TutorialMontevideo2017

Content:

• Introduction
• 1PMQ
• Setup
• Running BUSTER
• Visualisation of results
• Follow-up refinements
• Working with your own data

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Introduction

Remember that you have a BUSTER reference card ({file:buster_referene_card.pdf}) to get help - or search this Wiki (box on the upper left).

In the sections below, the commands you should type into a terminal are shown in green - as in

  ls -ltra

The main command you will be using is the command "refine". This comes with inline help via

  refine -h

which will also point you to the locally installed manual.

You can request a licence for BUSTER (free to academics) at https://www.globalphasing.com/buster/.

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1PMQ

A good example of using BUSTER (and associated tools) would be 1PMQ.

1. Setup

Create a fresh directory e.g. via

  mkdir ~/BUSTER-tutorial

and go there with

  cd ~/BUSTER-tutorial

(or use some other location if you want). You can get the required data files via

  fetch_PDB 1PMQ

(Note: this will not work at the moment as expected, since 1PMQ was superseded by 4Z9L).

This tool can be used to fetch any model/data from the PDB, resulting in two files:

  1PMQ/1pmq.pdb
  1PMQ/1pmq.mtz

Or save the following files:

• 1pmq.pdb
• 1pmq.mtz

You will also need a restraints dictionary for residue 880 - either from the Grade webserver or save the file

• 880.grade_PDB_ligand.cif

2. Running BUSTER

To just compute a map you would use the command (check your BUSTER reference card: buster_reference_card.pdf)

  refine -p 1pmq.pdb -m 1pmq.mtz -l 880.grade_PDB_ligand.cif -M MapOnly -d MapOnly | tee MapOnly.lis

and then visualise the results using

  cd MapOnly
  visualise-geometry-coot

Alternatively, you could run a full series of BUSTER refinements that are rather typical for getting started with BUSTER on a structure without NCS:

  refine -p 1pmq.pdb -m 1pmq.mtz -RB  -l 880.grade_PDB_ligand.cif -d 01 | tee 01.lis
  refine -p 01/refine.pdb -m 1pmq.mtz -M TLSbasic  -l 880.grade_PDB_ligand.cif -d 02 | tee 02.lis
  refine -p 02/refine.pdb -m 1pmq.mtz -M TLSalternate -TLS -M WaterUpdatePkmaps  -l 880.grade_PDB_ligand.cif -d 03 | tee 03.lis
 

The above is just an example of BUSTER refinements when starting from a deposited PDB structure or when switching from a different refinement program to BUSTER. Different starting points (poor initial model, domains missing, low-resolution etc) might need a slightly different approach. See the extensive material on this wiki for some pointers ...

3. Visualisation of results

The results of a BUSTER refinement can be loaded into Coot using e.g.

  cd 03
  coot --pdb refine.pdb --auto refine.mtz

But visualisation might be better done via

  cd 03
  visualise-geometry-coot

For details about this tool please see here. You will be presented with a box showing you so-called 'unhappy' atoms: these are atoms you can work through to see if there is something that needs manual attention.

Also, all other Coot tools can be used to analyse your structure - which would show you a potential issue at residue A45:

After correcting this, you can start a quick BUSTER job using the "BUSTER" button at the top right

After pointing to the correct MTZ file (1pmq.mtz) you can just compute a BUSTER map or do a short refinement:

4. Follow-up refinements

Following those initial steps, any subsequent BUSTER refinement would use commands like

  refine -p 03/refine-coot-0.pdb -m 1pmq.mtz -M TLSalternate -TLS -l 880.grade_PDB_ligand.cif -d 04 | tee 04.lis

Details:

• use the latest PDB file from manual adjustment in Coot
• no automatic water-update - since the solvent structure is probably already fairly complete and manual checking and adjustment is required to get to the final water structure (we want to avoid playing water "ping-pong" by adding/removing waters during each refinement cycle)
• the output of each subsequent run will go into a separate subdirectory and we are saving standard output as well (come up withe some meaningful system - here we're using an increasing 2-digit number)

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Working with your own data

If you want to work with your own structure:

• look at your BUSTER reference card buster_reference_card.pdf

• look at material on this wiki

• get ligand restraint dictionaries from http://grade.globalphasing.org/

• if you have NCS:
• start with -autoncs_noprune -sim_swap_equiv (to re-instate NCS relations that might have been lost)
• then switch to -autoncs (for all subsequent cycles of refinement)
• remember to not allow Coot to "fix" nomenclature, since this could destroy the correct NCS-relation between symmetrical side-chain residues (i.e. where atoms are chemically equivalent but have different atom names)

• if using TLS:
• start with -M TLSbasic
• then switch to -M TLSalternate -TLS

• for occupancy refinement, have a look at pdb2occ tool and some tutorial

• difference Fourier maps are described here

• if your data comes out of autoPROC you could switch on the computation and analysis of additional maps:

• F(early)-F(late) maps via BusterEarlyLate=yes
• anomalous Fourier maps via BusterAnoFourier=yes