Presentation of the problem

We use this dataset as an example of refinement in the SIRAS method.

The position of the unique Krypton site was determined without ambiguity on a Patterson map, that was made much more informative by the application of local scaling to the data (program SCALA, Evans 1993), and is used as a starting point for heavy-atom refinement.

Practical introduction (first-time users only)

SHARP is run through a graphical user interface. Although we think that this type of presentation will be a standard in the near future, it may still puzzle new users.

There are very few reflexes to acquire before getting started. In fact, only two actions are necessary :

• Finding the right place to click the mouse (anything underlined, and/or with a special colour, usually blue).
• Clicking the mouse (in case of doubt, click the leftmost button on the mouse) on that place.

The browser

The purpose of this system is to navigate from one document to another, just by clicking on a hyperlink that defines the network address of the next document (or 'page') to open.

When you call Netscape, you are prompted with a new window. This window is made of a static frame, with a title, a list of scrolling menus, a few buttons ; and of a mobile part that contains the hypertext document, located at the address you have asked for, either by specifying the address in full or by clicking on a previous hyperlink.

The static frame of the browser

• Back and Forward in the upper left corner of the frame. By clicking Back you go back to the previous document in the chronology of pages you have already accessed. By clicking Forward, you navigate the other way in the stream of documents already consulted.
• Reload (fourth button from the left). Extremely useful to examine the SHARP output. Whenever you are looking at a document while information is still added to it, you need to update this document by clicking on Reload.
• Print (seventh button from the left). To get a paper copy of the document in the mobile part. Of course, the window from which you have launched the browser must 'see' a printer (lpr -P<printer_name>).

The mobile part of the browser

Clicking a hyperlink or on a button in the SHARP output will sometimes trigger a 'helper application' (plotting program, Fourier transform, peak-search). The result may then be displayed in a separate window. But this will only work if the helper application is installed on your system ! (see helper applications page for the helper applications you need).

Frames and JavaScript

Warning windows, prompted by the JavaScript code, may also come up from time to time to protect you against clicking errors, or to help you check wether you have not typed incorrect values for parameters.

Preparing the refinement - first run

We will now describe the steps that you should follow before running SHARP.

The SHARP control panel

netscape http://<your.machine.name>:8080 &

Note : This login/passwd system is independent from the Unix login and password.

From here, you decide what action should be taken , namely START, but before pressing on the button, you get to choose an existing set of parameters, bearing close enough resemblance to your present problem so that you do not have to re-type everything. If you are running this tutorial for the first time, and want to go through all the form-filling (for your own good of course) select None in the list of project IDs, and start from scratch. If you are lazy, and if it exists, you can start with the existing KrEl.1.sin, which contains the 'solution' to the tutorial. Then press START.

Setting up the parameter hierarchy

Your browser is now divided into four frames. On the left, you can see the Table of Contents (ToC), that represents the present hierarchical organisation of parameters. You can interact with this structure, using the seven buttons provided in the top frame : Help, New, Copy, Delete, Submit, Down, Up. The meaning of these buttons should be clear, you can experiment with them, keeping in mind a few basic rules :

• The buttons in the top frame apply to the ToC only, they will never have an effect on the browser itself (for instance, Up will not take you to the Control Panel again).
• The action of these buttons apply only to the hierarchical level currently selected.

The last frame, in the upper right of the screen, contains the interface logo. The interface is called SUSHI (Simple to Use SHarp Interface) and these are the Japanese characters for it, kindly provided by Atsushi Nakagawa (Hokkaido University).

This tutorial will now take you through all hierarchical levels in the ToC, and tell you in detail what has to be done in each of them.

Global : Global Information Editor

All the hyperlinks in this page (Identification, Calculation Options, Datafile, Symmetry and Cell and Other information), will take you to the on-line documentation. You are welcome to read it. If you want immediate action, you can start filling or modifying the form.

The field after Project Name : is the name that will be given to all files and directories pertaining to this project. Once you have decided for a name, do not change it ! Documents produced by successive runs with the same project name 'TOTO' will be stored in directories called TOTO.1, TOTO.2 ...etc

Suggested name : KrEl

You are free to choose a title. But make the first ~30 characters in the title as informative as possible : they will be appended to the project name in + file listings, to help you remember which run did what.

For example : One isotropic Kr atom ; initial refinement.

NOTE : Maybe the meaning of 'appended to the project name in the file listings' is somewhat obscure. It will gradually become clear once you see what listings we are talking about

Under Calculation Options, activate all four tick boxes for Outlier rejection using likelihood histogram , ML parameter refinement , Residual (LLG Gradient) maps , and Centroid electron density map . The residual maps will show you if the model is incomplete, i.e. if there are any minor sites, or characteristics for the known sites that have not been modelled. Electron-density maps are not compulsory at this stage ; they should only be traced at the very end of the refinement.

If you have ticked ML parameter refinement, you will be asked a strategic choice between three options, start by refining scale, Lack-Of-Isomorphism (LOI) and Occupancy parameters only - choice 1, start by refining these and coordinates - choice 2, start by refining all parameters that have been marked for refinement - choice 3. You should select choice 1 if you are starting a refinement from values you are not very sure of, and choice 3 if your starting values are already coming from a previous SHARP refinement. See online doc for more details.

Recommended choice : scale, LOI, occ (1)

Under Datafile, Symmetry and Cell, you should start by selecting a datafile in the list.

Compulsory datafile :ElaKr.data.mtz

If you change the datafile, you will then be prompted with a JavaScript window asking you if you really want to change the crystallographic attributes attached to the datafile, i.e. the spacegroup and cell parameters as read from the header of the MTZ file. If you answer yes, these will be changes to be compatible with the MTZ file you just selected.

The Chemical Composition is not compulsory, but is useful to anchor the scale of your reference dataset to some quasi-absolute standard. The consequence is, that the refined values for occupancies will then be quasi-absolute.

Recommended chemical composition :

                              C 1135
                              N  329
                              O  465
                              S   11
                              CA   1

Now click on the next level in the ToC G-Sites, to define a list of coordinates for all sites.

G-site editor - just a list of sites

Depending on which template file you are using, there may be no G-site defined (in which case you click on the button Create), or there may be already one site (in which case you just modify its coordinates if needed), or you may find two or more sites, in which case you will have to mark them for deletion and press Delete. The Add facility is designed so that you can add a large number of sites from a file. See documentation for details.

Compulsory starting coordinates , site 1 : ( 0.285 ; 0.063 ; 0.488 )

Once this is done, you will enter the hierarchical part of the ToC, that consists in four nested levels :

• COMPOUND (C)
  • CRYSTAL (X)
    • WAVELENGTH (W)
      • BATCH (B)

Compound Editor

Compound 1 - both Reference and Native

Please first read the on-line documentation for this page "Help".

If the documentation is clear enough, you will have understood that a compound is either a native or a derivative. In this case, we start with a native.

Note : This is a new feature ! In SHARP, for a better generality, if there is a native it must be described (as a 'derivative without heavy-atom'). More explanations about the special role of COMPOUND 1 as a reference can be found if you click the documentation hyperlink "Reference" in the ToC.

If there is one or more C-site in the template, mark it/them for deletion and press Delete.

Note : Elastase has no heavy-atom in the native. That may sound obvious, but consider the case of an iron-containing protein. If you record the anomalous signal in the native, then you should enter the iron atom in the list of C-Sites of the native, as well as in the list of C-Sites for whatever derivatives there are.

Now we move down one hierarchical level, to the first crystal of the first compound : please activate X-1 just under C-1.

Crystal Editor

Compound 1 / Crystal 1

Please first read the on-line documentation for this page "Help".

If the documentation is clear enough, you will have understood that no T-site can appear in this site, because you have defined no C-site.

Please move on to the next level W-1.

Wavelength Editor

Compound 1 / Crystal 1 / Wavelength 1

Note : Why 'wavelength' ? This is not a multi-wavelength experiment !
Sure, but in the general setup of SHARP, even if you use only one wavelength per crystal (the usual case), you should describe its characteristics (here in terms of resolution limits). If it makes you more comfortable, you could think of a 'wavelength' as a 'dataset' in this case.

Recommended resolution limits : 25. to 1.85

Please move down to the bottom level of the hierarchy for this first compound by activating B-1.

Batch Editor

Compound 1 / Crystal 1 / Wavelength 1 / Batch 1

The button Select Columns will take you to our MTZ hyper-editor. The present version of this program does not enable you to select columns by clicking on them, but is a useful alternative to using 'mtzdump' in the Unix window if you have forgotten the names of the relevant columns in the MTZ datafile.

To know what FMID, SMID etc. mean, plese press on the hyperlink Assign columns from file. It will take you to the relevant part of the documentation.

Compulsory column assignments :

FMID = FP ; SMID = SIGFP

You then have to give initial values for scaling and non-isomorphism parameters.

Scaling parameters. In the absence of information about these parameters, we will start from (for example) K = 1. and B = 0. Because the first dataset is a reference for the scale, these values should NOT be refined.

Because we take the first dataset, by convention, as a reference for the scale, if you activate the Estimate first? tick box for the multiplier scale factor (K), SHARP will perform a pseudo-absolute scaling based on the chemical composition given in the first page 'General informations'. In case you request this, you should have previously made sure the chemical composition makes sense. Do not activate the Estimate first? tick box for the B scale factor : it would deny the atoms in the molecule any right to thermal disorder, and result in unphysical values for the heavy-atom B-factors.

Because we have to choose a reference dataset for `isomorphous' lack of isomorphism, Global non-isomorphism parameters and Model imperfection parameters for isomorphous differences should be all set to zero (0.) and not Refined nor Estimated. Conversely, the same parameters for anomalous differences should be both Estimated and Refined.

The sentence 'No heavy atom type defined' appears at the end of the page, meaning that no heavy-atom parameters need be refined here.

We have now finished parametrising COMPOUND 1, here assigned to be the native COMPOUND. Please have a look at the frame entitled 'Table of Contents' on the left-hand side of the browser window. If COMPOUND 2 is not defined, you should go and create it. Click on C-1 and click on button New in the top frame. This will create another COMPOUND (C-2), ready for parametrising. In the same way, any other level can be created by activating an existing item at that level and clicking New in the top frame.

If C-1 already exists in the Table of Contents, just activate it.

Compound Editor

Compound 2 - Krypton derivative

If there are too many C-sites in the template, click their tick boxes in the 'Delete ?' column and then on 'Delete C-sites marked below' until you are left with one only. You then should assign a Chemical name (in the 'Atom' column) to a G-site (in the G-site column).

Beware ! this is case-sensitive : the first letter should be uppercase, the other(s) should be lowercase. In any case, if you assign a name that is not present in the list of possible chemical types, the interface will refuse it, and you will be prompted with an Alert window.

Compulsory action , C-site 1 : write Kr in the field and select G-SITE-01 in the scrolling menu.

Then activate X-1 (under C-2) in the Table of Contents.

T-site editor

Compound 2 / Crystal 1

This time, one T-site appears, corresponding to the C-site you just defined. Because we have no prior knowledge of these values, we will keep the default and just activate the refine tick boxes for the occupancy and the isotropic temperature factor of this site.

Please click on X-1 (under C-2) in the Table of Contents.

Wavelength-level editor

Compound 2 / Crystal 1 / Wavelength 1

Recommended resolution limits : 25. to 1.85

Then click on B-1 (under C-2) in the Table of Contents.

Batch-level editor

Compound 2 / Crystal 1 / Wavelength 1 / Batch 1

Compulsory column assignments :

ISYM = ISYMKR56 ; FMID = FPKR56 ; SMID = SIGFPKR56 ; DANO = DANOKR56 ; SANO = SIGDANOKR56

Scaling parameters. This time all scaling parameters have to be both estimated and refined, because this scaling is relative to the first (here native) dataset.

LOI parameters. Similarly, they are now all estimated and refined.

Anomalous scattering parameters. Because this is carefully-measured, high-resolution dataset, we will take the risk of refining the imaginary anomalous scattering factor f''. The real anomalous scattering factor f' shoud never be refined in a SIRAS experiment, because it is completely correlated with the occupancy.

Recommended values for the anomalous scattering factors :

f' = -8. ; norefine
f" = 3.5 ; refine

Note : In normal circumstances you should not refine f'' unless you suspicious of the value given in the tables (i.e. you are close to an edge). But in this case we were justified (even if a posteriori for doing so, you will have the full explanation at the end of this tutorial.

The whole dataset has now been described in terms of its scaling, lack-of-isomorphism and heavy-atom parameters. We shall then press the button Submit (in the top frame) for the first refinement run.

Last check before job submission

If you have spotted an error, or if you want to exercise going through the forms again, click on Cancel and return to the start of this document. If you want to have a coffee, and submit the job when you come back, click on Save only ; you will have to go through the restart mechanism (see end of this document) to get the refinement going from these saved parameters.

In any other case, just click on Submit , to start the refinement.

You will be prompted by a confirmation message Your sharp job *** has been submitted, and a choice either to go to output or to close that window. If you click on Go to output, after five seconds that window will close, and the second window that had contained the input form, will now turn into a pointer to the logfiles directory. If you click on Close window, this third window disappears, and you are left with the Control Panel window and the input form window. Go to the Control Panel window, click on the hyperlink logfiles, and go the relevant logfile directory via this alternative route.

Examining the logfile - first run

We will comment on the logfile that you obtain if you followed exactly the recommended values in the input pages. If you have deviated from ideality (which is not advisable if you are a first-time user of this tutorial, but could be interesting for further tests), do not be surprised if the results are different from those mentioned here !

To access the main logfile (from which all other files in the directory can be consulted), click on the filename 'LIST.html', described as SHARP OUTPUT in the rightmost column.

Section 1 : PREPARATION OF DATA

Section 2 : ML REFINEMENT

The interesting phenomenon at this stage, is that the occupancy of the Krypton atom decreases very rapidly from 1. to 0.67 , while the anomalous lack-of-isomorphism parameters slowly adjust.

Then, we head on to BIG CYCLE 2, with more parameters being refined, namely the coordinates of the Krypton Geometric Site. Nothing changes very much during this Big Cycle, just small adjustements of occupancy and very small (but significant when compared to their standard deviations) changes to the coordinates.

At BIG CYCLE 3, the most sensitive parameters (heavy-atom temperature factor and anomalous scattering factors) are added to the list.

You can see a concerted shift in both occupancy and temperature factor, while anomalous lack-of-isomorphism parameters show an overall decrease, and the f'' parameters adjusts to a value larger than predicted by the tables (by 1 electron). Please feel free to click on all available hyperlinks, to look at the details of the refinement (first and second-order derivatives, eigenvalues of the hessian matrix), or at the agreement between our model of lack of isomorphism and the mean lack-of-closure error in resolution bins. In particular, you will notice that , whereas the isomorphous lack-of-closure statistics closely follow our parametric model, the anomalous lack of closure behaves very strangely, probably due to large uncertainties in the values of the standad deviations of the measured anomalous differences.

Section 3 : RESIDUAL (LLG gradient) MAPS

Before clicking on the View button, you have to choose the level of the residual map (see on-line documentation for details). In this case, the best choice is of course the isomorphous residual map for compound 2, because the very weak anomalous signal will not produce any reliable residual information at this stage.

Recommended level : iso/ano

You then select the line 'Compound2/Crystal1/Wavelength1/Batch1 ISO (2 1 1 1 ISO)' in the scrolling menu (our general way to specify an isomorphous residual map for the second compound), and click on Go to viewpoint selection.

The only options that are usable for the moment are to view the whole map (i.e. the whole asymmetric unit) with program PLUTO (npo), or with the program 'O'. Just click on Plot to trigger the Fourier transform and the plotting program. A new window will then appear, with five sections through the residual map. You can then click on Peak List to see the list of peaks and their coordinates. This enables you to note down coordinates of new peaks prior to entering them in the G-sites list of the graphical SHARP input.

If you choose to view Pluto maps (map sections), first click on relative in the new window, then click successively on 'Picture 1', 'Picture 2', .... To enlarge the pictures, as long as you are in 'relative size' mode, you can just enlarge the window where the section is displayed - the map should be enlarged to fill the window.
If you choose O as a plotter (recommended when possible), an O window appears, and you just have to do what is written there. The user menu is either displayed directly on the right side of the graphical O window (version 5), or as a sub-choice of the the menu 'menu', on the right side of the command bar.

In the present case, the only significant features in the map occur in the vicinity of the krypton site. It is very clearly surrounded by two positive peaks and two negative peaks, in opposition (like Bridge players around a table). This indicates anisotropic behaviour for the thermal motion of the Krypton atom. Two more (negative) peaks are in sight, but once again they are split by the limits of the crystallographic cell, and show up in symmetry-related locations along the edge. We will postpone interpretation of these other features until anisotropy has been refined, and see if they still show up then.

We can now move on to the next refinement run.

Preparing the refinement - second run

The SHARP control panel

To be on the safe side, please click on the Reload button (fourth from left in the Netscape frame), so that the list of files in the various scrolling menus get updated.

Depending on what name you gave to the first refinement run, you will find in the scrolling menu in front of the START button, a file called end_<name-of-run>.1.sin
This parameter file contains all the refined values at the end of refinement run 1. Except if you want to experiment with things, we advise you to highlight this file in the list, then click on the START button.

General information

For example : "One anisotropic krypton atom ; refinement of parameters."

You can look at the rest of the page, but it should remain unchanged.

Where do you go next ?

T-site editor

Now click on the button Submit in the top frame.

Last check before job submission

Examining the logfile - second run

Section 1 : PREPARATION OF DATA

Section 2 : ML REFINEMENT

They converge quickly (4 cycles), but you can observe that most of the six components of the anisotropy tensor (in fact, all but the last) have a significant departure from zero (4 to eight standard deviations). This confirms the validity of our interpretation of the residual maps produced by the previous run of SHARP.

You can also notice a slight decrease of the lack-of-isomorphism parameters as the refinement proceeds. This illustrates one of the main advantages of the maximum-likelihood refinement : it allows all parameters including those describing the lack of isomorphism to be refined together, so that uncertainty decreases at the same time as the information about the substitution gets more precise.

Section 3 : RESIDUAL (LLG gradient) MAPS

As for the first map, please select 'iso/ano' in the scrolling menu, and click on the button View.

On the next page, select 'Compound 2/Crystal 1/Wavelength 1/Batch 1 ISO (2 1 1 1 ISO) and click on the button Go to viewpoint selection. Then click on Plot.

You can notice that the refinement of anisotropic thermal motion for the krypton atom has effectively removed the pairs of positive and negative peaks close the the krypton site.

The other peaks remain, though. Just underneath the site, you can see a pair of peaks of opposite signs. We suggest that they reflect a small-amplitude movement of protein atoms upon fixation of the krypton atom. This hypothesis was confirmed by superimposing the map with the refined model, where the positive and negative peaks were on both sides of Valine 209.

The two other peaks (both negative, one large and one smaller) can probably be attributed to departing water molecules. The is no direct evidence for this, but another observation indirectly confirms the hypothesis. Why does f'' refine to a value larger than its theoretical value of 3.5 electrons ? The wavelength of the X-rays is far from any absorption edge of the Krypton atom, si that the theoretical value can be precisely relied upon. But what would happen if a (or two) water molecule(s) was removed or displaced upon binding of the Krypton atom ? The isomorphous differences would be caused by ( Kr - O ), whereas, since the oxygen atom has no significant anomalous diffraction properties in this wavelength range, it would produce full ( Kr ) anomalous differences. The refinement in SHARP would reconcile these conflicting source of information by having an occupancy for the krypton atom lower than its physical value in the derivative, but would compensate with an anomalous scattering parameter f'' larger than its physical value. This could explain both the unusual value for f'' and the negative residual peaks in the immediate neighbourhood of the Kr binding site.

If these features in the residual maps really are departing water molecules, should they be refined as oxygen atoms with negative occupancies ? This is possible in SHARP, and has been tried, but the improvement to the electron-density map, altough measurable, does not make much difference as far as map interpretation is concerned. Therefore, we will consider that refinement is complete.

Section 4 : ELECTRON DENSITY MAP (CENTROID MAP)

If you click on View to look at the SIRAS electron-density maps, you may be very disappointed. Due to important noise from the high-resolution data, this map looks fairly un-interpretable. Even molecular boundaries are not obvious to the eye.

You should nevertheless try solvent flattening on this map. The density modification program Solomon from CCP4 (Abrahams & Leslie, 1996) is triggered from the button Flatten. All you have to do is specify a solvent content.

Recommended solvent content : 0.43

Clicking on Flatten will take you to the solvent flattening page, from where you can follow the statistics of the density modification procedure, in the form of comparisons between the map at cycles N and N-1 , and you can also look at the electron-density map during the procedure. After 130 cycles of Solomon, you essentially obtain a perfect electron-density map, as described in (Schiltz et. al., 1997).

Trouble ?

If you go there (through the Go menu in the Netscape frame, or via a Bookmark, or by typing the URL in full, or by exiting Netscape and starting again), you see after the Refine and the Synthetise buttons, another button called Request. In front of this button, you find a first menu with a list of possible actions, and a second menu with a list of runs. If you want to restart the run, say, KrEl.2, select KrEl.2 in the rightmost menu, then select Restart in the middle menu, then click on the button Request button.

SHARP will always restart from the end of the last completed cycle, but will go through likelihood filtering before refinement. That means you do not restart exactly in the same conditions (in fact, you start in somewhat better conditions because the outlier rejection will be more precise if refinement is more advanced). But do not be surprised if the first cycle after restart does not behave exactly like the last uncompleted one.

Bibliography

Shotton, D. M., Watson, H. C. (1970). Nature 225, 811.
Meyer, E., Cole, G., Radhakrishnan, R., Epp, O. (1988). Acta Cryst. B44, 26.
Schiltz, M., Prangé, T. & Fourme, R. (1994). J. Appl. Cryst. 27, 950-960.
Schiltz, M., Shepard, W., Fourme, R., Prangé, T., La Fortelle, E. de & Bricogne, G. (1997). Acta Cryst. D53, 78-92.
Evans, P. R. (1993). Proceedings of CCP4 Study Weekend (Data Collection & Processing), 114-122.
Abrahams, J. P. & Leslie, A. G. W. (1996). Acta Cryst. D52, 30-42.