Standard Operating Procedure for Crystal Mounting, Centering, Unit Cell Determination, Data Collection, and Integration on the DUO
*** IF AT ANYTIME YOU DO NOT KNOW WHAT TO DO OR SOMETHING DOES NOT LOOK RIGHT CONSULT A CRYSTALLOGRAPHER***
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Checking the liquid nitrogen and starting a new project:
1. Make sure there is enough nitrogen in the big dewar to run your experiment (at least 1/3 full), if there is go to step 2; if not talk to a crystallographer. Also the red display on the white box behind the instrument tells you how much LN2 is in the small dewar.
2. Check to see if the instrument is at low temperature:
2.A. Behind the instrument look at the blue Cryostream Controller box:
2.A.i. The top TEMP display tells the current temperature, while the bottom STATUS display tells the current status of the Cryostream
2.A.i.a. If the temperature is where you want it (100K) go to step 3
2.A.i.b. If the temperature is not where you want it and it is cooling down, wait until the TEMP display is stable at 100K, then go to step 3
2.A.i.c. If the TEMP display says SHUTDOWN, the Cryostream is currently off. To start the Cryostream:
2.A.i.c.1. Press the blue START button once, wait until the STATUS display says Cool to 100K, and then press the START button again.
2.A.i.c.2. Wait until the TEMP display is stable at 100K to begin your experiment.
3. The APEX3 software should be open, if it is not, open it from the desktop (do not open APEX3 server software).
4. Click the Sample dropdown in the top left of the screen, select New
4.A. In the New Sample Window:
4.A.i. Name = MCL Project Number (insert a “d” between the first letter and number)
4.A.ii. Group = Users
4.A.iii. Folder = Same as the “Name”, make sure there is a ‘\’ between the drive and the folder name (if the box is pink something is wrong)
4.A.iv. Click OK
5. ALWAYS SAVE THE CURRENT PROJECT IF YOU ARE ASKED, THAT IS SOMEONE ELSE’S DATA
Once you have set up your new project:1. Click the Setup tab on the left and select Describe Sample
1.A. Fill in the empty boxes:
1.A.i. Compound: project name_user’s sample id from lab notebook_user’s name/advisor’s last name (example: bd83b JAB358 Jeff Bertke/Gray)
1.A.ii. Formula: anticipated chemical formula
1.A.iii. Crystal Color: you only need to fill in the Primary Color
1.A.iv. Crystal Dimensions: this will automatically be filled in after face indexing
1.A.v. Crystal Shape: the shape of your crystal (common shapes include: plate, needle, column, cube, block, prism, etc…; simple descriptors are ok too: square plate, hexagonal prism, etc…)
2. Centering your crystal:
2.A. Click Center Crystal in the Setup tab
2.A.i. Click the Center button in the bottom right corner, once the goniometer stops moving you can open the doors and mount your crystal on the magnet. To unlock the doors press the circular silver buttons at the end of each black handle and slide the doors apart.
2.A.ii. Use the tool to center your crystal in the crosshairs on the screen. Only adjust from the front of the goniometer to center the crystal. At this time make sure both the beam stop for the Cu tube and the Mo tube are in place. If you do not see two beam stops on the instrument find a crystallographer before proceeding.
2.A.iii. Center your crystal with a series of 180o and 90o rotations using the large buttons on the computer inside the enclosure. ONLY CLICK THE SPIN PHI 90/180 BUTTONS. If you try to move any axis other than phi with the doors open it will result in an instrument error.
Unit Cell Determination
3. Click on the Evaluate tab and select Determine Unit Cell
3.A. Click on the Collect Data button on the right side
3.A.i. Be sure that the correct source is displayed in the Anode dropdown box.It will almost always be Mo.
3.A.ii. You might need to edit the exposure time (default is 10.0 sec., most of the time this is enough)
3.A.iii. You might also want to edit the Image Base Name, particularly if you have already collected a matrix in this project and do not want to overwrite the frames. The default is matrix, for each additional matrix you collect after the first one you can change this name so it will not overwrite the frames. Example: matrix, matrix2, matrix3…
3.A.iv. Click on the collect button at the bottom of the page to begin the collection
3.A.iv.a. Mo: matrix consists of 2 sets of 12 frames
3.A.iv.b. Cu: matrix consists of 3 sets of 20 frames
3.B. After the collection is complete, click the Harvest Spots button on the right
3.B.i. Check the Minimum I/Sigma limit; the default is 20, if your data is weak you need to lower this typically 10 or 5 works
3.B.ii. Check that the Store dropdown is an empty group, you do not want to harvest spots into a group that already has spots
3.B.iii. Click the Harvest button at the bottom of the screen
3.C. After harvesting spots, click the Index button on the right
3.C.i. Check the Minimum I/Sigma, the default is set to the value you used on the harvest spots screen, you cannot go lower than this value but you can go higher
3.C.ii. Check that the Store dropdown is an empty group, you do not want to overwrite a unit cell that is already present
3.C.iii. Click the Index button at the bottom of the screen
3.C.iv. The program will give you two unit cell options; it will highlight the one it thinks is the better choice.If this cell is reasonable click the Accept button at the bottom. If the other cell is better click on that cell to highlight it and then click the Accept button at the bottom.To determine what is a good cell look at the score for each and the % of spots that fit the hkl.A high score is good and you want a high % of spots to fit hkl.
3.C.v. Click the Refine button, if the value of the RMS Angle is:
3.C.v.a. < 0.30 click Accept, skip to section 3.D.
3.C.v.b. > 0.30 scroll to the bottom of the Parameters window and check Goniometer Zeros, click the Refine button and look at the RMS Angle
3.C.v.b.1. If the RMS Angle is < 0.30 click Accept, skip to section 3.D.
3.C.v.b.2. If the RMS Angle is still > 0.30, your crystal might be twinned, cracked, actually more than one crystal, or just not good enough. Select another crystal and start over at section 2. Centering your crystal.If you repeatedly get this problem consult a crystallographer.
3.D. After refining the unit cell, click the Bravais button on the right
3.D.i. Select the appropriate lattice type; the program highlights the one it thinks is best, but this is not always the correct choice.To determine which is the best choice:
3.D.i.a. Look at the Figure of Merit (FOM) given for each lattice; it should be close to 1.00.Note that Triclinic will always be 1.00 even if that is not the one you should choose.
3.D.i.b. Look at the cell parameters (a, b, c, α, β, γ) and make sure the unit cell makes sense.
3.D.i.c. Highlight the lattice of your choice and click the Accept button
3.E.After accepting a Bravais lattice, click the Refine button on the right and follow the directions from section 3.C.v from above and then move on to section 3.F
3.F.Look at the Expected Resolution table at the bottom of the screen.You have to be able to get to 0.83Å resolution in a reasonable amount of time per frame:
3.F.i. If you can get good resolution in a relatively short time ( ~60 seconds or less) skip to section 4
3.F.ii. If the table tells you it will take a long time per frame (>60 seconds or more) to get good resolution:
3.F.ii.a. If your judgment of the matrix frames tells you the crystal will diffract strongly enough to high angle either run the crystal using a reasonable time per frame or consult a crystallographer.
3.F.ii.b. If your crystal does not diffract to high angle in the matrix frames, you will not get good enough resolution for publication.Try to find a better crystal and start over at section 2. Centering your crystal.
4. Click on the Collect tab on the left
4.A.i. Click on the Calculate Strategy button
4.A.ii. Make sure the correct source is displayed in the Anode dropdown box
4.A.iii. If you are collecting with Mo radiation, change the resolution to 0.75 for a crystal that diffracts well (the minimum publication limit is 0.83, so never use a value larger that that)
4.A.iv. If you are collecting with Cu radiation, be sure your resolution is set to at least 0.83.
4.A.v. In the Symmetry drop down, be sure the chiral laue group is displayed.This ensures enough data will be collected if your crystal is in a chiral space group.
4.A.vi. Click the Determine strategy… button
4.A.vii. A small window will pop-up; be sure the Shutterless data, low temp. safe scan, and min. multiplicity check boxes are checked; and change the value in the minimum multiplicity for 90% of the data to at least 4.00; also make sure the Strict efficiency theta limitations box is NOT checked; click ok
4.A.viii. The program will calculate a set of scans and it will sort the runs (this can take several minutes).
4.A.ix. Once the calculations are complete, click the Select scan parameters button at the bottom of the screen.
4.A.x. Another small window will pop-up; change the Frame angle to 0.5 (this is the scan width of each frame) and change the Frame time to the desired length (the minimum is 1.0 sec. the maximum is 60.0 sec.)
4.A.xi. Be sure the Shutterless scans checkbox is checked
4.A.xii. Check to see that the Expected end time is reasonable and click OK
4.A.xiii. Click the Run Experiment button on the left
4.A.xiv. In the second row click the Operation dropdown and select Crystal Video.This will record a video of your crystal for face indexing before the experiment begins. See section 5 for instructions on face indexing.
4.A.xv. In the third row click the Operation dropdown and select Fast Scan.
4.A.xv.a. The time you set for this Fast Scan depends on your crystal and how strongly it diffracts at low angle.The point of this scan is to collect the low angle spots with less intensity than they will be during the calculated experiment.Ex. 60s exp. à 10 or 20s Fast, 10s exp. à 2 or 3s Fast, etc…
4.A.xvi. Click the Append Strategy button on the bottom left and your calculated experiment will load.
4.A.xvii. In the Exposures dropdown at the top of the screen be sure to select shutterless.
4.A.xviii. Click the Validate button on the bottom
4.A.xix. If all operations are valid, click the Execute button and your experiment will begin.Once your collection is complete, see section 6 for instructions on how to integrate your data.
Face Indexing Your Crystal5. Click the Reduce Data tab on the left and select the Index Crystal Faces button
5.A. You’ll see the camera image of your crystal.To face index your crystal:
5.A.i. Rotate the image by scrolling with the mouse until a dotted line shows up which is perpendicular to one of the crystal faces
5.A.ii. Move the mouse so that the solid black line is on top of the crystal edge and click.On the right side you should see and (h, k, l) appear.
5.A.iii. Repeat this until you have defined all of the crystal faces and the crystal is outlined by the black box you have defined. (Most crystals have “normal” faces such as 100, 010, 001, etc…)
5.A.iii.a. If the background is dark, you might want to change the color of the box by right clicking on the background and selecting Configure overlay…A window will pop up and you can change the color.
5.A.iv. Once this is complete, rotate the image until one of the dotted lines runs down the spindle axis of loop.This is the spindle axis of your crystal.Place the cursor on this line and press the print screen button on the keyboard.Open paint on the computer and paste the screenshot, save the image in your project folder with the title bd##b_faces.
5.A.v. When the face indexing is complete, the crystal dimensions will automatically be loaded into the Describe section of your project mentioned in section 1.A.iv.
6.A. Click on the Reduce Data tab on the left and select Integrate Images
6.A.i. Click the Find Runs…button on the right
6.A.ii. In the Select Runs window check the boxes next to your runs (ex. bd##b_01, bd##b_02, etc…) and uncheck the boxes next to matrix runs and click OK
6.A.iii. On the top right of the screen change the Resolution limit to 0.83 or less.Most of the time you’ll set the resolution to the same value you entered when calculating the experiment in Section 5.A.ii.0.83 is the minimum publication limit, so do not go higher than this.In the unit cell box below make sure that only one unit cell is checked and that it is the correct unit cell.
6.A.iv. Click the Start Integration button at the bottom and your integration will start.Four boxes will pop up:
6.A.iv.a. The top left is the correlation between the observed spots and the expected spots.This should be high, typically it averages around 0.8, if it is significantly lower this is an indication that the unit cell might not be correct, the orientation of the crystal has changed since you indexed the cell, or you have weak data at high angle.
6.A.iv.b. The top right plots the average intensity and the I/Sigma.You want both of these things to be high, but more important is that the I/Sigma is high.
6.A.iv.c. The bottom left is a 3-D image of the spot shape, you can click and drag to rotate this image, it should be spherical.
6.A.iv.d. The bottom right is the error in x, y, and z. These errors should be small.