you go.
• Download the Experimental Safety Approval Form from the APS website or ask the CAT user representative to fax you a copy. Fill it out at least five days before your beamtime begins and fax it back to us.We need it so that we can assess how your experiment fits within the APS Safety Envelopes.
• Make travel arrangements. The APS is located at the south end of Argonne National Laboratory, about twenty-five miles southwest of the Chicago Loop, just south of the Stevenson Expressway (I-55). O'Hare and Midway Airports are both nearby, and many major airlines fly into each. There is no useful public transportation from either airport, but United Limousine, American Limousine, and several other on-road agencies can get you to Argonne for $25-$40 from either airport; numerous rental-car agencies are also available. Comfortable lodging is available for reasonable prices at the Argonne Guest House, walking distance from the APS. Other hotels are available within five miles of the Laboratory.
• If this is your first visit, you should register as a user at the APS. There is an on-line registration form available; use it. You can also call the APS User Office at 630-252-9090 and arrange for training for you and your colleagues. You should ask to be trained on the business day preceding the beginning of your beamtime.
• Get some sleep before you come. You may need to work odd hours when you're at the beamline, so you should come in fresh.
• Even if this is not your first visit, we recommend that you arrange to come in the day before your beamtime begins. You can get your samples set up in the chemistry laboratory or chill room, refamiliarize yourself with the facility, learn what has changed since your previous visit, and get some sleep. If the group preceding you finishes early, you may even be able to start a bit early!

Mounting Samples for the IMCA-CAT Beamlines

Sample-mounting for cryogenic-temperature experiments on the IMCA-CAT beamlines involves the same techniques you would use for any macromolecular crystallographic facility that operates near liquid-nitrogen temperatures. Each sample is mounted on a fiber loop, typically 0.1 - 1.5 mm in diameter, and held in place on the loop by the surface tension in the solvent surrounding the sample. The loop is attached to a pin, usually made of metal, that is attached to a metal base about 7 mm in diameter. This metal base is designed to snap over a powerful magnet that mounts onto a standard goniometer head. Thus when the sample has been impaled in the loop, the user can snap the base over the magnet, immobilizing the pin for the duration of the experiment. During data collection the loop is subjected to a stream of dry nitrogen that emerges from the Cryostream nozzle at roughly 100K; the temperature at the sample is probably closer to 120K.

Methods for crystal mounting vary substantially from group to group, and we at IMCA-CAT strive to accommodate a variety of styles. Please feel free to bring the mounting hardware with which you are most comfortable. If you do not have your own equipment, we will be happy to show you how to use ours. Two references on cryo-mounting techniques are available: Håkon Hope's notes and a video available from MacCHESS on cryogenic sample mounting. Crystals may be mounted onto the sample goniostat either directly from a crystallization dish or after storing them in a cryogenic vial of liquid nitrogen, liquid freon, or solid propane. If they are mounted in "popsicles" of propane, the APS requires that we be particularly careful in shipping the samples in and out. Now that the APS has obtained a small-quantity exception to the usual rules about shipments of flammable substances (like propane), it's easier than it used to be to get permission to ship propane-frozen samples, but you will still need to toe the line. Contact Lisa Keefe for details.

Crystals that are mounted directly from a crystallization dish must be cryo-protected unless they were grown in a medium that is already cryoprotective. Often cryoprotection can be accomplished by scooping up the crystal onto the loop, removing it from the droplet in which it is grown, and then dragging it through one or more solutions with increasing concentrations of cryoprotectant in them. Thus the crystal is gradually exposed to increasing amounts of cryoprotectant. You may wish to experiment with this technique on small, poorly-ordered crystals before you sacrifice your only good, large crystal to the cryoprotectant gods.

The aspects of sample mounting that are specific to the IMCA-CAT beamlines are few. The Mar system has only a single horizontal spindle, so it is not possible to rotate the goniometer head into a vertical position to handle pre-frozen crystals in cryovials. We are in the process of acquiring a head with a 90° arc on it to enable this, but you should plan to bring your own if you're coming soon; ours isn't here yet. The Mar has a long travel along the phi axis. If your crystal is mounted anywhere from about 60mm to 80mm from the base of the goniometer head, the spindle can be backed in or out to drive the beam through the sample.

The Bruker system has a very limited translation along the phi axis, so the crystal needs to be mounted at a specific height relative to the goniometer head. That distance is 70mm +/- 3 mm from the base of the goniometer head, or 25mm from the flat of the magnet; the distance from the base of a Huber head to the is 45mm. We do have an extender if you mount your sample too close to the base, but if you mount it too high, you're out of luck.

Updated by Ann Bertling on 07 Sept 2007.