Content:

• Introduction
• Quick example
• Running jobs
• Other Examples

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Introduction

Here are some maybe more site-specific notes for the SEA-COAST Workshop held at KMUTT/Bangkok in January 2020.

Remember to always run

module load ccp4
module load xds
module load gphl

whenever you open a new terminal or open a new tab on an existing terminal.

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Quick example

As part of the lecture/demo/tutorials, we're going to first create a new directory to work in:

mkdir ~/autoSHARP
cd ~/autoSHARP
module load ccp4
module load xds
module load gphl

We need the example data, namely:

• 1o22.seq (monomer sequence of the macromolecule crystallised)
• 1o22_peak.sca (scaled and merged reflection data, collected at the Se peak - in SCALEPACK format)

(right mouse click, "Save file as") and move both files into the current directory:

mv ~/Downloads/1o22* .

Now we can start autoSHARP via

run_autoSHARP.sh -fast \
  -seq 1o22.seq -ha Se \
  -wvl 0.9778 peak -7 5 -sca 1o22_peak.sca \
  -d 1o22.01 | tee 1o22.01.lis

and from a second terminal/tab open the results file with

firefox ~/autoSHARP/1o22.01/LISTautoSHARP.html

We will go through that result file as part of the tutorial in more detail: what is it telling us and what does it mean?

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Running jobs

Our software is usually driven by commands within a terminal (shell) - although there is a task for running autoSHARP within the CCP4i (not CCP4i2) interface. We recommend using that command-line interface, since it gives you full access to all features and tools of our software packages. Once you get used to this way of running programs it will become a very powerful way of running different programs (or different trials) on a large number of datasets.

You should be able to run jobs exactly as they are presented on the tutorial pages (for SHARP/autoSHARP or autoPROC) and described on the reference cards (which you should have as a print-out, see also autoSHARP, autoPROC) and in the manuals (SHARP/autoSHARP, autoPROC).

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Examples

Have a look at the examples we provide here: just pick one of those examples - maybe one that will run quite quickly - and see what steps the program is performing. You could also pick just one of the various wavelength datasets for the MAD examples: some will work even with a single wavelength (SAD), while some might not work as well. Why is that?

If you pick one of those examples and download the relevant files (right mouse-click the file name/link and select "Save Link As..."), remember that they will be saved by default in your ~/Downloads folder. You might need to move them over to your current working directory with a command like

mv ~/Downloads/1o22* .

A good way of getting started is to run

run_autoSHARP.sh -h

to get a help message. At the end it will show some example commands for typical situations (SAD, MAD, SIRAS, partial model etc) that you should be able to easily adapt to your situation. Please note:

• If you don't have a sequence file handy, just give it the number of amino-acid residues per monomer (via -nres <N> flag). Remember that you will have to specify the number of Se or S atoms (per monomer) if doing Se-MET or S-SAD in that case (since autoSHARP won't be able to determine the number of expected sites from the sequence)

• If you didn't do a fluorescence scan and you are not collecting close to the edge, just giving the wavelength value should be ok.

• To speed things up - or for testing different approaches - you could add the -nobuild flag to the command: autoSHARP will then stop after the initial density modification step. If everything worked well, you should have some interpretable electron density maps to see if the structure is solved. And if something (HA detection, hand determination etc) didn't work, no time will be wasted trying to build into an uninterpretable map. You can always re-run autoSHARP without this flag for successful jobs.
• Another option is to run with the -fast flag.

• Remember to load the LISTautoSHARP.html file (inside the directory created by the autoSHARP run - usually autoSHARP/LISTautoSHARP.html) into a browser (e.g. firefox) to follow the progress of the job - hitting the reload button from time to time.
• If you did run with the -d flag (telling autoSHARP to write all output into a subdirectory), the LISTautoSHARP.html file will obviously reside in that directory.