Hacking PDBs for fusion protein and missing loops

This is a How-to guide to make structures of fusion protein —the shoddy way.
For the proper way see the how to do it in Rosetta or Pyrosetta or by coercing the RosettaCM threader do it.
NB. This assumes medium–advanced PyMol competency.

Introduction

Representing the structures of fusion protein is a rather annoying task. There are many ways of doing this.
Contrary to expectations, submitting the sequence of a fusion protein to the Phyre server (threading/ab initio predictor) or similar will return spaghetti, just as happens with circular permutations (for which you get a cracked half open domain with spaghetti attached). But give it a go nevertheless —it may save you a lot of time!
Rosetta can be used, but the domains will not be photogenic and it is overkill for some operations.
So the best way is to simply hack the PDB files and some PyMol ninjutsu.

EDIT/WARNING: since writing this I have realised that one could add missing loops by threading as several algorithms have loop adding operations —in which case using PyMod may be the best approach for non-coders as, it is less straighforward than this, but utilises a proper loop modelling algorithm as it's a GUI wrapper for MODELLER. PyMod requires some easy installations (MODELLER and a PyMOL plug in) and the generation of the alignment (load the structure, run print cmd.get_fastastr(), get your full sequence from Uniprot, go to the online Muscle alignment tool and bingo) and does not require any awkward pulling of chains.

About

As an example I will make phusion, i.e. Pyrococcus furiosus DNA polymerase fused to Sulfolobus solfataricus 7D domain (Sso7), because its name basically says it is a fusion.

Rosetta easteregg

The guide to use Rosetta may be a bit, ehm, flaky, but I think it is made up for by this rather amusing comment header I found in one of its Perl script, kudos to the author.

###############################################################################
#
# MAKE_FRAGMENTS.PL 1.00 -- THE (PEN)ULTIMATE IN HOME FRAGMENT-PICKING SOFTWARE!
#
# CAUTION:  NO USER SERVICEABLE PARTS BELOW!
#
#           TO REDUCE RISK OF ELECTRIC SHOCK, DO NOT REMOVE THE COVER!
#           DO NOT ATTEMPT REPAIRS!  REFER SERVICING TO YOUR AUTHORIZED DEALER!
#           AVOID PROLONGED EXPOSURE TO HEAT OR SUNLIGHT!
#           TO REDUCE THE RISK OF FIRE OR ELECTRIC SHOCK, DO NOT EXPOSE THE
#            PRODUCT TO RAIN AND/OR MOISTURE!
#           DO NOT MOVE THE PRODUCT WHILE IN USE!
#           DO NOT LOOK AT THE PRODUCT WHILE IN USE!
#           DO NOT COMPLAIN ABOUT THE PRODUCT WHILE IN USE!
#           DO NOT DISCUSS THE PRODUCT WHILE IN USE!
#           DO NOT THINK ABOUT THE PRODUCT WHILE IN USE!
#           CLEAN ONLY WITH MILD DETERGENTS AND A SOFT CLOTH!
#           USE ONLY IN WELL-VENTILATED AREAS!
#
#           FOR EXTERNAL USE ONLY!  DO NOT TAKE INTERNALLY!
#           MAY PRODUCE STRONG MAGNETIC FIELDS!
#
#           DO NOT REMOVE THIS TAG UNDER PENALTY OF LAW.
#
#           THIS ARTICLE CONTAINS NEW MATERIAL ONLY.
#
#           THIS LABEL IS AFFIXED IN COMPLAINCE WITH THE UPHOLSTERED AND
#            STUFFED ARTICLES ACT.
#
#
# (IN OTHER WORDS:  DON'T EVEN *THINK* ABOUT CHANGING THINGS BELOW THIS POINT!)
#
###############################################################################

In vivo shuffling via heteroduplex amplicons

Bluescreen transilluminator photo showing
different shades of green.

In a previous post I discuss the heteroduplicity of epPCR plasmids. Namely often colonies in a library will have bases that have two equal variants. The most likely cause being that the amplicons do not anneal perfect and that the transformed plasmids are actually heteroduplexes. These can either divide before being corrected or are corrected by mismatch repair.
I did a sneaky experiment to see if this could be used to make protocol to shuffle mutations between variants and got a positive result, but possibly not as effective as hoped.

A note on Mutazyme and Manganese

Can Mutazyme be powered up?
Adding manganese works poorly, but stronger variants seem to be known.
(see also discussion about Mutazyme in Part I and II)

A simple hack for a phylogenetic Noah's ark dilemma

Ever had an endless list of bacterial names that needed a trim?
Ever see a tree where the bacterium chosen is not the famous one, but it's cousin? Or actually a tree where you don't recognise a single name?
The issue of picking bacteria from a list is what I call Noah's ark dilemma. This term is used generally for the biblical problem of the size of the boat required for all the animals in existence (except dinosaurs). Here I mean it picking the most meaningful bacteria from a list. In the past year, I have come to rely on a simple solution: Pubmed popularity.

Peak height variation

Sequencing a plasmid pool containing a sequence with a randomised codon can reveal the frequencies of the bases are (Acevedo-Rocha et al., 2015 ).
The problem is that sequence traces are not consistent. Some peaks are bigger than others and beyond a certain point the traces get messy. So how does that affect the prediction of the base frequencies?

Top 5 useful facts about the MDS42 strain

MDS42, also known as the Blattner strain, is a strain made in 2006 by deleting 13% of the genome of E. coli K-12 MG1655. Unfortunately, as tools go, it rivals IKEA in cryptic instruction manuals.