BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography. Practical 3 Data Processing and Reduction

Transcription

1 BCH 6744C: Macromolecular Structure Determination by X-ray Crystallography Practical 3 Data Processing and Reduction

2 Introduction The X-ray diffraction images obtain in last week s practical (P2) is the raw data for your experiment. Other than the phase information, all the data that you require for solving your lysozyme structure is contained within the diffraction patterns. In the interest of time, these images have already been transferred to the McKenna lab Silicon Graphics computers for you. The aim of this practical is to index each of the reflections in all the images collected, i.e, assign each reflection its HKL value, along with its associated intensity (or amplitude, F) and error ( ). You will also scale the indexed data set to refine the crystal unit cell parameters and crystal orientation parameters and you will end up with a reduced data set, i.e., equivalent reflections (symmetry related) will be compared and listed just once, along with the error associated with all compared measurements. Outliers will be rejected, i.e,. those symmetry related reflections whose intensities are too different from the others (either too high or too low). The indexing, processing and data reduction will use Silicon Graphics workstations on which the HKL Suite (1) has been installed. The HKL suite is a package of programs developed for the analysis of X-ray diffraction data collected from single crystals, and consists of three programs that you will use during this practical. They are XdisplayF, Denzo and and Scalepack. XdisplayF will allow you to display the first (and all other) diffraction image to be indexed and do a peak search for the indexing by Denzo. Denzo will also allow you to refine the data collection parameters for the first image and use this information to index and process (reduction and integration) all subsequent images collected. All images will be displayed on the XdisplayF window during indexing and processing. Scalepack will merge and scale the relection intensities obtained by Denzo to generate a data set for phasing (P4). PLEASE NOTE For this and all the remaining labs you have to work on pairs - so please choose a partner. Accounts have been created in the SGIs for each pair of students, lab1, lab2, lab3 and lab4. In the instructions on the commands to use during this lab, extra information will be given in square parenthesis []. 1. Z Otowinoski and W. Minor, Processing of X-ray Diffraction Data Collected in Oscillation Mode, Methods in Enzymology, Volume 276:Macromolecular Crystallography, part A, p , 1997, C.W. Carter, Jr. & R. M. Sweet, Eds., Academic Press. 2

3 P3 At A Glance ImageWindow Launch XdisplayF Load images onto disk DenzoWindow Edit all.dat Confirm Xbeam, Ybeam Launch denzo Peak Search Verify that preds match spots Peaks file HKL Adjust mosaicity, spot size, check profile fitting radius Verify enough preds match spots:overlaps HKL Watch frames process Screen output &.e files Scalepack Log file.sca file 3

4 Getting that First Orientation: Indexing Step 1 Log on to the SGIs using the username and password assigned to your group. labx>cd Lyso/Process [this command changes the directory to your process subdirectory] [ indicates carriage return or enter] Open a second window on the SGI and cd to the process directory. Note: Your images have already been transferred from the PC used for the data collection to a local disk (/xtal5/bch6744/lyso###.osc, for fast access). Note: Your input files have also already been created in your accounts ~labx/process. You should have four input files, all.dat,.dat, process.dat and scale.com - see back of notes. all.dat contains the information for running denzo,.dat is a series of commands (also for denzo), process.dat contains the information for the processing of all the diffraction images after the first one has been indexed (also for denzo) and scale.com is for scaling your data in scalepack. Step 2 On one window, edit the all.dat [use jot or nedit] input file to update the experimental parameters used for your data collection: detector type [entered], wavelength [entered], oscillation start, range and step (*** to be updated), raw data filenames [entered], output filenames [entered], sector range (entered], space group or lattice [P1 has been entered, to be updated after initial indexing], mosaicity [to be undated during indexing and/or after scaling], resolution limits [value to be updated after initial indexing and/or scaling], spot properties [entered - may need to be updated], cassette properties [entered], crystal to film distance [entered], beam position [entered], y scale [entered], film rotation [entered], skew [entered], crossfire [entered], positional error [already entered], niometer information [entered], offsets [entered], longest vector [entered], peak file name [entered]. 4

5 Step 3 Your two windows will now be referred to as the Imagewindow and the DenzoWindow. In DenzoWindow: >denzo [launches Denzo In ImageWindow: >raxis4 /xtal5/bch6744/lyso3001.osc [launches the display program for R-Axis frames, this will display the first image] Step 4 In ImageWindow: Adjust the display so that you can see the spots clearly. Click on the Peak search icon: see tiny circles appear for the selected peaks. Use the more peaks /fewer peaks icon to obtain the desired number of peaks (~2000) in peaks. file In [this will use the peaks and begin autoindexing] Step 5 In ImageWindow: Make sure green circles (corresponding to whole reflections) cover real reflections (the black spots on image). Partial reflections are described by yellow circles. In DenzoWindow: Examine distortion table. Note highest symmetry lattice with low distortion. Hopefully, this should corresponds to thep1 lattice type you entered in the file all.dat. Now select the correct lattice type, exit both windows, edit all.dat appropriately, and repeat from Step 3. Now you have your first set of parameters. 5

6 Mosaicity and Spot Shape in Refinement Step 6 In ImageWindow: Hit Zoom Wind. Select a region of your pattern to examine. Hit the Int. Box button. Now you are ing to make sure your integration box parameters are correctly set. This exploits the advantage of Denzo's interactive analysis. Examine the diffraction pattern to determine the resolution limits. The resolution at the position of the cursor is displayed in the green box (on the right side of the window). Let's say it's 2.2 Å. In DenzoWindow: resolution limits [changes the resolution range entered] FIT [fits all the parameters entered in all.dat] [command file that repeats the fitting process numerous times] In ImageWindow: Examine the diffraction pattern. Are all the real reflections covered by predicted reflections ( preds. )? If not, you will have to increase the mosaicity. If there are too many preds., you will have to decrease the mosaicity. You started with a mosaicity of 0.5 degrees (see all.dat input file) and if you need to decrease it a bit. Here's how to change it. In DenzoWindow: MOSAICITY 0.4 GO In ImageWindow: Check again. Let's assume you ve t it right now. Note the value that works. You should not have to to unreasonable values of mosaicity (>> 1 degree)! Look at the integration boxes in the zoom window. Zoom in enough to see the spots. Are preds. big enough to cover the spot? Are they too big? Are the reflections so close together that the boxes overlap adjacent spots (lots of red circles)? If you have overlaps that a small adjustment of the spot or box size is not ing to fix, then you will have to adjust your data collection, perhaps by changing the oscillation range or the crystal-to-film distance. Let's say that your reflections are smaller than the inner white jagged outline, which is the Denzo spot area, and that a slight adjustment in the box size will prevent most of your overlaps. In the all.dat file, the spot size was defined as a 0.8 x 0.8 mm ellipse and the box print was 2.3 mm on a side. Change that now: In DenzoWindow: SPOT ELLIPTICAL BOX GO GO GO

7 In ImageWindow: Hit Prof fit R (set to 8.0 in all.dat). The display will show the profile fitting radius as a white circle and put preds. only on those spots with I/s greater than the weak level (default value 5 - this has been set to 2.0 in all.dat ). If you don't have enough spots for profile fitting (say > 10), you may want to increase the profile fitting radius. Let's say you need to increase it slightly: In DenzoWindow: PROFILE FITTING RADIUS 10.0 GO Hopefully you get the idea of how to adjust your parameters. 7

8 Refinement of Images Step 7 In begins sequential refinement of frames specified in the sector n to m section of all.dat (31 to 98 in all.dat). Depending on how fast your computer is and how fast you can read, examine the screen output for the correct histogram shape (meaning you chose the mosaicity correctly, and for od 2 values and convergence). If your all.dat file was set up correctly, things should work. In ImageWindow: Watch refinement proceed. Little circles should line up on reflections. Step 8 In DenzoWindow: After last frame, type STOP. This ends the program. Files containing hkl, I and for each image are generated, lyso3###.e. In ImageWindow: Hit Close button to kill image. You're done! Understanding the Denzo Log file in Refinement Step 9 Analysis: Scroll backward through the DenzoWindow to examine the histogram and the final 2 values. If they are normal, you can proceed to Scalepack. If this scenario didn't work and you entered the correct values in the all.dat file, we have to look into the input file carefully. Possible errors could be in X BEAM and Y BEAM values. Scaling (with SCALEPACK) Step 10 Edit your input file scale.com (to input your lyso3###.e files) and parameters, use jot or nedit. >scalepack <scale.com>scale.log [to run the scalepack program] The program will scale and combine the data from each image and scale them into a 8 single data set. It will also refine you cell dimensions and other parameters. You now can examine the quality your data. You will need tables at the end of the output for your write up.

9 title 'Lysozyme' all.dat [detector parameters] format raxis4 100 [source parameters] Wavelength [data collection setup] oscillation start *** oscillation range *** step *** [to be updated] [to be updated] [file names] raw data file '/xtal5/bch6744/lyso3###.osc' film output file 'lyso3###.e' sector 31 to 98 [Crystal properties] space group p1 use partials position mosaicity 0.5 resolution limits [to be updated] [to be updated] [to be updated] [Spot properties] spot elliptical background elliptical box print overlap spot weak level 2.0 profile fitting radius 8.0 error positional [Camera properties] cassette rotx roty rotz theta 0.00 distance x beam y beam y scale skew crossfire y x xy [niometer] spindle axis vertical axis [rotations] radial offset angular offset Film rotation 0.0 [Indexing parameters & peak file] longest vector 300 peak search file 'peaks.file' [start indexing] write predictions.dat 9

10 process.dat [The processing loop start here] sector 31 to 98 start refinement write predictions fix all resolution limits fit crystal rotx roty rotz x beam y beam fit cell fit detector rotx roty radial offset angular offset crossfire x y xy res 20.0 *** [to be updated] fit all print profiles 1 1 calculate end of pack Scale.com space group **** number of zones 10 estimated error error scale factor 5.0 reference film 1 format denzo_ip [scale restrain 0.05] rejection probability postrefine 20 fit crystal a* 1 to 67 fit crystal c* 1 to 67 fit batch rotx 1 to 67 fit batch roty 1 to 67 fit batch mosaicity 1 to 67 output file lyso.int resolution 20 *** [@reject] write rejection file 0.9 add partials 1 to 67 sector 31 to 98 file 1 lyso3###.e 10