Home · About CCP4 · CCP4 Projects · Downloads · Documentation · Courses · Developers · CCP4 people · WG1/WG2 · Privacy. Other MR examples can be found at the end of this tutorial, and at: When this tutorial is obtained as part of the CCP4 distribution, $MR_TUTORIAL. Previously Lecture Notes and Tutorial Material. 1、 Lecture Notes and Tutorial Material. iMosflm training · Mosflm examples. 2、 Lecture Notes and .
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See also the accompanying document giving background information. In the following instructions, when you need to type something, or click on something, it will be shown in red. Output from the programs or text from the interface is given in green. MOSFLM can process diffraction images from a wide range of detectors and produces, as output, an MTZ file of reflection indices with their intensities and standard deviations.
Experience has shown, that most people benefit from being familiar with the program before learning about the STRATEGY option, which therefore in this tutorial features only after integration.
The tutorial also covers the effects of changing integration parameterslocating outliers and restarting an interactive Mosflm process. These are crystals of a small domain 91 amino acids that have been soaked in a mercury compound.
Alternatively, get the images directly from http: The input file runit contains the minimum information required to start analysing the data:. However, this depends on the software being correctly installed at the beamline, and the necessary communication between the program controlling data collection and the MAR software, and this is not always reliable.
Various processing parameters can be entered in the “Processing params” panel on the left. Within the image display section, there is a small “blow-up” window in the top left corner approximately 20mm square. When the mouse is in the image display, clicking middle mouse button will improve the resolution of this window, but it can no longer be moved.
The Zoom feature has been disabled on some SGIs because it can cause the terminal to freeze. Change the Max value in the image display window to and zoom an outer part of the image to get a better idea of the quality of the image.
Click on Circles in the Main menu to display resolution circles. The resolution of the circles is given in the Output panel. The window will be closed automatically when the task has been finished. The shape of the symbol depends on the terminal program you are running – it could be a rectangle seen on Unixa letter seen on Linuxor a Windows symbol.
The success of autoindexing is critically dependent on the accuracy of the direct beam coordinates. The required accuracy will depend on the spot spacing to avoid mis-indexingbut in general you should know the direct beam position to within 0. The best way to locate the direct beam position is to look at a powder diffraction ring e. In this example it is being used to define the direct beam position.
Read what is in the small window because you have the option of changing both the beam position and the position and size of the backstop; you only want to do the former in this case! If you want to remove the circle, select Circles and then Erase circles from the main menu. Auto-indexing works well with either single or multiple images, and with – spots: For these data, the true direct beam position is within 0. If it were much further away, autoindexing would not work. Look for a gap of around e.
NOTE that the true space group symmetry can only be established once data have been integrated. It is best to be conservative and use the lower symmetry, i. H3 in this case. The cell parameters derived from the autoindexing will now be refined using the observed spot positions note that the a and b cell parameters are not equal in the initial cell. The program first asks if you want to change the cutoff for rejecting individual reflections from the refinement:.
The cell parameters will be refined imposing the constraints for that spacegroup e. The final sd in spot positions should be one pixel or less.
If it is more than two pixels, the autoindexing may have failed if you do not know the pixel size, look in the “mosflm. Then the refined direct beam coordinates will be given, with the option of accepting them. In such cases you should check that you supplied the correct beam coordinates, to avoid the risk of mis-indexing the image. Finally you are given the option of selecting the solution default yes. If you want to try another solution from the list, reply N and you will be prompted to select a new solution.
Sometimes it is possible to distinguish between similar solutions e. If you need another image preferably at a very different phi value to get the autoindexing to work, click on Read image to get the new one, and then on Find spotsand then Autoindexing. By default all found spots from all images will be used in autoindexing, but any combination of images can be selected using Select images. Many images can be used in the autoindexing although there is a limit of spots.
For orthorhombic or lower symmetries, the use of two or more images will give better estimates of the cell parameters, and this is recommended. Try reading in another image, run Find spots and Autoindex again. See how much the cell parameters change. Note that when you run the autoindexing the second time, by default it will use the symmetry you chose H3 when you first ran the autoindexing.
If you want the complete list of possibilities again, give the reply N to the following question the second question that is asked when running the autoindexing:.
Look at the final sd in spot positions. It is not unusual for this to be higher for two images than for a single image, because the crystal orientation may have changed between collecting the two images. Click on Clear spots and hit Enter to remove the crosses from the image. The image will be displayed with the predicted pattern overlaid as rectangular boxes with the following colour codes:.
There will probably be some diffraction spots which are not covered by a prediction box; this means that with the current mosaic spread, these spots would not be predicted. Using a higher value for the mosaic spread will result in more boxes being overlaid, and some of the blue boxes will change to yellow.
It is also likely that the number of overlapped reflections will increase, as will the number of reflections with green prediction boxes. Those reflections which appear on the image near the rotation cfp4 cannot be measured accurately by any method the student is encouraged to become familiar with the Ewald Tutroial construction which explains why this is so.
This region of the detector is therefore called the “blind region”; because of its appearance in the image, it often also called the “bow-tie”, “apple-core” or “cusp” region.
Blind region reflections have green prediction boxes. Click left mouse on any reflection. Its indices, phi value and phi width will be displayed in the Output window of the GUI.
Look at the predictions of the reflections on the image.
They should agree with the positions of the measured reflections. If not, then the autoindexing has failed check the direct beam coordinates, wavelength, detector distance.
See how sensitive the autoindexing is to the main beam values. Try changing them by mm. The direct beam coordinates can be tutkrial in the Processing params panel Beam X and Y.
Check that this value is reasonable i. Try varying the mosaic spread Processing params panel and see the effect on the predicted pattern select Predict from the main menu after any change in mosaic spread. If the mosaic spread is very high 1 degree or more a significant number of reflections will be flagged as “too wide in phi” green boxes. The maximum reflection width can be changed as follows: It is possible to save the current values of the detector, beam and crystal parameters to a file, which can be executed by typing filename to restore tutorila values if something goes gutorial wrong or if the program crashes should never happen of tuyorial If you do exit, follow the instructions at the end of this section on how to restart from saved parameters.
Read what is in the small window because you have the option of changing both the beam position and the position and size of the backstop; you only want to do the latter! Find out the tutoiral and extent of the backstop shadow using the left gutorial button and looking at XC and YC in the Output panel.
Make a note of these values, then mask out the backstop shadow by clicking on Keyword input and typing in backstop centre X Y radius Z X, Y and Z are in tutodial and then run to get back to screen control. The masked area will come up as a red circle. It is assumed that the backstop shadow is circular. Irregular backstops can be dealt with using the NULLPIX keyword, providing all pixels in the shadow have pixel values less than any “active” part of the detector.
Giving the keyword NULLPIX will mean that any predicted reflection that has a pixel within its measurement box with a value less than will be rejected during integration. If you tugorial going to use more than one segment, ensure that the prediction is also OK for the first image of the other segments.
If it is not because the crystal has moved then get a new orientation matrix for each segment, using ONLY spots from cxp4 image Select images.
These matrices will be written to different files. When setting up the cell refinement, when asked “Use the current orientation” reply “no” and then supply the name of the appropriate matrix file. Before starting the cell refinement, read in image 1 if it is not tutoria, displayedselect Predict and make sure that the spot positions are correctly ttutorial.
If not, check the values for the following parameters in the Processing Parameters window and reset them if necessary:. Select Refine cell Main menu and use one segment of 2 or 3 images for orthorhombic or lower symmetries you should use 2 or 3 segments with images widely separated in phi.
Click on Continue adapted Main menuuntil you get back to the starting menu or else click the “Timeout mode” toggle in the Processing params panel. The program will now integrate these images, and use the resulting intensities to refine the cell parameters, crystal orientation and tutoril spread using the post-refinement procedure.
CCP4 Molecular Graphics – Tutorial Contents
As the images are integrated, tutorkal new window will pop up showing the average spot shape for spots near the centre of the detector, and giving the rms residual the positional error in predicting the spot positions and the weighted residual. Example of this ccp. Pixel values are represented by numbers and then letters A-Z.
Thus Z should be in the middle of the box. It is often necessary to integrate the images several times before the refinement converges; if the shift in cell parameters is more than 2.
When the refinement has finished, image 1 will be displayed with NO spots predicted and the message “Waiting for input” will appear over the image, and cxp4 summary of the refinement will be given in an output window:. Normally you will want to reset the misseting angles to those of the first image, because you will start integration with that image. Note the value for mosaic spread in the text window giving the refinement. This should be a more reliable value than that obtained earlier, but for mosaic spread tutorisl than 0.