User`s manual
Contents
1. HCib_UsersManual R02 docx January 2010 4 Getting started 4 1 Determination of the RH starting point Check that the Drop Size Tracking option is selected in the main window Center the loop perpendicular to the plane of the image and put a drop of the crystallization condition on tt Define the drop size selecting with the left button of the mouse the ROI region of interest Follow the drop size behavior If the drop increases the size decreases the RH value and if the drop decreases the size increase the RH value Repeat the procedure with a fresh drop until you reach a constant drop size r i i a 10 i Relativo Humidity Drop size 1 i heads Hae GAA Obi foi ititi Tera Drop Size Y1 Relative Humidity Y2 RH sapon Y Fi Drop size tracking EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 11 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 4 2 Dehydration standard protocol Set the RH value at the starting point determined as described before Mount and center a crystal removing the excess of mother liquor that surrounds it with a paper wick Take a diffraction image to characterize the initial state Space group unit cell mosaicity etc Decrease the RH value in steps of 1 letting the crystal reach the equilibrium 5 minutes approx and taking a dif2. J Huet S Russi 8 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1b UsersManual R02 docx 3 Software January 2010 To start the program double click in the HC1b icon placed at the Windows PC desktop 3 1 The main window Image of the drop including image processing overlays 4 EMBL HC Control Software 5 x File Help Monitoring A Relative Humidity 97 06 Relative Humidity Setpoint 97 00 Condenser Temperature 15 02 Temperature Setpoint Sample Temperature 25 01 Water Heater State Running Water Level Ox o Commands Humidity Temperature JV Drop Size Tracking Relative Humidity Scripts Run Stop Execution Time 00 00 33 10 i i i i i 15 01 10 15 01 20 15 01 30 15 01 40 15 01 50 15 02 00 Time Control j Condenser Temp Y1 Sample Temp Y1 Water Temp Y1 Stat Stop Relative Humidity Y2 ee toe Description I Session Folden Trp INFO x le ld i Ready Humidity and temperature controlers and script execution EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK Remote Host WARNING Jmulation M 14 34 26 INFO Control State Starti 14 34 26 INFO Contra Start control 14 34
3. 26 INFO iControl Set water heatWa temperature 29 0 15 02 08 Time plot graph of humidity drop size and or temperature 9 15 HCib_UsersManual R02 docx January 2010 3 2 Selecting the RH percentage Use the HUMIDITY button on the main window to enter the RH value required A EMBL HC Control Software f T File Help jee BE Sere Soe Ses ee Relative Humidity 3 Relative Humidity Setpoint Condenser Temperature Temperature Setpoint D s 3 n Sample Temperature 20 r Water Heater State oO Running Water Level x Temperature Relative Humidity IV Drop Size Tracking r Scripts Run Execution Time Control i0 i i Stop 17 57 50 17 58 00 17 58 10 i 17 58 20 17 58 30 17 58 40 ime Condenser Temp Y1 Relative Humidity Y2 Das tme tea oon F Session Folder Sample Temp Y1 Water Temp Y1 tl Remote Host 3 3 Running a script To run a script click on the main window RUN button select the script that you d like to execute and enter the parameters required leaving a space between each one EMBL HC Control Software H jaj xj File Help Monitoring Relative Humidity 92 19 Relative Humidity Setpoint Ol E ee saan econ ani a R E E E IREE ae E ERER cn PE E R EIE ETOR R B DE ROE RRE E BE ORR PR E BEI E RTI E RE ORR R E BE RR ER E REE E E R E EEI EE
4. relies on the fact that we can couple it with data collection and we can precisely control the speed at which the experiment happens In this way you have a direct measurement of the effect dehydration is having on your diffraction with no added treatment like cryo protection prior to data collection Why use this device and not others Currently there are other options to carry out this sort of experiments Acta Cryst D61 1173 1180 amp Applied Cryst 33 5 1223 1230 Despite being perfectly suitable to perform dehydration they are not very user friendly and are not available for use at a synchrotron beamline The new HC1b device is easy to mount on a standard EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 13 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 beamline without disturbing any of the other equipment It is easy to use and to monitor progress to anyone used to normal operation within a standard beamline Why perform this experiment in a beamline rather than at my home source In principle your homes source is perfectly good for this sort of experiments will definitely pose less threat to your crystals in terms of radiation damage and you will not waste a day of good beamtime Despite this many bad diffracting crystals only give any hint of diffraction once they are tested on a synchrotron and thus will hav
5. HC1ib_UsersManual R02 docx January 2010 HC1b Humidity Control Device User s manual EMBL 2223222 Grenoble EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 1 15 http www embl fr research unit cipriani index html Current address Daimond Ligth Source UK HCib_UsersManual R02 docx January 2010 Before you start It is important that a number of points are considered before using the HC 1b e Crystals need to be brought in their plates to the synchrotron or crystallised onsite a good stock of crystals is necessary e Crystals that have been cryo cooled already are not suitable for these experiments e The crystallisation condition cannot include high amounts of volatile compounds e Due to the experiments are carried out at room temperature crystals need to be stable between 20 and 25 C e Extremely delicate crystals photosensitive O2 sensitive radiation sensitive may have problems e Meshed loops must be used to mount the crystals because they allow removing the excess of mother liquor that surrounds the crystals without damaging them e Exposure time and flux need to be minimised to prevent radiation damage e Crystals need to have enough scattering power to be able to index the image s e The outcome of the experiment is totally empirical and dependant on the intrinsic nature of the individual crystals These general guidelines are given as a tool to help the experiment but they do not guaran
6. R E EE E Condenser Temperature Temperature Setpoint aa 3200 1508 I Sample Temperature 25 03 Dr p Water Heater State C Unknown Temperature Setpoint Commands Humidity Temperature IV Drop Size Tracking Single Gradient olL i 90 1 wy Parameters 0 50 00 10 50 30 Final RH Relative Humidity Y2 Step size Description Step time seconds Enter khe script s parameters Cif anyi oz 1 30 10 47 57 728 ARNING ji ace Manual command to start control 26 10 09 10 47 57 728 INFO Control Set session null 26 10 09 10 47 57 728 INFO o Starting 26 10 09 10 47 57 728 INFO Control Start control 26 10 09 10 47 57 728 INFO Contral a Ready 26 10 09 10 48 13 9 WARNING interface Manual command control 26 10 09 10 48 13 9 INFO Control control lx ALE Te gt ne T lt lt J 7 Ready 10 50 55 start Beaea8Gou s Inbox Thunderbird E presentations _juan 9 srussi E HC_statistics_of_ ohe_1 4 1 E Microsoft PowerP EMBL HC Contr en Vg 10 50 automatizaci n For detailed information about the software see the HC1x Software Manual EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 10 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK
7. S Russi 5 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HCib_UsersManual R02 docx January 2010 Power supply Connect the power plug at the back into a standard wall socket Serial connector Pee Connections at the back of the HC1b All you need plugged into the HC 1b The humidifier nozzle head Mounting In order to mount the humidifier Nozzle you first need to remove the cryo stream Before remove the cryo strem unscrews the annealer and removes it gently off the nozzle then unscrew the two allen screws that hold the nozzle in place and once they are loose gently remove the cryo stream There is a special holder at the back of the beamline where you can place it The nozzle can be left in this place indefinitely whit the cryo stream still in operation Alignement Adjust manually the vertical height with the screw at the top of the holder If needed readjust the horizontal and vertical position of the nozzle to ensure a centred crystal is fully covered by the air flow Once the nozzle is in place mount a loop centre it and then attach the alignment tool to the nozzle If required readjust the position of the support to ensure the loop is in the centre of the air flow EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 6 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 doc
8. e not opportunity at a home lab Furthermore the time required for collecting a single frame in house 5 30 minutess gives very little opportunity of recording changes that happen within a shorter time span At a beamline the typical exposure time 0 1 2 seconds permits accurately characterising the most subtle change Lastly despite requiring a long time these experiments are often performed as part of a broader approach to obtain suitable data In many important cases a number of synchrotron shifts is required to test hundreds of crystals that have been harvested over weeks or months of careful work in order to obtain that elusive dataset If this beamtime is used responsibly it can only help in achieving that final goal What do I need to look for in my crystals This is a difficult question as no thorough work has ever been undertaken There are several pointers that may hint about the likeliness of your crystals changing In principle the solvent content may be a good indication that there is room for improvement The more solvent in your molecules the more solvent can be excluded and the more space there is for the molecules to move Despite this keep in mind that if the crystals harbour a membrane protein with some form of detergent or membrane between the different protein molecules there is not as much removable solvent and thus may not permit the required rearrangement The second thing to look for is a protein capable of crystallising i
9. fraction image to compare with the previous ones In case that an improvement is observed further optimization will be needed EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 12 15 http www embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 Appendix FAQ How does the machine work The machine produces an airstream around the sample at controlled relative humidity RH that can be used to change the hydration state of macromolecular crystals This airstream is generated by using DewPoint to remove water from a hot and saturated airstream so that it will be at the correct RH when it reaches the crystal In practice what you will see at the beamline is a device next to the diffractometer and a nozzle that looks like a standard cryo stream Your crystals can be directly mounted by hand as you would normally if you where cryo cooling directly on the beamline and from that point onwards nothing much changes from a standard data collection Why use dehydration Dehydration is a known factor to affect crystal lattice The changes it produces in the packing can in some cases lead to improved diffraction properties Unit cell contraction decreased mosaicity space group changes and even improved diffraction limit are among the documented effects Why use a device and not chemical methods The advantage of using a device to control dehydration
10. n a number of alternative spacegroups and or unit cells more than 1 Interesting also are those crystals capable of changing packing upon the addition of substrate additives cryos Or that change with different time of harvesting soaking or cryo protection All this sort of changes is indicative of the possibility of movement within the crystal and thus potentially very useful Symmetry may also be important In order to improve the internal order of the crystal local rearrangements are needed to realign the molecules or to stabilise flexible EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 14 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 areas of the individual proteins If the arrangement of the molecules is such that they establish a great number of equivalent crystal contacts there may be very little hope for such a high number of contacts to be broken and reformed differently On the contrary if the symmetry of the crystal is such that a with small compression or rearrangement new symmetry relations can be created a greater number of previously unrelated molecules can now co contribute to the overall scattering thus increasing the diffraction power order of the crystal Furthermore a similar effect may be triggered by dehydration if the asymmetric unit is constituted by a number of non symmetric monomers What do do af
11. nal Air Flow WAGO Meter Controll Ne Internal Peristaltic 44S eee DC O rl o E 7 A ee gt ee el Air Flow Pumps Adi aT X Meter a i ee Tene pa 5 Water Reservoir Hot Water Bottle EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 4 15 Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 The 5 water reservoir and the hot water bottle are filled with destilled water and the minumun and maximun levels are indicated If any of them need refilling and you re unsure of how to proceed contact a member of the Diffraction Instrumentation Team at the EMBL for assistance 2 Installation 2 1 Connections Air supply Connect the 6 mm tubing attached to the device to the manual valve connected to the air or nitrogen supply in the beamline blue or yellow tubing respectively Once its Connected you can open the valve The air connectors lock by simple pressure Make sure they are tight before you open the valve To disconnect them you need to push the blue ring at the same time you pull the tubing apart 6mm tubing amp Connecting Disconnecting connector I O serial port There is an I O serial port at the back of the humidifier find the one attached to the back of the Windows PC in the hutch Plug the I O serial cable to both the device and the computer respecting male female compatibility EMBL Grenoble Outstation J Sanchez Weatherby J Huet
12. ter dehydrating Dehydration may actually be an alternative way of cryo protection Dehydration will exclude water while increasing the concentration of the buffer within the solvent channels and can thus prevent ice formation Furthermore much of the mechanical stress crystals undergo upon flash cooling can be minimised by having contracted the lattice to a minimum prior to cryo cooling What if cannot cryo cool my crystals after dehydration lf all attempts to cryo cool the crystals fail to yield a suitable dataset an alternative option is to collect at room temperature Despite the device being stable enough to permit collecting a full dataset it will have to be collected with great caution Firstly radiation damage is a mayor worry and thus the data collection strategy has to be meticulously optimised It may even be necessary to collect data from several crystals in order to get a full dataset Secondly as crystals are not cooled they may move complicating data processing EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 15 15 http www embl fr research unit cipriani index html Current address Daimond Ligth Source UK
13. ty success EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 2 15 http www embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 1 The HC1b 1 1 The casing The HC1b is mounted on wheels so it can be easily transported between beamlines You can pull and steer by using the front handle There are brakes on both front wheels that are operated with your feet Brake off Brake on HC1b Handle There are two serial I O connections two air inlet connections an Ethernet connection and a warning ligth at the back of the device There are two air flow meters an emergency stop button and on off button at the front Front View of HC1b Stop Back View of HC1b ON OFF Switch EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 3 15 http www embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 The casing device can be opened by unscrewing two central allen screws at the top of the device Once they are unscrewed the side panels can be lifted by hand For safety reasons the two covers and the two top screws should always be mounted when the device is running Do not unscrew the four screws at the sides Removal of the cover 1 2 Internal components DG4 DewPoint Aa ER Controller UF E ee i OE naor Exter
14. x January 2010 Nozzle head into the HC1b support Nozzle mounted onto the cryo support EMBL Grenoble Outstation J Sanchez Weatherby J Huet S Russi 7 15 http Awww embl fr research unit cipriani index html Current address Daimond Ligth Source UK HC1ib_UsersManual R02 docx January 2010 2 2 Checking that everything is working OK Warning light Switch on the machine with the on off button placed at the front side green light in button become on and check that the state light on the back side is green and blinks Wait approximately 15 20 minutes for the stabilization of the device and check if the state light became fixed and green If the light turns yellow or red you must contact a member of the Diffraction Instrumentation Team at the EMBL for assistance Air flow The external protective air flow should be at around 10 l min and the internal air flow at about 5 l min Check the flow meters at the front of the device to ensure the flow is correct The correct flow should produce an even rotation of the fan too fast will become unstable and too slow will not move fast enough If the two air flow are zero after a rotation of adjustment button check air pressure connection 2 1 or contact a member of the Diffraction Instrumentation Team at the EMBL for assistance External Air Flow 10 I min Internal Air Flow 5 I min Control of the internal and external air flow EMBL Grenoble Outstation J Sanchez Weatherby
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