Manual

X-RAY CRYSTALLOGRAPHY FACILITY, KASHA LABORATORY

INSTITUTE OF MOLECULAR BIOPHYSICS

Florida State University, Tallahassee, FL 32306-4380

Data Collection & Processing Guide

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M A C R O M O L E C U L A R X - R A Y F A C I L I T Y C R Y S T A L L O G R A P H Y F A C I L I T Y

Data Collection & Processing Guide

Copyright © 2000-2004 Thayumanasamy Somasundaram 410-414 Kasha Laboratory • Institute of Molecular Biophysics

Florida State University, Tallahassee, FL 32306-4380 E-mail: soma@sb.fsu.edu • URL: http://www.sb.fsu.edu/~soma

http://www.sb.fsu.edu/~xrayPhone 850.644.6448 • Fax 850.644.7244

September 28, 2004

Table of Contents

INTRODUCTION I

FACILITY INFORMATION 1

SERVICE 2

LAYOUT 3

CONTACT 3

VENDORS & SUPPLIERS 4

PURCHASE ORDERS & BLANKET ORDERS 5

1. GENERATOR START-UP 6

2. GENERATOR SHUTDOWN 12

3. REGULAR DATA COLLECTION PROCEDURE 13

IP (WINDOWS NT, ANACONDA.CHEM.FSU.EDU) 14 CCD (LINUX, SPRUCE.CHEM.FSU.EDU) 14

4. HIGH-& LOW-RESOLUTION DATA COLLECTION PROCEDURES 15

5. LOW (CRYO) TEMPERATURE DATA COLLECTION PROCEDURE 16

DATA COLLECTION SET-UP 18

IP DETECTOR 18 CCD DETECTOR 20

1. ARCHIVING DATA 27

LINUX/UNIX 27 WRITING A TAPE USING DDS3 TAPE DRIVE (RACCOON.CHEM.FSU.EDU) 27 WINDOWS NT 28

2 EXTRACTING DATA 30

EXTRACTING (READING) FROM TAPE 30

Chapter

1 Facility Information & Contact Numbers This chapter deals with the Information about the X-Ray Crystallography Facility and the names and contact numbers of relevant people.

Facilityhe

Information X- Ray Facility (XRF) established in 1993 is located on the fourth floor of, Kasha

Laboratory, Institute of Molecular Biophysics. It occupies ~1200 ft2 of space spread among four different rooms KLB 410, 411, 412, & 413. It is a multi user macro-molecular x-ray diffraction laboratory equipped with three copper rotating anode

generators (Rigaku H2Rs and Elliott GX-20), one R-Axis IIc image plate (IP) detector and one Mar165 charge-coupled device (CCD) detector. The x-ray generators are coupled to either Osmic Confocal Max Flux or Supper total reflection double mirror systems. Automated data collection and detector control are handled by dedicated Windows NT and Linux computers. X-ray diffraction data can be collected at variety of temperatures including cryo temperatures. Crystallographers and scientists from the Structural Biology Program at the Florida State University campus and State University System use the facility to collect x-ray diffraction data from single crystals of biological macromolecules. Dedicated and shared computer resources in several platforms (HP-Alphas, Linux, WinNT, and WinXP) are available for data manipulation & modeling. Popular software packages like HKL2000, Mosflm, CCP4, X-PLOR, CNS, ‘O’, RsRef, TNT, ShelX, and XtalView running under different operating systems are available. Data archiving using DDS-4, DDS-3, and DVD±R, ±RW media is supported. The facility has a precession camera and several optical microscopes including an Olympus Stereo Zoom Microscope SZ-6045 with SZX-Ill Illuminator base for crystal mounting purposes and a 1.3 Mega pixel Motic digital camera with a Leica S8 Apochromatic Optic System with Windows 2000 computer. The facility has state of the art low temperature system for collecting cryo crystallographic data consisting of two Oxford Cryosystems' Cryo Stream cryo coolers and an American Magnetics' auto refill system. The facility has access to a machine shop and an electronics shop. The facility has a low vibration reach-in Crystallization Chamber for growing crystals at temperatures in the range 10-26° C. We have an Environmental Monitoring Unit for

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measuring the temperature and relative humidity conditions of a crystal set-up and detector rooms.

SERVICE The facility provides the following services:

1. Single crystal (capillary or loop mounted) x-ray diffraction data collection from proteins, nucleic acids, viruses and complexes of macromolecules using automated image plate or CCD detector.

2. Macromolecular crystallographic data collection at temperatures from 80 to 400 K using gaseous nitrogen stream including cryo data collection using cryo loops.

3. Low- and high-resolution data collection using large sample to detector distances (w/ Helium path) and non-zero 2-theta angles.

4. Preliminary data processing (indexing, merging and scaling) of single crystal data sets using popular data processing software.

5. Fiber diffraction data collection from fibrous proteins and viruses using either standalone image plate or x-ray film with double mirror focusing system.

6. Two individual dedicated areas (one for ambient temperature and the other at 4°C) for setting-up, monitoring, documenting and mounting crystals of macromolecules.

7. Facility and hardware to flash-cool, store and transport macromolecular crystals for synchrotron beamline data collection.

8. Co-ordinate “our” beam-time at SER-CAT at APS by requesting time-slots, provide expertise in data collection and processing of the samples.

9. Provide both archiving and retrieval of archived data to and from diverse media and platforms.

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Layout

Clickable layout of X-Ray Facility. Facility Manager’s is Room KLB414.

Contact The contact for day-to-day activities of the Facility is Dr. Thayumanasamy Somasundaram [aka, Soma; soma@sb.fsu.edu]. For computer related problems, contact Dr. Mike Zawrotny. In case of emergency involving x-rays or radiation, please contact Mr. Jason Johnson, Radiation Safety Officer (4-8802). If the emergency involved is not radiation but chemical or biological, contact Mr. Tom Jacobson, Director of Environmental Health & Safety (4-7687).

More information about the facility can be found in the web pages of the Facility or the Facility Manager.

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Telephone numbers* (updated May 2004): I C O N K E Y

Contacts

Data Collection

Tools

Data Archiving

Data Processing

References

Thayumanasamy Somasundaram (KLB 414) 644 6448 (Office)

X-Ray Facility (KLB 410) 645-1333

Soma’s Residence (for emergencies only) Inside the Facility

Wade Baggett (for Building & Amenities) 4-1419

Mike Zawrotny (KLB 415) 644-0069

Jason Johnson, Radiation Safety Officer 644 8802

Tom Jacobson, Director, EH & S 644 6895

Emergency/Urgency (Police/Fire) 911 /311

* If calling from a campus phone replace 644 by 4 and 645 by 5. To call outside first dial 9 then the 7 digit number

Vendors & suppliers X-Ray Facility's two main generators, Image plate detector system, and CrystalClear processing software were all installed and tested by Rigaku/Molecular Structure Corporation (Rigaku/MSC), The Woodlands, Texas. Rigaku/MSC remains one of the important contacts in terms of basic trouble shooting and advice. The x-ray generator manufacturer Rigaku/MSC needs to be contacted if the problem is from the high voltage, vacuum or power. Mar/USA Inc is the contact for marCCD165 and the marCCD control software. Haskris is the contact for the water chillers. Oxford Cryosystems, UK needs to be contacted for the CryoStream cooling system. Osmic is the manufacturer of the confocal mirror systems. Hampton Research supplies CryoLoops, CryoCaps and other cryo accessories.

Vendor & Supplier Telephone Number:

Rigaku/MSC USA 800 543 2379/281 363 1033

Mar USA Inc 877-627-9729

Haskris 847 956 6420

Oxford Cryosystems 866 OXCRYO8 /781 8435900

Osmic 800 366 1299/248 232 6400

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Hampton Research 800 452 3899

A complete list of all the vendors and suppliers can be found in the Standard Operating Procedure file-folder and is periodically updated.

Purchase Orders & Blanket Orders X-Ray Facility purchase orders are handled by Soma with the permission of the Director of the Institute and coordinated with IMB Fiscal Office (Alice G. Connor). The fiscal year starts July 1stof every calendar year and ends with June 30thof the following year. The director of the Institute with the consultation of other faculty members using the facility approves a budget for every year. Purchases, repairs, and services for the facility are allocated from the budget. So far the Facility does not charge any users for the use of Facility.

Several blanket purchase orders are drawn at the beginning of each fiscal year for vendors with whom the Facility deals frequently. Purchases with these vendors can be made simply by calling in the items and providing the vendor with blanket purchase-order number. Individual purchase order number needs to be taken from the Fiscal office for all other vendors and services. The following is the list of vendors with whom the Facility has blanket purchase orders:

℡No Vendor Information Phone Material/Service

1 Chemistry Stockroom

1-850-644-3429 Glassware, liquid nitrogen, helium gas, & miscellaneous

2 Grainger 1-850-575-4137 Hardware supplies

3 CDW-G 1-800-547-4239 Media, computer accessories

4 Hampton Research 1-949-425-1321 Cryo crystallographic supplies

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Chapter

2 Generator Start-up & Data Collection

1. Generator start-up The following is a standard operating procedure for powering up the generator. For the detailed procedure please read the x-ray generator manual kept at Room Number KLB 410A. Contact Soma (by phone at 644-6448 (Room Number KLB 414) or by e-mail at soma@sb.fsu.edu.) to schedule a User Training before actually using the instrument. All the users of the X-Ray Facility are required to complete the Radiation Safety Training offered by the Radiation Safety Office.

• Wear radiation safety badge (body and/or finger badges). • If you do not have a radiation badge please contact the Radiation Safety

office by telephone at 644-8801 or 644-8802. Users can also submit a web form and get a dosimeter (radiation badge) using the link:

http://www.safety.fsu.edu/radbadge_form.html

There are two logbooks: one for the generator and one for data collection.

• Enter in the generator logbook, the date, time, your name, and a general description of your experiment (e.g., low temperature, high-resolution, etc.)

• Open the inlet & outlet valves in the building chilled water supply by turning the handles to 'OPEN' position. The handles are located just behind Haskris water chiller and on the chilled water pipes (identifiable by the silver insulation wrapped around the pipes; see Figure 1.1).

• Press the 'ON' switch in Haskris water chiller. Ensure that the 'PUMP SELECTION SWITCH' is in the center position [@ BOTH] and 'TANK WATER LEVEL INDICATOR' lamp is 'ON' (See Figure 1.2).

Figure 1.1. Chilled water supply, valves and controls. Figure 1.2. Haskris water chiller with dials and flow

meters.

• In the front panel of the Haskris water chiller, you will notice two dials and three flow meters. The left-hand side (LHS) dial and flow meter give information about the pressure, temperature and flow rate of the internal chilled water to the target. The right-hand side (RHS) dial and flow meters give the same information for the tube housing and turbo molecular pump.

• The Haskris chiller is a water-to-water type and will take several minutes of external chilled water flow to produce the desired internal temperature. Therefore, at the start of the chiller operation temperature reading on the both dials will be close to the ambient (~70°F). The pressure gauges should be reading ~38 psi (LHS) and ~28 psi (RHS). The flow rate should be ~3.5 GPM (LHS) and ~20 GPH & ~1.6 GPM (RHS). Look for the pre-scored lines on top of the meters.

• After several minutes of chiller operation, the temperature dials should read 48-52°F, indicating that the internal water supply has reached equilibrium with the building chilled water.

Figure 1.3. Circuit breaker box and the handle. Figure 1.4. Helium cylinder with gas regulator.

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• Flip the handle in the circuit breaker box on the wall to ‘ON’ position

(gray box with a red handle; see Figure 1.3). This will supply power to the x-ray generator and vacuum system.

Haskris water chiller should be running before flipping the ‘ON’ handle in the breaker box. Failure to do so will disable the generator.

Figure 1.5. Helium cylinder switcher. Figure 1.6. Helium flow control box

• Next step is to ensure that helium gas is flowing from the cylinder (Figure 1.4) to the Osmic confocal mirror system (Figure 1.9). Two helium cylinders are strapped to the wall and both have a two-stage regulator on them. One of the cylinders is labeled ‘P’ for Primary and the other ‘R’ for Reserve (Figure 1.4). Open the valve on both the cylinders.

• The high-pressure stage at the regulator should read between 1000-2500 psi and the low-pressure stage should read 5-10 psi. When the Primary cylinder runs out of helium the Reserve will supply the gas.

There should be one nylon hose each from the Primary and Reserve regulators to the Cylinder Switcher. However, only one white braided hose out of it.

• Now ensure that a white nylon hose is connected from both the helium regulators to the Helium Cylinder Switcher (Figure 1.5) control box and another white braided hose is connected from Helium Cylinder Switcher to the Helium flow control box (Figure 1.6).

• Ensure that the ‘Power’ switch on Helium Cylinder Switcher is in upward (‘ON’) position and either of Primary or Reserve red lights is ‘ON’. Ensure also that there is no audible alarm.

• Finally ensure that the flow meter is in 'SLOW PURGE' mode (at 3 O' Clock position as shown in Figure 1.6) and the flow indicator is indicating a value of 55.

• Walk to the back of the generator to ensure the following (default) conditions are met:

1. (LHS): In Vacuum controller panel yellow POWER lamp is lit (Figure 1.7).

Failure in any one of the conditions will prevent the generator from starting.

2. (LHS): In TMP Drive Unit both POWER and READY orange lamps are lit.

3. (LHS): In the bottom panel big LINE orange lamp is lit and switch marked ELB1 is up and on 'ON' position.

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4. (RHS): All four lamps marked Temp, Flow, HP, and Flow (Target) is lit (green color). Figure 1.8 shows when conditions are met.

Figure 1.7. Vacuum control panel at the back of the generator.

Figure 1.8. Water-flow and temp sensors.

Figure 1.9. Osmic mirror system. Figure 1.10. Vacuum control panel. • After about 20 minutes of chiller operation water temperature and

pressure should remain stable. • Press the 'START' button in the 'VACUUM' panel in the x-ray generator

(see Figure 1.10). This should execute an automated sequence of engaging the rotary pump, the turbo molecular pump, and the ion gauge, indicated by the activation of respective green lamps. This sequence will take ~12 minutes to complete.

• Press the 'ON' button in the X-RAY panel (Figure 1.11). One should see two LED's light up below 'TUBE VOLTAGE' and 'TUBE CURRENT' each indicating a value of zero (see Figure 1.12).

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Figure 1.11. Front X-Ray Control Panel. Figure 1.12. Tube Voltage and Current Indicators.

• Confirm that the amber light at VACUUM panel is activated indicating it

is ready to ‘OPERATE’ At this point, check to see the digital multi meter (located at far left in the front panel) reads a value of 0.200V or less (Figure 1.13)

• Wait until the digital multi meter reading falls below 0.120, preferably below 0.100. Then switch the 'ON' button below X-RAY. Now the red light below X-RAY will be activated and the red light in the light tower will be activated. You should notice the slow increase in the values shown in the LED's until a value of 20 is reached in TUBE VOLTAGE and 10 in TUBE CURRENT.

• If the OPERATE amber light is not lit it indicates the following: 1) the 12V/110 mA Ready light in the lamp post has burnt out, or 2) the OL-387 lamp has burnt out, or 3) both lamps are burnt out. Replace them first before proceeding.

Figure 1.13. Vacuum Digital Multi Meter. Figure 1.14. Lamp Post (Light Tower).

• Wait until the digital multi meter's reading to fall below 0.150. Then press the 'ON' button located just below TARGET. You should see the amber

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light just below READY at the X-RAY panel is activated. You should see the target starting to rotate. Simultaneously a green light in the light tower will also be activated (see Figure 1.14).

• Allow 10 minutes for warm-up at 20 kV and 10 mA. Then slowly increase the TUBE VOLTAGE and TUBE CURRENT following the sequence described below (Make certain that you DO NOT operate above the maximum power of the generator for a particular filament):

1. Increase the voltage by six (6) kV; Wait for two minutes, 2. Increase the current by ten (10) mA; Wait for two minutes 3. Repeat SStteeppss 11--22, until desired or the maximum allowable power is

reached. Maximum load for our generator (w/ 0.3 mm cathode) is 5.4 kW.

• The maximum working power (load capacity) of the generator for a particular filament is a fixed value and should never be crossed. For example, for a 0.3 x 3.0 mm2 filament, that value is 5.4 kW (e.g., 40 kV and 125 mA) and for 0.2 x 2.0 mm2 filament, it is 2.8 kW. The applied load (power) for any filament can be calculated by simply multiplying the TUBE VOLTAGE and TUBE CURRENT.

• Enter the voltage, tube current, filament-current, hour meter reading, time of the day, vacuum, filament used and other pertinent details in the logbook.

• To carry out an experiment shutter of the x-ray port on the x-ray generator need to be opened.

• Only the Right Hand Port of the generator is coupled to an Osmic confocal mirror system (Figure 1.9) on both the Rigaku generators. However, they are controlled differently.

• For R-Axis IP detector the shutter is opened by the data collection computer anaconda.sb.fsu.edu. For marCCD detector the shutter is opened manually. Here lies the major difference.

• If you are using R-Axis, the 3-position shutter switch should be in 'EXT' (Figure 1.15a). If you are using marCCD then it should be in 'OPEN' (Figure 1.15b) position while collecting data.

Figure 1.15a. Shutter Position for R-Axis IP. Figure 1.15b. Shutter Position for marCCD.

• Note: Before opening the shutters please read the manual

thoroughly. Once the shutters are opened and if there is inadequate

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shielding, there is nothing to prevent you from accidentally being exposed to the radiation.

• The amber lights at the light tower indicate which x-ray port is open. • Proceed with the experiments.

2. Generator shutdown The following is a standard operating procedure intended to be an introduction for powering down the generator. For the detailed procedure please read the x-ray generator manual kept in KLB410A. In addition, get trained by Soma before actually using the instrument.

• The red light at the light tower (aka Lamp Post; Figure 1.14) indicates that x-rays are being produced and the amber lights indicate which x-ray port is open for your experiment.

• Carry out your experiment(s). • Close the shutters after your experiment is finished. If you are using R-Axis

the shutters will be closed when the data collection ends in normal mode. • Reduce the TUBE VOLTAGE and the TUBE CURRENT in steps. They can

be brought down rather quickly unlike the power-up procedure: 1. Decrease the voltage by six (6) kV; Wait for two minutes, 2. Increase the current by ten (10) mA; Wait for two minutes 3. Repeat SStteeppss 11--22, until minimum value of 20 kV and 10 mA is

reached. • After reaching 20 kV and 10 mA, press the white X-RAY 'OFF' button

(Figure 1.11). • Press the red 'OFF' button in the X-RAY panel. Wait for five (5) minutes. • Press the red STOP button in the VACUUM panel (Figure 1.10). • Wait for 15 minutes for the generator and the target to cool down. • Flip the handle to OFF position in the circuit breaker on the wall (Figure 1.3). • Turn the handle back to ‘OFF’ position on Helium Control Box (Figure 1.6) • Close the inlet and outlet valves to the chilled water. • Switch ‘OFF’ the Helium Cylinder Switcher (Figure 1.5) • Close the helium cylinder valve. • Switch OFF the water chiller. • Enter the final value of hour meter and time of the day in the generator

logbook. • If you have encountered any problems or difficulties during data collection

please report it to Soma. This now concludes the section describing how to power-up and power-down the generator for data collection.

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3. Regular data collection procedure Regular data collection consists of several simple but important steps that depend upon whether it is: 1) low resolution or high resolution data collection, 2) helium-filled beam paths, or 3) non-zero 2-theta, etc. If a user wants to collect room temperature data for a known crystal (i.e., the user is fairly familiar with the space group and cell constants) the following procedure is followed:

CAUTION

X-RAY GENERATORS PRODUCE COLLIMATED IONIZING RADIATION. PROPER PROCEDURES NEED TO BE FOLLOWED TO AVOID SERIOUS INJURY AND PHYSICAL HARM.

EVERYONE USING THE X-RAY FACILITY IS REQUIRED TO TAKE RADIATION SAFETY COURSE OFFERED BY RADIATION SAFETY OFFICE BEFORE USING THE INSTRUMENT.

• The Facility has an Image plate detector and a Charge-coupled device (CCD) detector. The user contacts Soma via telephone, e-mail or in person and requests time for the data collection on a specific or either of the detectors.

Regular Data Collection Normal or room temperature data-collection procedure is simple.

• For most of the samples, a tentative date and time is assigned for the data collection. For fragile or one of a kind sample, a particular date and time can be assigned. Users can look at the current and upcoming schedules at the following XRF URL:

http://www.sb.fsu.edu/~soma/XraySchedules/schfrmset.html

• A day prior to the scheduled time, user mounts the crystal in a glass or a quartz capillary (0.3-1.0 mm diameter). The exact placement of the crystal from at least one end of the capillary should be between 12 and 18 mm.

• On the day of the data collection, capillary containing the crystal is mounted onto a goniometer at the x-ray laboratory and the crystal position adjusted to match the beam center.

• Based on the prior experience with a similar crystal, the sample-Image Plate (IP) or CCD distance is set. In case of IP several 2-theta angles can also be set. If needed a helium-filled beam path or additional beam-stop are introduced (it usually takes additional an hour for this modifications).

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• At the computer (Windows NT anaconda.sb.fsu.edu for IP and Linux, spruce.sb.fsu.edu for CCD), the user logs in with their username and password. If it is the first time data collection, username and password can be obtained from Soma.

IP (Windows NT, anaconda.sb.fsu.edu) •

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Log in with username and password to the Windows NT machine anaconda.sb.fsu.edu. Launch CrystalClear by clicking the icon (see sidebar) or use the run command with the following argument: C:\Program files\Rigaku MSC\CrystalClear\CrystalClear.exe

Log in to Crystal Clear program with username and password.

Typical user directory name: TSMay00-1M, MSCDec99-10R, SBJul01-3M, etc.,

• You will be put in a data directory (either D:\data\username or E:\data\username. Create a sample directory preferably with the following name convention.: AAJun01-1R (two or three user initials in caps, three letter abbreviation of month with first letter cap, two digit year, a ‘-’, one or two digit experiment number, and finally R for R-Axis or M for MarCCD.

• Start the data collection. More detailed information about the data collection is provided in Chapter 3.

• You need to specify the starting phi, delta phi, and ending phi angles. Exposure time and number of frames are also need to be specified.

• Begin the data collection and wait for exposure time + eight (8) minutes to see the first frame. Confirm that every thing looks okay and allow the data collection to proceed. In case of failure modify one or more of the following: crystal, sample-IP distance, beam-stop, helium flow, phi rotation, delta phi, or exposure time.

CCD (Linux, spruce.sb.fsu.edu) • Log in to the Linux machine spruce.sb.fsu.edu with username marccd and

password will be provided by Soma. Go to the directories (either /d1/ or /d2/ in spruce.sb.fsu.edu).

• Create a user directory preferably with the following name convention: SIBMar01-1M (two or three user initials in caps, three letter abbreviation of month with first letter cap, two digit year, a ‘-’, one or two digit experiment number, and finally R for R-Axis or M for MarCCD.

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• If you prefer you can create sub-directories called Data, Denzo, Mosflm, etc. From the Data directory launch marccd program by issuing marccd command (do NOT put it in background).

• Specify starting, ending, and delta phi., sample-CCD distance, and time of exposure.

• Begin the data collection and wait for the first frame to appear. Unlike IP detector, it takes only three seconds for the data to be read. Confirm that every thing is okay. If not adjust one of the following: crystal, phi values, exposure time, or distance.

• More detailed data collection information will be given in Chapter 3.

4. High-& Low-resolution data collection procedures For crystals that require high-resolution data collection (i.e., over and above achievable with smallest sample-IP distance of 65 mm; 1. 7 Å at the edges or CCD distance of 35 mm and 1.35 Å at the edges or crystals with larger than 100 Å cell edges), 2-theta stage has to be rotated with respect to the phi axis. The extent of 2-theta stage rotation depends upon the level of high-resolution required (consult separate tables, kept at the XRF, for IP and CCD detectors). When the 2-theta stage is rotated the direct beam strikes the detector at a location different from the physical center of the detector. For IP detector rotation of 30° is achievable for our 2-theta stage. For CCD, limited 2-theta off-sets are available by physically removing the aluminum blocks on which the CCD itself sits. Tables available in the X-Ray Facility consisting of the three related parameters namely: 2-theta swing, sample-detector distance, and the highest achievable resolution (for Copper Kα radiation) can be consulted for the correct 2-theta swing angle for both IP and CCD.

Special Procedure High- or low-resolution data collection is special a procedure and therefore, requires additional planning.

However, the increased resolution comes at the cost of decreased amount of total coverage of the diffraction data because the detector is now off-centered. This means the data needs to be collected for larger phi angles. Larger off-centered detector may require the use of beam-stop mounted on the collimator rather than the one mounted in front of the detector. Collimator mounted beam-stop casts a larger shadow on the detector than the beam-stop mounted in front of the detector. High-resolution data collection may also require the use of a low-temperature device (cryo crystallography) discussed later in this Chapter.

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During a low-resolution data collection, the sample-IP distance is increased to a high value, say 400.00 mm. Due to the increased distance x-ray beam traverses between sample and IP there is increased air (non-Bragg) scatter. This scatter produces a higher background in the images. Therefore, it is essential to introduce the flexible helium tunnel equipped with a beryllium window between the sample and the detector (currently supported only on IP) to reduce the air scatter. However, addition of helium tunnel introduces four additional reflecting surfaces (two beryllium and two Mylar) for x-rays while reducing the air scatter. Each interface reflects approximately four (4%) of the intensity. It has been estimated that replacing one centimeter of air by helium decreases the air scatter by one percent. The beneficial effect of helium tunnel, therefore, overcomes the intensity reduction due to reflection for sample-IP distances ≥ 150.0 mm. Low-resolution data collection may also require a new and bigger beam-stop to capture the diverging straight beam and increased exposure time.

5. Low (cryo) temperature data collection procedure For low temperature (cryo), data collection we routinely use Oxford CryoSystems Cryo Stream cooler. First the reservoir for the cryo cooler needs to be filled with liquid nitrogen (~ 50 L). Then an appropriate temperature suitable for the crystal (between 95-100°K) is selected. Cryo cooler controller is then programmed to pre-cool to the selected temperature. In consultation with Soma, the user decides upon the height and diameter of the Cryo Loop™ for his/her crystal. The selected Cryo Loop is glued to a Cryo Pin and the Cryo Pin in turn is glued on to a Cryo Cap. A special magnetic holder is inserted into the goniometer's central hole and tightened. The Cryo Cap Loop is then placed on top of the magnet and the goniometer is adjusted to coincide with the x-ray beam with the center of the loop. Meanwhile, the user serially equilibrates his/her crystal in an appropriate cryo protectant. Then when the pre-set temperature is reached at the cryo cooler, the user brings in his/her crystal immersed in the cryo protectant to the Facility to be mounted using the Cryo Loop.

Pictures of Cryo accessories Pictures of Cryo Loop, Cryo Pin and Cryo Cap are shown in Figure 1.

• User contacts Soma via telephone, e-mail or in person and fixes a tentative date and time for the data collection.

• User selects an appropriate Cryo Loop (diameters 0.2-1.0mm range) depending upon the size of the crystal.

• The selected Cryo Loop is attached to a Cryo Cap™ and the mounted on top of a goniometer with a help of a magnet. The center of the loop is adjusted to beam center.

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• User picks the crystal with the Cryo Loop, temporarily blocks the flow of the cryo stream and very quickly mounts the crystal on the goniometer. Flow of the cryo stream is restored to flash cool the crystal in the loop. The finer adjustment of the well frozen (vitreous ice and NOT crystal ice formation) crystal is quickly performed.

Cryo Data Collection Cryo data collection procedure requires prior planning both by the user and by the Facility.

• At the computer, starting phi, delta phi, and ending phi angles are set. Exposure time and number of frames are also set.

• Begin the data collection. Collect the first frame and confirm that every thing is okay. Allow the data collection to proceed.

• After approximately 36 h of data collection, liquid nitrogen reservoir needs to be manually refilled with liquid nitrogen. Cryostream Cooler attached to MarCCD165 has an auto-refill system and therefore there is no need for manual refill as long as there is enough liquid nitrogen in supply tank, the supply tank is connected to the refill system, and the refill system is ON.

• Upon completion of the data collection, bring the temperature to ambient, switch off the cryo cooler, switch off the external dry air supply.

Pictures of some of the cryo components used are shown below (Courtesy of Hampton Research ):

CryoCap CryoLoop Mounted CryoLoop

CryoTong CrystalWand VialClamp Figure 5.1 Pictures of cryo accessories

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Chapter

3 Data Collection Details of data collection

Data collection set-up IP Detector In the previous section, the data collection procedure has been outlined and the details are elaborated in this section. Login with username and password (Windows NT default: press Ctrl, Alt and Delete together to get login box) to anaconda.sb.fsu.edu. Double click Crystal Clear icon or use the Run command with the following argument: C:\Program files\Rigaku MSC\Crystal Clear\Crystalclear.exe. The user will see another log in screen with Crystal Clear logo on it (Figure 3.1). Log in again with username and password.

Fig. 3.1. Crystal clear log-in screen.

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Crystal Clear program opens with a full screen (see Fig. 3.2). This screen has several sections: 1) toolbar section, 2) task window 3) icon section, 4) flow bar window, 5) messages window, 6) Stop/Abort button, and 7) Info window.

Fig 3.2. Full screen crystal clear window.

Following the full screen window, crystal clear will prompt for a new project and/or new sample name, depending upon whether it is a new project or a new sample (see Fig. 3.3). Provide the project/sample name such a way that they are consistent with

Fig. 3.3. New sample/project wizard

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UNIX conventions (we will be using both Windows NT and UNIX operating systems). This means no spaces, no special characters like ‘#, /, \, *, @, !,?, &’. The program will then ask for a Task with the possible selection includes: Screen Collect and Process, Collect and Process, Process, etc. Depending upon the nature of data collection select the appropriate task, for example, if you want only collect the data, select Collect. But if you wish to first screen several samples before collection, select Screen Collect and Process.

Fig 3.4. Task selection wizard.

Next, the Image Files dialog will open up. In this you will see your default data directory and possibly the correct disk name, for example, D:\data\username \projname\xtalname\images. If this is incorrect, use the Browse button and select the correct directory for the images (see Fig. 3.5). Finally click Finish.

Fig. 3.5. Image directory dialog.

CCD Detector For collecting data using marCCD165 first login to spruce.sb.fsu.edu (Linux machine) with username ‘marccd’ and Soma will provide you with a password.. Change either to

20

/d1 or /d2 directory and create sub-directories (follow the rules of suggested directory names) called Data and Denzo. Change to Data directory and issue command marccd to start the data collection GUI (see Figure 3.6). But DON’T run marccd in the background.

Figure 3.6 MarCCD 165 data collection GUI. Legend: 1=File Menu, 2=Status Menu, 3=File Name, 4=Data, 5=Magnified Data, 6=Cursor Information, and 7= Goniometer Information.

Now the user has to decide either to collect a single frame (more suitable for screening the sample) or a data set. If one desires to screen samples before the actual data collection, go to Acquire menu and select sub menu Single Frame (Acquire>Single Frame). A small window pops open on top of the main GUI asking for details of the single frame data collection (see Figure 3.7).

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Figure 3.7 Single Frame CCD data collection GUI. Legend: 1=Goniometer Information, 2=Data Collection Information, 3=Start & Stop Commands, 4= Directory Information, 5=Auto-Save Function, and 6=File Name.

In the Acquire Single Frame sub-menu user specifies Phi, Distance (sample-CCD) in mm, Time (Exposure) in seconds, Scan (delta phi) in degrees and Clicks the Start button to start the exposure and click the Dismiss button to clear the sub menu GUI. One important difference between data collection using CCD and IP is that in the case of CCD the user has to physically open the shutter switch. During IP data collection the instrument opens the shutter automatically. This difference is shown in Figure 3.8a (marCCD) and Figure 3.8b (R-Axis IP).

Fig 3.8a The Right Port shutter is shown in OPEN position for normal operation of marCCD

Fig 3.8bThe Right Port shutter is shown in EXT. position for normal operation of R-Axis IP

CAUTION

THIS MEANS THAT THERE IS A SIGNIFICANT DIFFERENCE IN THE X-RAY SHUTTER POSITIONS BETWEEN NORMAL marCCD AND R-Axis

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IP OPERATIONS.

EVERYONE USING THESE INSTRUMENTS SHOULD BE WELL AWARE OF THIS TO AVOID ACCIDENTAL EXPOSURE TO X-RAYS.

Therefore, during the actual x-ray exposure the x-ray shutter in the marCCD base will open to allow the x-rays to fall on the sample. An overall and close-up views of this shutter is shown in Figure 3.9a & b. Once again users should be aware of the difference between marCCD and R-Axis IP operation, as these instruments were manufactured by two different vendors.

Figure 3.9a Normal marCCD data collection: Lamp post amber & red lights ON; shutter red light ON and mar shutter red light ON.

Figure 3.9b Close-up view mar shutter red light ON for normal x-ray exposure

If the exposure is going to last more than 120 seconds, it is advisable to select Multi-Read in the GUI. If selected, Multi-Read = 2 (radio button turned orange), will split the Time (Exposure) into two halves and read the CCD two times, thus eliminating any zingers. Zingers are eliminated by comparing the first half to the second and removing any differences between the two, assuming zingers are random events. During data collection top window will be updated every ten seconds or so with the following details 1) name of current protocol, 2) time left in current exposure, 3) Intensity value 4) Shutter status, 5) Temperature of CCD, 6) Pressure of CCD and 7) Status of cooler (Figure 3.10). After the exposure time is over, CCD takes about 3 seconds to complete the data reading and displays it in the main window (Figure 3.11).

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Figure 3.10 marCCD status window

After screening the samples for the best one, the user can then start with their regular data collection by selecting Acquire>Dataset sub menu. A new data-set window will open up (Figure 3.12). The user can complete the required information and start the data collection. Consult the marCCD manual for further details.

Figure 3.11 Image of the data collected using marCCD165. Central white spot is from the lead beam-stop

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Figure 3.12 Data set window. See manual for more details. Legend: 1=File Info., 2=Comments, 3=Directory Info., 4=Multi-read, 5=Data Collection Dialog, 6=Auto-Check Button, and 7=Start & Stop Function.

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Chapter

4

Archiving data Long-term storage and retrieval of data in/out of magnetic tapes and recordable CDs (CD-R)

Archiving and long-term storage of the data is an essential component of any successful data collection procedure. The responsibility lies heavily on the users to ensure that the data is archived and accessible to them whenever they need it. Upon completion of an experiment, the data will remain in the hard disk for two weeks. During this period the user should back-up data by writing to DDS

Tape Back-up Archiving data involves both writing as well extracting the data to and from 4mm tape, 8mm tape or a CD-R.

-4 or DDS-3 tapes (sometimes wrongly referred to as DAT tapes). Alternatively the user can write the data to a CD-R or CD-RW (maximum capacity ~700 MB/CD). Another possible but not a recommended option is to copy the data to another hard disk via the network. The Facility has DDS-4 drives connected to anaconda and spruce via SCSI cables (DDS4 media capacity for native/compressed format is 20/40 Gb, for a 155 m tape), DDS-3 tape drive connected to raccoon via SCSI cables (DDS3 media native/compressed capacity is 12/24 Gb, for a 125 m tape). One copy of all the data is kept in the Facility either in DDS-3 or DDS-4 format tapes before it is removed from the hard disk. Due to the larger capacity of DDS-4 and -3 tapes and due to the unreliability of our older 8mm tape-drive, beginning April 2000 the Facility’s archives will be kept only in DDS-4 or -3 formats (data prior to year 2000 are archived in 8mm or DDS formats). These archived tapes are meant for emergencies and never for regular retrieval of user's data.

Compressed capacity quoted assumes 1:2 compression.

Our Certance DDS-4 & -3 drives (formerly Seagate) are backward compatible and therefore can read and write tapes with DDS, DDS-2 and DDS-3 specifications (look for logos shown below).

Figure 4.1 Logos identifying DDS, DDS2, and DDS3 tape media.

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1. Archiving data Linux/UNIX

Log in with username and password. If you don’t have username and password ask Soma for one. Change to a directory one level above your data directory. This will usually be either /spruce/d1/ or /spruce/d2 or /raccoon/d3/ or /raccoon/d4. Confirm the size of the directory you are planning to archive (total number bytes of all data) using the command du -ks dir_name. Decide on whether to archive the data in a DDS4 or DDS3 tape ensuring that the amount of data being archived is not larger than the capacity of the tape (uncompressed capacity for DDS4 and DDS3 are 20 and 12 GB, respectively). Insert a fresh tape into the appropriate tape drive with the tape's write-protection tab moved out of way. For DDS4 and DDS3 tapes a closed-tab allows writing.

Issue the tar command in the generic format tar -cvf /dev/st0 directory_name and this will start archiving the directory and all its sub-directories.

tar -cvf /dev/st0 dir_name/ &

Shown below is a typical session for DDS4 tape writing and reading (commands that you issue are shown in italic courier font, the computer response is shown in Arial Narrow font and comments in regular Times New Roman font).

Writing a tape using DDS3 tape drive (raccoon.sb.fsu.edu) We will first consider writing

to DDS3 tape in this section.

>pwd ↵ [Check the directory]

raccoon>/d3/Ip/soma

>ls -ltFa ↵ [Checking the list of sub directories]

total 48

drwxr-xr-x 5 soma users 4096 Sep 18 16:17 DDSRestore/

drwxrwxr-x 16 xray users 4096 Sep 18 10:49 ../

drwxr-xr-x 12 soma users 4096 Sep 18 10:46 ./

drwxr-xr-x 9 soma users 4096 Aug 31 11:58 E225QTsac/

drwxr-xr-x 4 soma users 4096 Aug 25 15:50 ApoAkAug01/

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drwxr-xr-x 2 soma users 4096 Jun 20 15:34 LysDenzoHDisk/

>du -ks E225QTsac/ ↵ [Checking the size of the directory to be archived]

1298648 E225QTsac / [~1.2 Gigabytes, a small directory]

>mt -f /dev/st0 status ↵ [Checking status of tape and drive]

SCSI 2 tape drive:

File number=0, block number=0, partition=0.

Tape block size 512 bytes. Density code 0x25 (DDS-3).

Soft error count since last status=0

General status bits on (41010000):

BOT ONLINE IM_REP_EN Controller: SCSI

>tar -cvf /dev/st0 E225QTsac/ & ↵

[1] 3470

or

>tar -cvf /dev/st0 E225QTsac/ | & tee ~/Tapes/dds3-01.list & ↵

[Writing tape as well as listing it into a file simultaneously]

[1] 3478 3479 Writing to DDS4 drive is exactly the same except for their higher capacity.

DDS4 drives are backward compatible with DDS3 DDS2 and DDS formats. Similarly DDS3 drives are backward compatible with DDS2 and DDS formats. Further information about writing to tape can be found in the Linux/UNIX man pages, Facility web manual pages or by consulting with people more familiar with the operating system.

http://www.sb.fsu.edu/~xray/Manuals/misc_man.html

Windows NT

28

Login with username and password. From the Start menu select Programs> Administrative Tools (Common)>Backup (see Figure 4.2). This will open up Windows NT back up GUI (see Figure 4.3a). Now select one of the disks shown on the GUI (in the example shown, drive D: has been selected). Then click Backup on the menu button. Another window pops open (Figure 4.3a) and in this window specify what exactly needs to be backed up (archived). There are several options, e.g., does the backup is replacing (new) or appending (add to old), does one need full details, or less detail, does one need daily, differential, incremental or full backup. After selecting all these options, click on OK. This starts the back-up process. More details can be found in the XRF Web page link to the Manuals page:

http://www.sb.fsu.edu/~xray/Manuals/misc_man.html

Alternatively Windows NT Back-up book or manual can be consulted for in-depth understanding of the complete procedure of back-up and restoring.

Figure 4.2 Invoking Windows NT backup program (DDS4 drive) on anaconda.sb.fsu.edu

29

Figure 4.3 Window NT Backup GUI

2 Extracting data

Extracting (reading) from tape In this section we will look at the procedure to extract the archived data back to hard disk. We can extract either all the contents of the directory or only part of the tape. The latter procedure is probably used most of the time since tapes usually contain more than one data set. However, in order to extract a particular directory it is essential to know the exact path name of that directory while the data was archived. It is, therefore, recommended that the user makes the listing of the contents of the tape as and when they are archiving their data (see the special command described in the earlier section, look for the following sign in the left hand margin)

In this section, we will discuss extraction of data from tape.

>pwd ↵ [Checking the present working directory]

/d3/Ccd/soma

>df -k . ↵ [Checking the amount of available free-space in current directory {.}]

Filesystem 1k-blocks Used Available Use% Mounted on

/dev/sda1 17639220 8004440 8738760 48% /d3

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>mkdir new_dir ↵ [Making a new directory for extraction]

>cd new_dir ↵ [Moving to the new directory]

>tar-xvf /dev/st0 & ↵

[Extract all the contents of tape]

>tar -xvf /dev/st0 | & tee extract.list & ↵

[Extracting and simultaneously writing the lists of extracted files into a file. "|" for pipe, first "&" for standard output, "tee" for one input to two output redirect, and the final "&" to put the job in background]

or

>tar -xvRf /dev/st0 a/data/proc/dir_name3 & ↵

[Extracting only contents of one directory {dir_name3}. Note here, there is no leading or succeeding slash in the directory designation].

The data will now be extracted to the hard drive.

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32

Index

# H

Hampton Research, 4, 5, 17 ©, i Haskris, 4, 5, 7, 8 helium, 8 A

anaconda, i, 12, 14, 19, 25, 28 I Archiving data, i, 25, 26

Image plate, 4, 13 Institute of Molecular Biophysics, 0, i B IP, i, 14, 15, 16, 19

Backup, 28, 29 J

C John Beidler, 4

CCD, i, 13, 14, 15, 22, 23 Charge-coupled device, 13 L chilled water, 8

Linux/UNIX, 26, 27 circuit breaker, 8 load capacity, 11 cryo, i, 2, 4, 16, 17, 18

CryoCap, 18 CryoLoop, 16, 17, 18 M Cryostream, 17

Mar/USA Inc, 4 CryoVial, 18 MarCCD, 14, 15, 22 CryoWand, 18 marCCD165, 4, 22, 23 Crystal Clear, 14, 19, 20 Mike Zawrotny, 4 CrystalClear, 4, 14 Mosflm, 1, 15

D O

data collection, i, 2, 7, 12, 13, 14, 15, 16, 17, 19, 21, 22, 23, 25 OPERATE, 10

Osmic, 1, 4, 5, 8, 9, 12 DDS4, 25, 26, 27, 28 Oxford Cryosystems, 4, 5 Denzo, 15, 22

R E

R-Axis, 1, 8, 12, 14, 15 Emergency, 4 Rigaku/MSC, 4 Extracting data, i, 29

S F

shutter, 11 Facility Information, 1 Soma, 3, 5, 7, 12, 13, 14, 15, 16, 17, 22, 26 filament, 11 spruce, i, 14, 15, 22, 25, 26 Florida State University, 0, i STOP button, 12

G T

Generator shutdown, i, 12 Table of Contents, i Generator start-up, i, 7 Telephone numbers, 4

33

Thayumanasamy Somasundaram, 4 X TMP, 9

X-RaY FACILITY, 13 XRF

V Floor plan, 3 XRF URL, 13 Vacuum, 7, 9, 10, 11

W

Windows NT, i, 14, 19, 21, 27, 28 1, 1

34

35