CRYO-Xe-SITER OPERATOR AND SERVICE MAN UAL

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1 CRYO-Xe-SITER OPERATOR AND SERVICE MAN UAL October 1998 M OLECU LAR STRUCTURE C OR PO RA TION a Ri gaku com pany

2 Copyright Molecular Structure Corporation All rights reserved. Reproduction, adaptation, or translation without prior written permission is prohibited, except as allowed under copyright laws.

3 Table of Con tents Chapter 1 General Information 1.1 Sys tem Requirements Setup Chapter 2 Operation Chapter 3 Troubleshooting 3.1 Gas Leak age Me chani cal Problems Chapter 4 Maintenance 4.1 Rou tine Maintenance Re plac ing Magnetic Pin Holder O-Rings Re plac ing Window O- Rings Appendix A Condensed Operating Instructions Glossary Index Cryo- Xe- Siter Op era tor and Serv ice Man ual i Oct. 30, 1998; Rev. A

4 Table of Con tents Figures 1-1 Cham ber Alignment Tool (As sem bled) Cham ber Alignment Tool (Sepa rated) Mag netic Pin Hold ers Yale- style Crystal Mounting Pin Hampton-style Crystal Mount ing Pin Cryocup Cryocup Po si tioner Ball Valve Copper Cold Fin ger Cryo tongs Dewar and Plastic Cover Ex tra De war With Vial Stor age Rack Vial Stor age Rack Styrofoam Cup Cryovial For ceps Chain Place ment for Bottle HPLC Tubing Preparation HPLC Tubing Connections HPLC Tube Flush With the Nut Fill ing the Bal loon Bal loon Placed on a Centrifuge Tube Carbon Tetrafluoride Condensing Liq uid Carbon Tetrafluoride Align ment Tool In serted into Top Cham ber Align ment Tool In serted to the Knurl Align ment Tool in Both Cham bers Placing Cryocup into Top Chamber Cryocup Po si tioned in Center of Window Top Clamp Lever Re lease Pin Brake Knob Top Clamp D- ring Par tially Ro tated Cold Fin ger Submerged Cold Fin ger in Cover Styrofoam Cup Gas ket Placement Dewar in Place on Elevator Loosen Brake Op er at ing Lever Up Pull Out Cryo cup Oct. 30, 1998; Rev. A ii Cryo- Xe- Siter Op era tor and Serv ice Man ual

5 Table of Con tents Figures (Continued) 2-23 Pull Out Lever Re lease Pin Pull Lever Down Push Magnet Re lease But ton Re mov ing the Cryovial Ball Valve Set Screw Acorn Nuts Lu bri ca tion Points Mag netic Pin Holder As sem bly Tables 1-1 Common Cryo liq uid Melting and Boil ing Points Cryo- Xe- Siter Op era tor and Serv ice Man ual iii Oct. 30, 1998; Rev. A

6 This page in ten tion ally left blank. Oct. 30, 1998; Rev. A iv Cryo- Xe- Siter Op era tor and Serv ice Man ual

7 Chap ter1 General Information The X-ray crystallographic structure determination of a macromolecule frequently requires that isomorphous heavy atom derivative crystals be prepared for phasing calculations. The Cryo-Xe-Siter is designed to produce xenon (or other gas) derivatives of protein crystals at cryogenic temperatures. (Refer to Table 1-1, for the melting and boiling points for common cryoliquids.) It allows the user to equilibrate crystals with a gas, under high pressure, then freeze the crystals without releasing the gas pressure. This technique may be useful in introducing heavy atoms into crystals in order to determine phases with the isomorphous replacement method. Cryoliquid Melting Point Boil ing Point Warnings K o C K o C N Boils too eas ily Propane Flammable CF None Ethane Flammable Methane Flammable CCl 3 F CFC, melt ing point too high BrCF Melting point too high Isopentane Flammable Xenon Krypton Water Table 1-1. Com mon Cryo liq uid Melt ing and Boil ing Points. Cryo- Xe- Siter Op era tor and Serv ice Man ual 1-1 Oct. 30, 1998; Rev. A

8 Gen eral In for ma tion Chap ter System Requirements The primary Cryo- Xe- Siter components used and tools re quired for op era tion are de scribed in the fol low ing sec tion. Cham ber Alignment Tool (Fig ures 1-1 and 1-2): A two-piece tool used to align the top and bot tom cham bers so the magnetic pin holder moves freely through both chambers. Figure 1-1. Chamber Alignment Tool (assembled). Figure 1-2. Chamber Alignment Tool (separated). Mag netic Pin Holder (Fig ure 1-3): An ap pa ra tus, ei ther Hampton- or Yale- style, used to hold and place the crys tal mount ing pin with the crys tal into the Cryo-Xe-Siter. Figure 1-3. Magnetic Pin Holders. Oct. 30, 1998; Rev. A 1-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

9 Chap ter 1 Gen eral In for ma tion Crys tal Mounting Pin (Figures 1-4 and 1-5): Pins, either Hamptonor Yale- style, held in place by a magnet at the end of the mag netic pin holder which are used to hold the crystal for freez ing. Figure 1-4. Yale-style Crystal Mounting Pin. Figure 1-5. Hampton-style Crystal Mountin Pin. Cryocup (Fig ure 1-6): A small cryocup filled with a cryo pro tec tant liquid and placed in the top cham ber to pre vent crystal dehydration. Figure 1-6. Cryo cup. Cryo- Xe- Siter Op era tor and Serv ice Man ual 1-3 Oct. 30, 1998; Rev. A

10 Gen eral In for ma tion Chap ter 1 Cryocup Positioner (Figure 1-7): The slide- handle which al lows the cryocup to be moved in and out of the top cham ber by mag netic coupling. Figure 1-7. Cryocup Positioner. Cryo pro tec tant: A solution which helps pre vent the for ma tion of ice on a crys tal while freez ing at cryogenic tem pera tures. Pi pette: Ap pa ra tus used to place the cryo pro tec tant liq uid into the cryocup. Crys tal: The protein sample to be frozen at cryogenic temperatures. Ball Valve (Fig ure 1-8): The large metal knob located be tween the top and bot tom cham bers. In the Close position (turned all the way clock - wise), the ball valve sepa rates the bottom cham ber from the top cham - ber. In the Open position (turned all the way coun ter clock wise), the crys tal may be lowered into the bottom chamber. Figure 1-8. Ball Valve. Oct. 30, 1998; Rev. A 1-4 Cryo- Xe- Siter Op era tor and Serv ice Man ual

11 Chap ter 1 Gen eral In for ma tion Copper Cold Fin ger (Figure 1-9): Ap pa ra tus used to hold the cryo - vial of carbon tetrafluoride which is placed inside the bottom cham - ber to freeze the c rystal. Figure 1-9. Copper Cold Finger. Cryo tongs (1 supplied, 3 op tional; Fig ure 1-10): Tongs used to move the cryo vi als be tween dewars and to the copper cold fin ger. Figure Cryotongs. Dewar and Plas tic Cover (Figure 1-11): A con tainer filled with liq - uid nitrogen which keeps the cop per cold fin ger at a cryogenic tem - pera ture when it is placed in the plastic cover. Figure Dewar and Plastic Cover. Cryo- Xe- Siter Op era tor and Serv ice Man ual 1-5 Oct. 30, 1998; Rev. A

12 Gen eral In for ma tion Chap ter 1 Ex tra De war (op tional; Fig ure 1-12): A container, also filled with liq uid nitrogen, which is used for cryovial stor age. (The vial storage rack may be used in this de war.) Figure Extra dewar with vial storage rack. Liq uid Carbon Tetrafluoride (not supplied): The cryoliquid used to freeze the crys tals. (Refer to Chap ter 2, Operation, step 1, for one pro - ce dure used to con dense carbon tetrafluoride gas into a liquid.) Vial Stor age Rack (op tional; Fig ure 1-13): A rack used to hold cryovi - als of car bon tetrafluoride in liquid nitrogen un til needed. Figure Vial Storage Rack. Oct. 30, 1998; Rev. A 1-6 Cryo- Xe- Siter Op era tor and Serv ice Man ual

13 Chap ter 1 Gen eral In for ma tion Styrofoam Cup (Figure 1-14): A cup used to cover the cryovial of carbon tetrafluoride af ter it is placed in the copper cold fin ger, un til it is ready to be in serted into the bottom chamber. Figure Styrofoam Cup. Cryovial For ceps (Fig ure 1-15): For ceps used to remove the cryo - vial from the copper cold fin ger af ter freezing. Figure Cryovial Forceps. Tank of Xenon (not sup plied). WARNING! THIS EQUIP MENT IS NOT TO BE USED WITH PRO PANE. WARNING! MAXI MUM SYSTEM PRES SURE IS 300 PSI. Cryo- Xe- Siter Op era tor and Serv ice Man ual 1-7 Oct. 30, 1998; Rev. A

14 Gen eral In for ma tion Chap ter Setup NOTE: Read all instructions carefully before beginning the setup. Items required: Hex wrench (met ric set; not supplied) Adjustable wrench (not supplied) Tank of xenon (not supplied) The Cryo- Xe- Siter weighs approximately 30 kg; or 66 lbs. For this rea son, it requires two people to remove the equipment from the ship ping crate. 1) Remove the Cryo- Xe- Siter from the crate by us ing the four alu mi num han - dles placed at the top and bottom (two on each side) of the equipment. The han dles may be removed after positioning the equipment by unscrewing them coun ter clock wise from the post. 2) Remove all pack ing ma te rial and tie wraps. 3) Care fully place the unit in an eas ily ac ces si ble area. The sur face area re - quired for the equipment is approximately 24 inches by 24 inches, or centimeters by centimeters. NOTE: Normal light ing, ventilation, and ambient laboratory conditions are acceptable for operating the Cryo-Xe-Siter. 4) Place the base of the xenon bottle into the recess on the base plate. 5) Loosen the screw on the chain clamp and ad just the length of the chain to accommodate the bottle. Wrap the chain around the bottle and at tach the chain to the pin on the clamp. Ref er to Figure 1-16 for placement of the chain. Tighten the nut which secures the gas bottle. Figure Chain placement for bottle. Oct. 30, 1998; Rev. A 1-8 Cryo- Xe- Siter Op era tor and Serv ice Man ual

15 Chap ter 1 Gen eral In for ma tion 6) Remove the plug in the xe non bot tle with a hex wrench. 7) At tach the pres sure regulator, verify it is fully en gaged, and tighten with an ad just able wrench. Ref er to Fig ure 1-16 for placement of the regulator. 8) Prepare the HPLC tub ing, as shown in Figure 1-17, us ing the fol low ing steps: Figure HPLC Tubing Preparation. a) Place a f inger-tight nut on each end of the tub ing, with the threaded por tion fac ing the end of the tub ing. b) Slide a ferrule on each end of the tub ing, with the conical sur face fac - ing the nut. The tub ing should not ex tend past the ferrule, but should be flush with the end. c) In sert one end of the tub ing into a port, slide the nut to the port and screw it in until firmly en gaged. Re peat this step with the other end of the tub ing. d) Re peat steps a) through c) for each piece of tub ing. 9) At tach the HPLC tub ing as fol lows, ref er to Fig ure 1-18: Figure HPLC Tubing Connections. Cryo- Xe- Siter Op era tor and Serv ice Man ual 1-9 Oct. 30, 1998; Rev. A

16 Gen eral In for ma tion Chap ter 1 a) At tach one HPLC tube from the regulator to the con trol box port labeled Inlet. NOTE: If the tub ing is pushed too far into the top cham ber in the next step, it will in ter fere with the cryocup po si tioner. To avoid this problem, slide the fer rule flush with the end of the tubing, then slide the nut over the fer rule. Us ing a pen or marker, mark the tub ing where the nut ends, as shown in Figure When inserting the tubing into the top chamber, the mark should line up with the end of the nut; do not insert past this mark. Figure HPLC tube flush with the ferrule. b) At tach one HPLC tube from the con trol box port la beled Top to the port on the back of the top chamber. c) At tach one HPLC tube from the con trol box port la beled Bottom to the port on the left side of the bot tom cham ber. 10) Purge all tub ing with xe non prior to ini tial use by performing the following steps: a) Set the gas con trol valve to Vent Top Chamber. b) Open the xenon gas tank by turn ing the top knob one full turn counterclockwise and open the regu la tor. c) Turn the Fill Rate needle one full turn coun ter clock wise. d) Set the gas con trol valve to Fill Top Chamber for ap proxi mately 5 seconds; fill the bot tom cham ber in the same manner. e) Turn the Fill Rate needle fully clockwise and turn the gas control valve to Vent Bottom Chamber for a few sec onds and then to Vent Top Chamber. Oct. 30, 1998; Rev. A 1-10 Cryo- Xe- Siter Op era tor and Serv ice Man ual

17 Chap ter 2 Op era tion When the system has been purged with xenon, the Cryo- Xe- Siter is ready for op era tion. To operate the Cryo-Xe-Siter, complete the fol low ing steps: NOTE: It is recommended that the following steps be per formed once with - out a crystal. WARNING: Skin or eye contact with liquid nitrogen or carbon tetrafluoride may cause serious freezing in jury. Use appropriate safety meas - ures, such as wear ing in su lated gloves (appropriate for tem pera - tures of -100 o C or colder) and safety goggles with side shields, when working with liquid nitrogen, car bon te trafluo ride, or other cryoliquids. 1) Prepare two 1.5 ml vials of liq uid carbon tetrafluoride in a cryovial (to be thawed in step 13) by performing the fol low ing steps: a) Fill a small bal loon with approximately one liter of carbon te trafluo - ride gas, Fig ure 2-1. Figure 2-1. Filling the balloon. Figure 2-2. Balloon placed on a centrifuge tube. b) Place the end of the bal loon on a disposable centrifuge tube, Fig ure 2-2. c) Place the tube with the bal loon into a de war of liquid nitrogen un til the carbon tetrafluoride condenses into a liq uid, Fig ure 2-3. Cryo- Xe- Siter Op era tor and Serv ice Man ual 2-1 Oct. 30, 1998; Rev. A

18 Op era tion Chap ter 2 Figure 2-3. Carbon tetrafluoride condensing. Figure 2-4. Liquid car bon te trafluo ride. d) Once the liquid state has been achieved, Figure 2-4, pour the carbon tetrafluoride into the cryo vi als. Store the cryo vi als in liq uid nitrogen. 2) Us ing the cham ber alignment tool, verify the top and bottom cham bers are prop erly aligned by performing the fol low ing steps: a) Turn the ball valve to the Open po si tion. b) Us ing the cryocup po si tioner, re tract the cryocup to the side of the top chamber. c) In sert the longer part of the cham ber alignment tool into the top cham - ber, Fig ure 2-5. The tool should slide all the way to the knurl with ease, Fig ure 2-6. If the tool does not move freely in and out of the top chamber, ref er to Sec tion 3.2, Mechanical Prob lems. Figure 2-5. Alignment tool inserted into top chamber. Figure 2-6. Alignment tool inserted to the knurl. Oct. 30, 1998; Rev. A 2-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

19 Chap ter 2 Op era tion d) In sert the smaller part of the alignment tool into the bot tom cham ber and into the larger part of the tool, Fig ure 2-7. Figure 2-7. Alignment tool in both chambers. e) Remove the align ment tool and close and open the ball valve. f) In sert both parts of the alignment tool again. The tool should still move in and out of the chambers with relative ease. If not, refer to Section 3.2, Mechanical Prob lems. 3) Set the gas con trol valve to Vent Top Chamber and open the xenon gas tank by turn ing the knob on top one full turn counterclockwise. 4) Flush gas through the tub ing by set ting the gas con trol valve to Fill Top Chamber for ap proxi mately 5 sec onds, then return it to Vent Top Cham - ber. 5) Ver ify the ball valve is in the Close position, fully clockwise, and the Fill Rate needle is turned fully clockwise. 6) Place approximately µl of cryoprotectant in the cryocup and place the cryocup into the holder in the top chamber, Figure 2-8. Ver ify the cryocup positioner is pushed in, so the cryocup is po si tioned in the cen ter of the top cham ber window, Figure 2-9. NOTE: If the cryocup positioner is pulled out too far, the magnetic contact on the cryo cup will be lost. To regain mag netic pull, slide the O- ring closest to the up per cham ber as far as possible toward the chamber. Then, slide the cryo cup positioner all the way over to the O-ring to magnetically catch the cryocup. Cryo- Xe- Siter Op era tor and Serv ice Man ual 2-3 Oct. 30, 1998; Rev. A

20 Op era tion Chap ter 2 Figure 2-8. Placing cryocup into top chamber. Figure 2-9. Cryocup positioned in center of window. NOTE: When attaching a Hampton-style crys tal mounting pin in the fol - lowing step, ensure the tim ing mark on the pin is aligned with the center of the flat side of the D-ring. 7) Po si tion the op er at ing lever before mounting the crystal by attaching the crys tal mount ing pin to the mag netic pin holder and in sert ing the as sem bly into the top chamber. Loosely clamp the mag netic pin holder in place, Fig - ure Lower the crystal mount ing pin into the top cham ber manually by slowly push ing down on the rod. En sure the pin is cen tered in the cryo - cup and just above the meniscus of the cryo pro tec tant. Pull out the lever re lease pin, Figure 2-11, and lower the op er at ing lever to the proper po si - tion (the large D- ring of the mag netic pin holder should align with the D- ring slot when rotated in step 9). Tighten the lever brake knob to the left of the op er at ing lever, Fig ure Figure Top clamp. Figure Lever release pin. Figure Brake knob. Oct. 30, 1998; Rev. A 2-4 Cryo- Xe- Siter Op era tor and Serv ice Man ual

21 Chap ter 2 Op era tion NOTE: A bent crystal mount ing pin will cause an align ment problem. To avoid this prob lem, verify the crystal mounting pin is straight. 8) Mount a crys tal in the loop. Then at tach the pin and crystal to the mag netic pin holder. The pin base must make full con tact with the holder. 9) Care fully in sert the mag netic pin holder with the crys tal into the top cham - ber. The crystal should be just above the meniscus of the cryoprotectant in the cryocup. Tighten the top clamp enough to compress the gasket and pro- 13. Rotate the D-ring of the magnetic pin holder into the D-ring slot, Figure (This allows the operating lever to raise and lower the mag - netic pin holder and prevents the mag netic pin holder from ris ing unexpect - edly when the top cham ber is pressurized.) Figure Top clamp. Figure D-ring partially rotated. 10) Set the gas con trol valve to Fill Top Chamber. The pres sure is controlled by adjusting the pressure regulator on the gas tank. The fill rate is con - trolled by ad just ing the Fill Rate needle valve to the right of the top cham - ber gauge. After fill ing the top cham ber with gas, return the Fill Rate nee - dle to the lowest set ting. Allow the top chamber to equili brate for approxi - mately 10 minutes. 11) While the system is equilibrating, submerge (DO NOT DROP) the vial holder end of the copper cold fin ger into the dewar of liq uid nitrogen, Fig - ure 2-15, for ap proxi mately 8 minutes to pre chill. Do not submerge the gas ket with the cop per cold finger. Cryo- Xe- Siter Op era tor and Serv ice Man ual 2-5 Oct. 30, 1998; Rev. A

22 Op era tion Chap ter 2 12) After prechilling, place the copper cold fin ger into the dewar plastic cover and place the plastic cover on top of the de war of liq uid nitrogen, Fig ure The cop per cold finger should be kept in contact with the liquid nitro - gen. Cover the vial holder to prevent a build-up of frost; a Styrofoam cup works well, Fig ure Figure Cold finger submerged. Figure Cold finger in cover. Figure Styrofoam cup. 13) Thaw two cryo vi als of fro zen carbon tetrafluoride (by removing them from the liq uid ni tro gen for 30 sec. to 1 min.). Pour a small amount of carbon tetrafluoride into the copper cold finger vial holder to prevent freez ing of the cryo vial to the cold finger, then in sert a full cryovial of carbon te - trafluoride into the vial holder. NOTE: Applying too much vacuum grease in step 14 may pre vent a good seal. 14) Ver ify the black gasket is in place on the flange of the bot tom chamber. If the gasket is not there, place the gasket on the bot tom cham ber flange, Fig - ure It may be nec es sary to place a small amount of vac uum grease on the O-ring por tion of the gas ket, using just enough to se cure it. Figure Gasket placement. Figure Dewar in place on elevator. Oct. 30, 1998; Rev. A 2-6 Cryo- Xe- Siter Op era tor and Serv ice Man ual

23 Chap ter 2 Op era tion NOTES: If the lower gas ket be comes too cold it will not seal properly. Step 15 should be completed quickly to avoid this prob lem. When us ing Hampton- style crystal mounting pins, ensure the timing mark on the cryovial is centered in the bottom chamber window when performing step ) Po si tion the dewar so the cop per cold fin ger and cryovial are inside the bottom cham ber and the cold finger flange aligns with the bot tom cham ber flange, then raise the elevator by rotating the black knob, to the right of the elevator clock wise, to hold the de war in place, Fig ure Clamp the copper cold finger in place on the bot tom chamber. Tighten the clamp to pro vide a seal on the gasket. 16) Set the gas con trol valve to Fill Bottom Chamber. Al low the bot tom cham - ber pressure to equilibrate with the top cham ber pressure. 17) Once the bot tom chamber and top chamber are at equal pressures, the fol - low ing five steps must be com pleted in order and within 30 seconds to freeze the crys tal. F1 Turn the ball valve to the Open po si tion by turn ing it counterclock - wise, so the word Open is fac ing for ward. F2 Loosen the lever brake knob, Figure 2-20, and raise the op er at ing lever to the top stop position, Fig ure Figure Loosen brake. Figure Operating lever up. Cryo- Xe- Siter Op era tor and Serv ice Man ual 2-7 Oct. 30, 1998; Rev. A

24 Op era tion Chap ter 2 F3 Pull the cryo cup positioner out, Figure Figure Pull out cryocup. F4 Release the operating lever from the top stop by pulling out on the lever re lease pin, Fig ure (Once the operating lever is in mo tion, there is no need to hold the lever re lease pin.) Lower the op er at ing lever to the bottom stop position, Figure Figure Pull out lever release pin. Figure Pull lever down. F5 Quickly depress the magnet re lease button, on the top of the mag netic pin holder, to release the crystal and pin into the vial of carbon te - trafluoride, Figure Figure Push magnet release button. Oct. 30, 1998; Rev. A 2-8 Cryo- Xe- Siter Op era tor and Serv ice Man ual

25 Chap ter 2 Op era tion NOTE: The vent rate is controlled by adjusting the vent rate needle valve. 18) Set the gas con trol valve to Vent Bottom Chamber When the gas pres sure has dropped near 0, re lease the bot tom clamp and lower the dewar and vial holder on the ele va tor. 19) Care fully remove the dewar, with the copper cold finger in place, by slid - ing it for ward. Slightly raise the lead ing edge of the de war to remove it from the re cess in the plate. 20) Care fully remove the cryo vial, with the pin and loop, us ing cryo vial for - ceps, Fig ure 2-26, and place the cryovial in liq uid nitrogen to maintain its tem pera ture. Figure Removing the cryovial. Cryo- Xe- Siter Op era tor and Serv ice Man ual 2-9 Oct. 30, 1998; Rev. A

26 Op era tion Chap ter 2 This page intentionally left blank. Oct. 30, 1998; Rev. A 2-10 Cryo- Xe- Siter Op era tor and Serv ice Man ual

27 Chap ter3 Troubleshooting 3.1 Gas Leakage If a system gas leak is suspected, conduct a pressure test by per form ing the fol - low ing steps: NOTE: Due to the high cost of xenon, it is recommended that compressed air, nitrogen, or he lium be used to conduct the pressure test. 1) Set the gas con trol valve to FILL TOP CHAMBER. 2) Ver ify the ball valve is in the Open po si tion. 3) In sert the magnetic pin holder and clamp it into place to pro vide a seal for the top chamber. 4) In sert the copper cold fin ger and clamp it into place to provide a seal for the bot tom cham ber. (The cold fin ger should be at room temperature.) 5) Open the gas tank by turn ing the knob on top one full turn counterclock - wise. Turn the regulator knob clockwise to fill the chambers to 300 psi. 6) Close the valve on the gas tank and close the regulator by turn ing the knob all the way coun ter clock wise. 7) If the read ing on the control box gauge shows a decrease in pressure, there is a gas leak in the system. If a de crease in pressure is observed, con tinue with step 9. 8) If the system main tains a consistent pres sure, there are no gas leaks. Purge the system with xenon prior to use. Gas leak age may oc cur for a number of rea sons. If the equipment is ex pe ri enc - ing a decrease in pressure, perform the following: 9) Ver ify all gaskets are seal ing prop erly. If not sealed, confirm the gaskets and mat ing sur faces are clean and free of de bris. NOTE: If the gas ket be tween the cold fin ger and the bottom cham ber becomes too cold it will not seal properly. Cryo- Xe- Siter Op era tor and Serv ice Man ual 3-1 Oct. 30, 1998; Rev. A

28 Troubleshooting Chap ter 3 10) Tighten the nuts on all clamps. 11) Ver ify all HPLC tube fittings are as sem bled cor rectly, are in place, and are tight. 12) Tighten the nut that at taches the regulator to the gas bottle with an ad just - able wrench if leakage is suspected. 13) Us ing a leak detection fluid, check the O- ring seals on the magnetic pin holder. Place a small amount of detection fluid around the top of the up per flange and the top of the D-ring. If leak age is detected, ref er to Sec tion 4.2, Re plac ing Mag netic Pin Holder O-Rings, for de tails on accessing and re - placing the O-ring seals. NOTE: Soapy wa ter may be used as a leak detection fluid. 14) Check the O-ring seals around the windows by us ing a small amount of leak detection fluid around the seal. If leak age is detected, ref er to Sec tion 4.3, Re plac ing Window O-Rings, for details on accessing and replacing the O-ring seals. 15) Apply a small amount of leak detection fluid around the square flange on the top chamber and around the ball valve knob. If a leak is detected, con - tact MSC. 16) If there is still evidence of gas leak age, af ter all seals and connections have tested negative with the leak de tec tion fluid, it is prob able there is a leak within the con trol box. Contact MSC. 17) Clean ex cess leak detection fluid from all surfaces when test ing is com - plete. 3.2 Mechanical Problems If the cham ber align ment tool does not move freely in and out of the top cham - ber when in serted, per form the following steps: 1) Ad just the ball valve back and forth until the tool slides in all the way to the knurl. In sert the smaller part of the tool into the bottom cham ber and into the larger part. If the alignment tool still does not move freely, con - tinue with the next step. If, at this point the tool moves freely, con tinue with step 5. Oct. 30, 1998; Rev. A 3-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

29 Chap ter 3 Troubleshooting 2) Us ing a 1.5 mm hex wrench, adjust the set screw on the ball valve handle, Figure 3-1, while rock ing the handle until the tool slides in and out easily. If the tool does not move freely, continue with the next step. If the align - ment tool moves freely, continue with step 5. Figure 3-1. Ball valve set screw. 3) Us ing an 8 mm hex wrench, loosen the four acorn nuts on the top of the top chamber, Fig ure 3-2, and again adjust the ball valve back and forth until the tool moves freely. If the tool still does not move freely, con tact MSC. Figure 3-2. Acorn nuts. 4) Us ing a figure-eight pattern, tighten the acorn nuts. (Do not tighten one nut completely with out partially tightening all other nuts in a rotation pat - tern.) 5) Remove the align ment tool and close and open the ball valve. 6) In sert the tool again to verify chamber alignment; the tool should move in and out with relative ease. If the tool does not move eas ily, re peat these steps; if not suc cess ful, con tact MSC. Cryo- Xe- Siter Op era tor and Serv ice Man ual 3-3 Oct. 30, 1998; Rev. A

30 Troubleshooting Chap ter 3 If the elevator will not stay in the raised position, adjust the ten sion screws located on the back of the ele va tor with a hex wrench. Contact MSC for the fol low ing mechanical prob lems: If the mag netic pin holder seems to be dragging through the ball valve, the problem may be in alignment. Ver ify the pin be ing used and the magnetic pin hlo der shaft are straight. If both the pin and the magnetic pin holder shaft are straight, the problem is in the alignment of the body. If rotating the elevator knob does not move the elevator, the gear may be at fault. If the lever brake knob will not lock the op er at ing lever, it is pos si ble the knob has been over-tightened or the brake pad may be worn and needs replacement. Oct. 30, 1998; Rev. A 3-4 Cryo- Xe- Siter Op era tor and Serv ice Man ual

31 Chap ter4 Main te nance WARNING! The control box pressure gauges should read 0 to ensure the cham bers are not pressurized before per form ing maintenance procedures; always use appropriate safety measures, such as wear ing protective eyewear. 4.1 Routine Maintenance Monthly Wipe all ex te rior surfaces with a soft, lint-free cloth. (Do not use harsh soaps or clean ers.) Apply 1 to 2 drops of light lu bri cat ing oil to the operating lever slots (1), the op er at ing lever pivot point (2), the mag netic pin holder stem tubes (3), and to the elevator drive shaft (4). Ref er to Fig ure 4-1 for oil place ment. Figure 4-1. Lubrication Points. Cryo- Xe- Siter Op era tor and Serv ice Man ual 4-1 Oct. 30, 1998; Rev. A

32 Main te nance Chap ter 4 Yearly Tighten all mount ing screws. 4.2 Replacing Magnetic Pin Holder O-Rings If it is de ter mined that one of the O-rings in the magnetic pin holder is the cause of a gas leak, the following sec tion describes how to ac cess and replace the O- rings. It is advisable to change both O- rings at the same time. Ref er to Fig ure 4-2 for ad di tional information. Figure 4-2. Magnetic Pin Holder Assembly. Items nec es sary to change the stem as sem bly O-rings: Internal snap ring pli ers Light lubricant 1) Remove the mag netic pin holder from the unit. 2) Remove the mag net release washer using the following steps: a) Depress the mag net re lease but ton on the top of the magnetic pin holder and hold it down. Oct. 30, 1998; Rev. A 4-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

33 Chap ter 4 Main te nance b) With the but ton held down, unscrew the mag net re lease washer located at the bot tom of the mag netic pin holder. c) Care fully re lease the spring tension. 3) Pull the in ter nal stem out through the top of the as sem bly, being careful not to lose the spring. 4) Care fully remove the small O- ring on the end of the magnet re lease stem and discard. 5) Apply a small amount of light lubricant to the O-ring or the shaft. Care - fully stretch the new O-ring over the shaft un til it is en gaged firmly in the groove. 6) Hold ing the D-ring, slide the top flange up enough to access the magnet car rier. 7) Hold ing the stem tube, unscrew the magnet carrier, be ing careful not to lose any small com po nents. 8) Remove the top flange from the stem tube. WARNING! Wearing safety glasses is highly recommended while per form ing the following step. 9) Us ing the internal snap ring pliers, care fully remove the snap ring located at the large end of the top flange. 10) Remove the plastic spacer ring, located di rectly un der the snap ring, then carefully remove the O- ring and discard. Do not scratch seal sur faces. 11) Clean all com po nents with a soft, lint- free cloth before reassembly. 12) Apply light lu bri cant to the O-ring and press it into the groove until fully engaged. If the O-ring is not en gaged properly, the plastic spacer ring will not seat fully. 13) Place the plastic spacer ring into place and verify it is fully en gaged. 14) Re in stall the snap ring us ing the in ter nal snap ring pliers. Cryo- Xe- Siter Op era tor and Serv ice Man ual 4-3 Oct. 30, 1998; Rev. A

34 Main te nance Chap ter 4 15) Apply a small amount of light lu bri cant to the stem tube and care fully slide it through the top flange and all com po nents. 16) Hold ing the stem tube, care fully thread the mag net carrier into place. Avoid cross- threading dam age, as this will cause mis-alignment. 17) Install the spring over the mag net re lease stem. 18) Apply light lubricant to the shaft and O- ring and care fully in stall the shaft through the top of the D- ring. Press down on the shaft un til the shaft ap - pears through the mag net carrier. 19) While maintaining compression on the spring, thread the magnet re lease washer on the tip. 20) Depress the pin release button to verify the mag net release washer is fully engaged and flat against the face of the mag net carrier. NOTE: Perform a pressure test of the Cryo-Xe-Siter system af ter replacing seals. Ref er to Chap ter 3, Troubleshooting, for instructions. 4.3 Replacing Window O- Rings If a gas leak has been de tected around the windows, the leak is likely in the window O- ring. This section de scribes how to access and change the window O- rings. Items nec es sary to change the window O-rings: Hex wrench (met ric set; not supplied) Light lubricant (not supplied) 1) Care fully re move the six M3 screws hold ing the window bezel in place. 2) Remove the bezel by pull ing it straight away from the window. 3) Remove the window by pull ing it straight away. The O- ring will ei ther come off with the window or remain en gaged in the body. 4) Care fully remove the O-ring and discard. Do not scratch seal sur faces of the body or win dow. Oct. 30, 1998; Rev. A 4-4 Cryo- Xe- Siter Op era tor and Serv ice Man ual

35 Chap ter 4 Main te nance 5) Clean all com po nents with a soft, lint- free cloth. 6) Apply a small amount of light lu bri cant to the O- ring and place it in the body. 7) Re in stall the window. 8) Re in stall the bezel over the window, replace the six M3 screws, and fingertighten only until all screws are in place. 9) Us ing a cross ing pattern, tighten all six screws with a hex wrench. NOTE: Perform a pressure test of the Cryo-Xe-Siter system af ter replacing seals. Ref er to Chap ter 3, Troubleshooting, for instructions. Cryo- Xe- Siter Op era tor and Serv ice Man ual 4-5 Oct. 30, 1998; Rev. A

36 This page intentionally left blank. Oct. 30, 1998; Rev. A 4-6 Cryo- Xe- Siter Op era tor and Serv ice Man ual

37 AppendixA Con densed Operating In struc tions WARNING: Skin or eye con tact with liq uid nitrogen or car bon te trafluo ride may cause se - ri ous freez ing injury. Use appropriate safety measures, such as wear ing in su - lated gloves (appropriate for temperatures of o C or colder) and safety gog gles with side shields, when working with liq uid nitrogen, car bon te - trafluo ride, or other cryo liq uids. 1) Pre pare two cryo vi als of fro zen car bon te trafluo ride (to be thawed in step 12). 2) Us ing the cham ber align ment tool, verify the top and bot tom cham bers are prop erly aligned. 3) Set the gas con trol valve to Vent Top Chamber and open the xe non gas tank by turn ing the knob on top one full turn coun ter clock wise. 4) Ver ify the ball valve is in the Close po si tion and the Fill Rate nee dle is turned fully clock - wise. 5) Place ap proxi mately µl of cryo pro tec tant in the cryo cup and place the cryo cup into the holder in the top cham ber. The cryo cup should be po si tioned in the cen ter of the top cham ber window. 6) Loosely clamp the mag netic pin holder in place and slowly push the rod into the chamber to check the align ment of the crys tal mount ing pin. En sure the pin is cen tered in the cryo - cup and just above the me nis cus of the cryo pro tec tant. Pull out the lever re lease pin and lower the op er at ing lever to the proper po si tion (the large D- ring of the mag netic pin holder should align with the D- ring slot when in serted in step 8). Tighten the lever brake knob to the left of the op er at ing lever. 7) Mount a crys tal in the loop. Then at tach the pin, with the crys tal, to the mag netic pin holder. The pin base must make full con tact with the holder. 8) Care fully in sert the mag netic pin holder with the crys tal into the top cham ber. The crys tal should be just above the me nis cus of the cryo pro tec tant. Tighten the top clamp. Rotate the D- ring of the mag netic pin holder into the D- ring slot. 9) Set the gas con trol valve to Fill Top Cham ber. After fill ing the top cham ber with gas, re - turn the Fill Rate nee dle to the lowest set ting. Al low the top cham ber to equili brate for ap proxi mately 10 min utes. 10) While the sys tem is equili brat ing, submerge (DO NOT DROP) the vial holder end of the cop per cold fin ger into the de war of liq uid ni tro gen for ap proxi mately 8 min utes to pre - chill. Do not submerge the gas ket with the cop per cold fin ger. Cryo- Xe- Siter Op era tor and Serv ice Man ual A-1 Oct. 30, 1998; Rev. A

38 Ap pen dixa 11) Af ter pre chilling, place the cop per cold fin ger into the de war plas tic cover and place the plas tic cover on top of the de war of liq uid ni tro gen. The cop per cold fin ger should be kept in con tact with the liquid ni tro gen. Cover the vial holder to pre vent a build-up of frost; a Sty ro foam cup works well. 12) Thaw two cryo vi als of frozen car bon te trafluo ride (by re mov ing them from the liquid ni - tro gen for 30 sec. to 1 min.). Pour a small amount of car bon te trafluo ride into the cop per cold fin ger vial holder to pre vent freezing of the cryo vial to the cop per cold finger, then in sert the cryo vial of car bon te trafluo ride into the vial holder. 13) Po si tion the de war so the cop per cold fin ger and the cryo vial are in side the bot tom cham - ber and the cold fin ger flange aligns with the bottom cham ber flange, then raise the ele va - tor to hold the de war in place. Clamp the cop per cold fin ger in place on the bot tom cham - ber. Tighten the clamp to pro vide a seal on the gas ket. 14) Set the gas con trol valve to Fill Bot tom Chamber. Al low the bot tom cham ber pres sure to equili brate with the top cham ber pres sure. 15) Once the bot tom cham ber and top cham ber are at equal pres sures, the fol low ing five steps must be com pleted in or der and within 30 sec onds to freeze the crys tal. F1 F2 Turn the ball valve to the Open po si tion. Loosen the lever brake knob and raise the op er at ing lever to the top stop po si tion. F3 Pull the cryo cup po si tioner out. F4 F5 Re lease the lever from the top stop by pulling out on the lever re lease pin. Lower the op er at ing lever to the bot tom stop po si tion. Quickly de press the mag net re lease but ton, on the top of the mag netic pin holder, to re lease the crys tal and pin into the vial of car bon te trafluo ride. 16) Set the gas con trol valve to VENT BOTTOM CHAM BER. When the gas pres sure has dropped near 0, re lease the bot tom clamp and lower the de war and vial holder on the ele - vator. 17) Care fully re move the dewar, with the cop per cold fin ger in place, by slid ing it for ward. Slightly raise the lead ing edge of the dewar to re move it from the re cess in the plate. 18) Care fully re move the cryo vial, with the pin and loop, us ing cryo vial for ceps, and place the cryo vial in liq uid ni tro gen to main tain the tem pera ture. Oct. 30, 1998; Rev. A A-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

39 Glossary Ball valve - A valve operated by the large metal knob located be tween the top and bot tom cham bers. Chamber alignment tool - A tool used to align the top and bottom cham bers. Copper cold finger - The tool which sits in the dewar plastic cover and holds the cryo vial of liq uid carbon tetrafluoride. Cryo cup - The cup placed in the top chamber which holds the cryo pro tec tant. Cryocup po si tioner - A slide-handle outside the top chamber which allows the cryocup to be moved in and out of the top cham ber by magnetic cou - pling. Cryo genic temperature - A tem pera ture far below 0 o C. Cryo liq uids - Any sub stance which is a liq uid at cryogenic tem pera tures. Cryoprotectant - A so lu tion which helps prevent the for ma tion of ice while freez ing at cryo genic tem pera tures. Cryovial - A small vial designed to hold cryo liq uids. Crys tal - A protein sam ple which is fro zen at cryo genic temperatures. Crys tal mount ing pin - Pins used to hold the crystal for freez ing. D-ring - The large metal ring located near the top of the magnetic pin holder. D-ring slot - The open ing in the op er at ing lever as sem bly where the D-ring slides into place. De war - A con tainer used to hold liq uid nitrogen. Dewar plastic cover - The plastic lid for a dewar that holds the cop per cold fin - ger. Elevator - The plate where the de war is placed during op era tion. Cryo- Xe- Siter Op era tor and Serv ice Man ual G-1 Oct. 30, 1998, Rev. A

40 Glos sary Fer rule - A tool pro vid ing a seal for the gas tub ing, be tween the system and the nut. HPLC tubing - High Per form ance Liq uid Chro ma tog ra phy tubing for the gas flow between the gas tank, control box, and top and bottom cham bers. Lever brake knob - The large black knob to the left of the op er at ing lever, used to lock the operating lever in place. Lever re lease pin - A hand operated spring plunger which pro vides up per and lower lock ing positions for the operating lever. Mag net re lease button - The but ton on the top of the magnetic pin holder used to re lease the pin into the cryovial of carbon te trafluo ride. Mag netic pin holder - The ap pa ra tus used to hold and place the crystal mount - ing pin with the crystal into the Cryo- Xe- Siter. Op er at ing lever - A handle to raise and lower the magnetic pin holder. Pressure regu la tor - A valve to regulate gas pressure to the sys tem. Oct. 30, 1998, Rev. A G-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual

41 Index B Ball Valve, 1-4, , 2-7, , 3-4, A-1 - A-2, G-1 Bottom chamber, 1-2, , 1-10, 2-3, , 3-1, A-2 C Carbon tetrafluoride, 1-1, , , 2-6, 2-8, A-1 - A-2, G-1 Centrifuge tube, 2-1 Chamber alignment tool, 1-2, , 3-3, A-1, G-1 Copper cold finger, , , 2-9, 3-1, A-1 - A-2, G-1 Vial holder, , A-1 - A-2 Cryocup, , , A-1, G-1 Cryocup positioner, 1-4, , 2-8, A-2, G-1 Cryogenic temperature, 1-1, , G-1 Cryoliquid, 1-1, 1-6, 2-1, G-1 Cryoprotectant, , , A-1, G-1 Cryotongs, 1-5 Cryovial, , , , 2-9, A-1 - A-2, G-1 Cryovial forceps, 1-7, 2-9, A-2 Crystal, , , , A-1 - A-2, G-1 Crystal mounting pin, , 2-4, A-1, G-1 D-ring, , 3-2, , A-1, G-1 D-ring slot, , A-1, G-1 Decrease in pressure, Dewar, 1-5, , 2-9, A-1 - A-2, G-1 Dewar plastic cover, 1-5, 2-6, A-2, G-1 D E Elevator, 2-7, 2-9, 3-4, 4-1, A-2, G-1 Ferrule, 1-9, G-2 Fill bottom chamber, 2-7, A-2 Fill rate, 2-3, 2-5, A-1 Fill top chamber, 1-10, 2-3, 2-5, 3-1, A-1 F Gas control valve Fill bottom chamber, 2-7, A-2 Fill top chamber, 1-10, 2-3, 2-5, 3-1, A-1 Vent bottom chamber, 2-9, A-2 Vent top chamber, 1-10, 2-3, A-1 Gas leakage, , 4-2 G H HPLC tubing, , 3-2, G-2 Ferrule, , G-2 Finger-tight nut, 1-8 L Leak detection fluid, 3-2 Lever brake knob, 2-4, 2-7, 3-4, A-1 - A-2, G-2 Lever release pin, 2-4, 2-8, A-1 - A-2, G-2 Liquid nitrogen, , 2-1, , 2-9, A-1 - A-2 M Magnet release button, 2-8, A-2, G-2 Magnetic pin holder, , , 2-8, 3-1, 3-4, , A-1 - A-2, G-2 Maintenance Magnet car rier, Magnet release button, 4-2 Magnet release stem, Magnet release washer, O-ring, O-rings, 4-2 Snap ring, 4-3 Spacer ring, 4-3 Stem tube, Window bezel, O Operating lever, , , 3-4, 4-1, A-1 - A-2, G-2 Cryo- Xe- Siter Op era tor and Serv ice Man ual i Oct. 30, 1998; Rev. A

42 In dex P Pipette, 1-4 Pressure regulator, 1-8, 1-10, 2-5, 3-1, G-2 Pressure test, 3-1 Propane, 1-7 S V Vent bottom chamber, 2-9, A-2 Vent top chamber, 1-10, 2-3, A-1 Vial storage rack, 1-6 W Safety measures, 2-1 Styrofoam cup, 1-7, 2-6, A-2 Surface area, 1-7 System pressure, 1-7 Weight, 1-7 X T Xenon, 1-1, , 1-10, 3-1, A-1 Top chamber, , 1-10, , 2-7, 3-1, A-1 - A-2 Oct. 30, 1998; Rev. A ii Cryo- Xe- Siter Op era tor and Serv ice Man ual

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