Remote Access using samples at non-cryogenic conditions

Using the SSRL Automated Mounting (SAM) system

Overview

SAM is a completely integrated hardware and software system for mounting and dismounting pre-frozen protein crystals and screening samples for x-ray diffraction quality in a fully automated or semi-automated fashion. SAM is installed on all of the SSRL macromolecular crystallography beam lines and is seamlessly integrated into the beamline control and data analysis software. Since upgrading the robot system in 2014, the screening sequence (comprising crystal mounting, automatic sample loop centering in the x-ray beam, video and diffraction image acquisition at 0 and 90 degrees, and dismounting) may take less than one minute per crystal. The diffraction images are analyzed and autoindexed on the fly.

Samples may be stored in either SSRL cassettes or Uni-Pucks for use with SAM. If you are scheduled beam time with use of the robot sample mounting system, a cassette kit will be lent to you.

The following sections describe how to prepare your crystals for data collection using the SSRL robotic sample mounting system. Follow these instructions to prepare the sample pins, mount them in an SSRL cassette or Uni-Puck and ship them to the SSRL.

For additional information on the SAM system, see also the guide to automated sample screening. There are also video tutorials illustrating the sample preparation, sample container loading using SSRL cassettes, and Unipucks and use of the SAM system for data collection.

For information about the cassette kit tools, including drawings and vendor information, see the SAM hardware web pages

**Figure 8:** The two types of container for automated sample mounting at SSRL

Sample pin selection and preparation

To have a successful experiment proper sample pin preparation is essential. The majority of problems we have observed with SAM have been related to use of improper sample pins. To avoid these problems please read the following directions.

Allowed types of pins

The SSRL system supports only Hampton-style CrystalCap Copper Magnetic pins or CrystalCap Magnetic pins. The allowed pins sizes are 16 or 18 mm. The 18 mm size copper pin is preferred.

**Figure 9:** Preferred size of pin. Hampton cryo-loops should be cut at the segment closest to the loop for use with the CrystalCap Copper pin bases, provided with the cassette kits. **Do not exceed the maximum MicroTube length shown in the diagram!** Unacceptable lengths are marked with a red x. The 11 mm MiTeGen Micromounts and meshes should be used with the CrystalCap Copper pin base; longer Micromounts will need to be cut to the proper length before inserting into this pin base.

Compatible Hampton-style sample pins may be purchased from a number of vendors including Hampton Research, MiTeGen, Crystal Positioning Systems or Molecular Dimensions.

If sample pins are purchased from MiTeGen, use the B1, B1A or B3S with the 18mm MicroMounts, MicroLoops, MicroMeshes or MicroGrippers. The B1, B1A, B3A and B3 bases can also be used with 19 mm mounts. The older B2 base can be used with 11 mm Micromounts and 10 mm nylon loop mounts. Please note that the SSRL loop-centering routine, based on visual analysis, is not yet optimized for use with the MiTeGen MicroMount, these mounts, as well as the loops are well suited for rastering. SPINE-standard pins can not be used with SAM.

**Figure 10:** Commonly available pin types

We recommend using Hampton-style Copper Magnetic pins because no laser etched lines are exposed. These are the pins supplied with the cassette kit. The microtubes used with Magnetic pins can sometimes break off if bumped.

**Figure 11:** Broken microtubes used with Magnetic pins can make the pin jam inside the cassette port, stopping the robot.

Pin preparation

Microtubes should be affixed inside sample pins using epoxy. Any epoxy with a curing time between 5 minutes and 24 hours should work well for this purpose. We have found that superglue and superglue gel is less reliable than epoxy for affixing microtubes. A number of failures with the SAM system have been attributed to using other types of adhesives on sample pins such as wax, nail polish, and Duco cement. These should not be used.

**Figure 12:** Use Epoxy to affix the microtubes to the sample pins. Glues other than Epoxy have been observed to cause sample mounting errors.
$\includegraphics[width=0.6\textwidth]{images/epoxy.ps}$ $\includegraphics[width=0.6\textwidth]{images/glues.ps}$

If an adhesive other than Epoxy has been used to affix the microtubes to your sample pins, please let your user-support person know in advance of using them. These pins may cause problems with SAM.

Be careful not to use pins with excess epoxy on the pin base or postpin-excess and do not get grease or excess cryo-protectant on the pin body. At liquid nitrogen temperatures, grease from crystallization trays gets rock hard. If you continually reuse your pins, please also inspect them for corrosion and loose microtubes. Anything that changes the outside form factor of the sample pin could cause the pin not to fit properly in the SAM robot tongs.

Warning: Do not use pins with excess epoxy on the copper post.

If you would like to mark your sample pins different colors, use permanent marker for this purpose. Paint or nail polish should not be used as this can change the form-factor of the pin or be sticky. Hampton Research now sells a new pre-assembled Copper Magnetic sample pin (catalog number HR5-112) which is already color coded according to the size of the nylon loop attached. It also has an alpha numeric code and bar code.

Pin testing

All pins must be tested to ensure they fall within the allowed tolerances. We have found some irregular pins that if used would damage the SSRL sample mounting system. To test your pins, place them on the end of the Pin Tester magnetic tool on the red line. Only use pins that completely cover the red mark without forcing. Pins that fail this test should not be used!

**Figure 13:** Use this tool (included with the SAM kit) to test all your pins before use with the SAM robot

Re-using pins

Broken micro-tubes and torn nylon loops are usually a result of mishandling sample pins in preparation for reuse. In particular pins should not be piled together in a container when washing and drying them. To wash pins, place them individually on a magnetic tray. Hang the tray upside down to dry the pins. It is important to store your sample pins in a safe place when they are not in use. The microtube storage rack is a useful storage location for extra pins.

**Figure 14:** Storing pins for re-use.

Loading and shipping SSRL cassettes

SSRL Cassette kit

If you are scheduled beamtime with use of the robot sample mounting system, a cassette kit will be lent to you.

For additional information about the kit tools, including drawings and vendor information, please see the SAM hardware web pages.

**Figure 15:** Cassette Kit: (A) Sample Cassette and 96 Hampton pins (microtubes and loops not included) (B) Dewar Canister - replaces stock canister in dry shipping dewars (C) Teflon Ring - to support the canister in the shipping dewar (D) Transfer Handle - for transferring cold cassettes (E) Magnet Tool - to mount pins in cassette and to the test size of pins (F) Guide Tool - to aid in mounting pins into cassettes with the magnetic tool (G) Styrofoam Spacer - to keep the cassette in place when shipping one cassette; not shown: A dewar for mounting crystals into the cassette.

The cassette contains 96 sample ports each port contains a ring magnet which holds in the sample pin. A cutaway view of the cassette is shown below. The ring magnets are shown in green. The magnets are held in place by a polycarbonate washer shown in white. The washers are removable so any broken ring magnets may be replaced.

**Figure 16:** Cassette diagram. The ring magnets are shown in green and the washers holding the magnet are shown in white
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Cassettes should not be stored where magnetic debris can get inside the ports. Before using your cassette, it should be inspected to ensure the ports are all empty.

The transfer handle is used to safely transport cold cassettes. To attach the transfer handle (D) to a sample cassette (A) first place the locking pins into the slots at the top of the cassette. Then push down the handle, and rotate clockwise until the handle locks in place.

**Figure 17:** Attaching transfer handle to cassette
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The slotted guide tool consists of a long magnetic wand with two slots in the middle and a loading guide with a track on the handle that accommodates the slots on the wand tool. To flash freeze samples, the wand may be inserted into the top of the loading guide. To transfer pre-frozen samples it is important to keep the samples at liquid nitrogen temperatures during transport into the cassette. The wand should be inserted from the side of the slotted guide tool to keep the sample under liquid nitrogen during transfer. Use the slot closest to the red (or maroon) side of wand (weak magnet) for loading samples.

**Figure 18:** Guide tool
$\includegraphics[width=0.85\textwidth]{images/guide_tool.ps}$

When unloading samples the wand is turned around and the opposite slot and stronger magnet is used.

The circular cutout in the loading dewar may be used to hold samples in cryo-vials or pucks for transfer.

Avoiding ice

The most common problems we see observe with cassette loading is the accumulation of ice in the liquid nitrogen bath. Ice will stick to your sample as it is transferred through the liquid nitrogen on the way to the cassette port. It can also fall inside the cassette ports. If excess ice is observed in the bath, the cassette should be stored in a cold dry-shipping dewar and the loading dewar emptied, dried and refilled with liquid nitrogen before proceeding.

Important: To prevent ice from accumulating in the liquid nitrogen, the cassette should not be stored in the loading dewar for more than 20 minutes without exchanging the liquid nitrogen.

To avoid ice accumulation, it may be useful to to load cassettes under a fume hood or a dry box (as an example, see a description of Nham Nguyen's setup at
http://smb.slac.stanford.edu/facilities/hardware/cassette_kit/Ice free_Nham.pdf). If not using a fume hood or a dry box, cover the dewar with the lid when you are not mounting crystals. To prevent ice from falling into empty cassette ports, some users advise filling empty ports with blank pins. Each blank pin is removed just before inserting a sample pin into the port.

Loading samples in the SSRL cassette

Important: Please read the safety notes before working with liquid nitrogen and make sure you are familiar with the liquid nitrogen safety procedures at your institution.

The custom foam dewar should be filled up to the internal indicator ledge This takes about 4 Liters of liquid nitrogen; it will be necessary to top off the liquid nitrogen level after inserting a warm cassette.

Figure 19: Filling the custom foam dewar

$\includegraphics[width=0.9\textwidth]{images/dewar_filling.ps}$
Once the dewar is filled and equilibrated, place the cassette in the dewar and tilt the transfer handle until it rests inside the side notch. Then push the cassette forward until the bottom of the cassette is touching the edge of the dewar.
The guide tool may be pre-cooled on the side dewar shelf. To prevent condensation on the metal portions of the guide, it may be stored on the shelf between loading samples.

Figure 20: a. Placing the cassette in the foam dewar. b. Storing the guide tool.

$\includegraphics[width=0.4\textwidth]{images/foam_dewar.ps}$ $\includegraphics[width=0.35\textwidth]{images/precool.ps}$
Rotate the sample cassette with the transfer handle to access the desired cassette port.
Place the guide tool (F) on the cassette centered on the port.

Figure 21: a. Placing the guide tool into the cassette. b. Putting the pin on magnet tool. c. Picking the crystal.

$\includegraphics[width=0.8\textwidth]{images/p10.ps}$
To flash freeze a sample:
1. Put a pin onto the red side of the magnet tool (E) and pick up a crystal.
2. Flash-freeze the crystal by placing the pin through the handle of guide tool and into the cassette port, minimizing the time the crystal is in the air.
Figure 22: Flash freezing the crystal

$\includegraphics[width=0.2\textwidth]{images/p12.ps}$
To transfer a pre-frozen sample from a vial (or puck):
1. Tilt the vial and use the red side of the magnetic wand tool to remove the sample pin. Be careful to keep the sample under liquid nitrogen at all times. (Vials or pucks may be placed in the round cutout inside the dewar.)
  
  Figure 23: a. Placing the guide on the cassette. b. Picking the pin. c. Transferring the pin
  
  $\includegraphics[width=0.9\textwidth]{images/guide_dewar_placement.ps}$
2. Slide the lower slot of the magnetic wand tool into the guide tool while keeping the sample under liquid nitrogen.
  
  Figure 24: Using the guide slot under liquid nitrogen.
  
  $\includegraphics[width=0.6\textwidth]{images/guide_slot.ps}$
3. Once the magnetic wand tool is in the center of the guide tool, the wand tool may be pressed against the back of the guide and pushed downward placing the pin into the port. (It is sometimes helpful to rotate the wand while it is pushed down to break any ice that may have formed between the magnetic wand tool and sample pin.) The magnetic tool may then be pulled up, removed and the next sample transferred.

As you fill the cassette we recommend you record the location of each crystal as described in the sample Excel spreadsheet documentation. A separate file should be created for each cassette.

Preparing cassettes for shipment

Important: It is strongly recommended to test the shipping dewar prior to shipping samples to make sure that the samples will be kept cold before arrival.

Prepare the dewar for shipping; place the Teflon support ring (C) inside the shipping dewar before inserting the canister (G) and fill the dewar with liquid nitrogen in the usual manner.

Figure 25: Placing the Teflon support ring (C) inside the shipping dewar

$\includegraphics[width=0.2\textwidth]{images/p15.ps}$
Transfer the cassette into the canister in the dewar, minimizing the time the cassette is in the air. Remove the transfer handle from the cassette by pushing down on handle and turning counter-clockwise to release it.
Two cassettes may be shipped inside one canister. When shipping just one cassette, place the Styrofoam spacer (F) on top of the cassette to keep cassette in place during shipment.
The cassettes are compatible with most dry shipping dewars. We recommend using the combination of a MVE model SC4/2V, Taylor Wharton CX100 or CXR100R cryogenic shipping dewar with a Taylor Wharton TAY CX10-8C00 dewar container.

$\includegraphics[width=0.2\textwidth]{images/p16.ps}$

Styrofoam spacer

For information on how to ship dewars to and from SSRL see the User Shipments web page.

Loading and shipping Uni-Pucks

For information about loading and shipping samples in the Uni-Puck see:
http://smb.slac.stanford.edu/facilities/hardware/cryotools/Uni-puck/Uni-puck_Directions.pdf

Important: It is strongly recommended to test the shipping dewar prior to shipping samples to make sure that the samples will be kept cold before arrival.

Storing sample information: The Excel spreadsheet

Sample information is entered into a Excel spreadsheet file. The spreadsheet has a specific format for use with the SSRL Sample Database and data collection applications.

The following instructions explain how to download, fill up and upload a spreadsheet.

You can download a spreadsheet template for the SSRL cassette, Uni-puck adapter (holding up to four pucks) or SSRL plate from the SSRL Sample Database (New Spreadsheet tab).
If your browser cannot display Excel spreadsheets, click the Save to file button in the pop-up menu; navigate in the dialog box to the directory of choice and click Save.
Type in the information for the samples in the cassette. Important: Make sure that ''text'' format is used.
If you are sending more than one cassettes, use one spreadsheet per cassette. The Uni-puck spreadsheet holds information for four pucks (labeled A, B, C and D).
- The ContainerID field is used to uniquely identify the cassette. You should enter the number engraved on your cassette or puck in this column; this is useful to verify that the spreadsheet position specified in the software matches the actual position of the cassette in the robot dewar. An ever safer method to prevent against container misidentification is the use of a barcoded pin in the A1 position of the SSRL cassette or any Uni-puck.
- The Port field indicates the position of the sample in the SSRL cassette. ID and Directory are used by the screening software to identify and store files. The other fields are not used by the system, but they are intended to help track and identify each sample.
  
  Important: Use only alphanumeric characters with no blank spaces for the Crystal ID. Special characters, brackets and spaces are not valid in image file names and will cause the screening to stop.
- After downloading or editing the spreadsheet, verify that it is saved as a 'Microsoft Excel Worksheet' or *.xls.
Figure 26: Formatted Screening System Excel Template.

See also information on editing the spreadsheet.

Uploading an Excel spreadsheet

Uploading the spreadsheet from a Web browser

Once the Excel spreadsheet has been filled out it can be transferred to the Sample Database at the URL:

http://smb.slac.stanford.edu/crystal-server/

Log in to the database interface using your account name and password. You also need cookies and javascript enabled in your browser to log in and upload the spreadsheet
Once you are logged into the system you should see the page shown below.

Figure 27: The Sample Database interface.
Click on Upload Spreadsheet. You will be directed to the Upload Excel File page.
Enter the name of the spreadsheet (you will need the full directory path, for example: /home/yourid/filename.xls) or click on the Browse button to search for the file. If you have trouble locating the file, verify that the filter for types of files is *.* or *.xls in the Choose File dialog box. Once the file is located in the dialog box, select Open.
Enter the Cassette number in the Cassette Pin box. If the Excel file does not contain a ContainerID, it will be generated and assigned the value you enter. On the other hand, if the spreadsheet already contains a ContainerID, the value you enter here will be ignored. Do not modify the default Spreadsheet name unless you changed it in your Excel file.
To finish uploading the file to the Database, click Upload. If your spreadsheet is not in a standard format (e.g., empty or duplicated CrystalID, no ContainerID) the interface will apply and display the necessary corrections in a separate page. Scroll to the bottom of this page and click on Display Cassettes. This will take you back to the home page, displaying the new spreadsheet entry.

Figure 28: Uploaded spreadsheet information.
To screen more than one cassette, click on "Create New Entry" again and upload the corresponding spreadsheet, etc.
Entries can be removed from the database by clicking Delete entry.

Important: If you do not wish to fill up the spreadsheet, it is possible to use a Default Spreadsheet.

Uploading the spreadsheet from Blu-Ice

Once you beamtime has started and you have been enabled to start the experiment, it is possible to access the Sample Database directly from the screening tab in Blu-Ice: Clicking the Web button will launch a web browser and you can upload the file as described in the previous section.

Assigning the Excel spreadsheet to a beamline

The information in the database may be assigned to a beamline once beamline access has been permitted. On the Sample Database page, the last entry (Beamline column) is used to assign the Excel spreadsheet to a particular beamline or to a particular cassette location. A total of 3 cassettes can be placed in the cassette storage dewar and are labeled 'left', 'middle' and 'right'. Select the appropriate position (staff will let you know which one to use) and beamline by using the drop down menu. If the assignment fails, ask the support staff to verify that your account has beamline access.

The spreadsheet assignment can also be done directly from Blu-Ice using the Cassette drop-down menu.

Using the Default spreadsheet

Sample screening without a spreadsheet is possible, but the results of the screening will not be saved. To avoid this problem, log in to the Sample Database interface as described above and click Use Default Spreadsheet. Select the cassette type (SSRL or Puck adapter), type in the cassette number if known and click Submit. A cassette named cassette_template.xls will appear in your spreadsheet list. The spreadsheet can be assigned to a beamline and dewar position as usual.

Editing the spreadsheet

The easiest way to make extensive changes to an uploaded Excel spreadsheet is to download the file to the local computer, then edit it and upload the modified file as explained in the previous section. The link Download Original Excel file can be used to retrieve the original uploaded file, without any edits or results. If you wish to modify the file after screening results are available, use the Download Results link instead.

You can edit the Excel file on the Unix beamline computer with the LibreOffice software. To run LibreOffice from the beamline computers type:

% libreoffice filename.xls

It is also possible to edit selected fields for a sample by clicking on View/Edit. This displays an HTML version of the spreadsheet

**Figure 29:** Editable HTML version of the spreadsheet.

Check the Port containing the sample you wish to edit
Click on the Edit Crystal button on top of the spreadsheet.
Edit the information in the field(s) of your choice
Click on Save changes to save your edits or Cancel to reset the old values.
Download the modified spreadsheet to the local computer by clicking on the Download Results link.

During the experiment, Blu-Ice can also be used to edit selected fields. Consult the Blu-Ice documentation to find out how to edit the spreadsheet on these applications.

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