JiaLabData

03/18/13

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Cancer Project Details

Main Plasmid Collection

Cancer Plasmid Collection

BRI Plasmids

Pichia strains

E. coli strains

 

Target Status (shows who is working on what, and their current progress)

Cloning Information (protein size, expression trial information, etc)

Source of Clones (IMAGE IDs and Genbank accession numbers of targets)

Purification Protocols:

Solution Recipes:

Purification of GST-tagged proteins from E. coli

Purification of HIS-tagged proteins from E.coli

Small-scale growth/purification of His/GST tagged proteins from E. coli (for expression trials, PDF, UPDATED)

Terrific Broth (TB)

Luria-Bertani Broth (LB) (Low Salt)

LeMaster media (for selenomethione incorporation)

YPD (for Pichia growth, NEW)

2X Laemlli SDS-PAGE Sample Buffer (NEW)

SDS-PAGE and Western Blot solutions

Agarose Gel Electrophoresis (includes TBE recipe and 6X DNA Loading Buffer recipes, NEW)

Cloning Methods

Jia Lab Cloning Manual (PDF) NEW, Work in progress

Ligating a linker (annealled oligonucleotide) in to a plasmid (NEW)

Ligation (of a cDNA clone in to a plasmid vector, NEW)

Lysozyme and MBP fusion vector maps

E. coli manipulation methods

Preparing Chemically Competent E. coli

Transformation of E. coli by heat-shock (UPDATED)

Plasmid Miniprep (alkaline lysis method, NEW)

Crystallography

Overview of x-ray data collection at Queen's home source

Requirements for making a trip to BNL (Brookhaven National Laboratory)

X8-C Beamline Parameters (NEW)

Pichia methods:

Electroporation of DNA in to Pichia (NEW)

PCR screening of Pichia

Small-scale purification of His-tagged proteins from Pichia

 

Software and Scripts

Click on the program name to go to the software's website for instructions and other information

ARP/wARP 6.0
"For improvement and objective interpretation of crystallographic electron density maps and automatic construction and refinement of macromolecular models". Accessable through the CCP4 interface.

CCP4

command: "ccp4i"

"A collection of disparate programs covering most of the computations required for macromolecular crystallography".

CNS 1.1

command: "cns < input.inp > output.out &"

Our lab's most used program for refinement of models. "A flexible multi-level hierachical approach for the most commonly used algorithms in macromolecular structure determination. including heavy atom searching, experimental phasing (including MAD and MIR), density modification, crystallographic refinement with maximum likelihood targets".

DENZO 1.97.9 

commands: "xdisp", "denzo", "scalepack", etc..
A program package for the visualization of X-ray diffraction images ("xdisp"), for data reduction and integration ("denzo"), and for merging and scaling of the intensities obtained by Denzo ("scalepack"). "denzo" and "scalepack" are command line executable. For fully integrated graphical interface, see "HKL2000".

EDEN 4.2

command: "eden"
A holographic method for solving electron density in real-space.

EPMR 2.5

command: "epmr cell.cel model.pdb data.hkl > epmr.log &"
An easy to use and relatively fast program for solving cyrstallographic molecular replacement (MR) solutions using an evolutionary search algorithm. To generate the EPMR-required hkl file, run "mtz2vaious" from CCP4 using the "USER" Fortran format of '(3i4,2F8.2)' and then add two column labels for "FP" and "SIGFP".

HKL2000 (temporary license)

command: "hkl2000"
A graphical version of Denzo/Scalepack. Several new subprograms are incorporated, including strategy and simulation, 3-D processing, mosaicity refinement during processing, variable spot size, easy change of the space group, report generation, etc.

HySS

command: "hyss"
A submodule of the future PHENIX cystallographic program package. HySS is "a highly-automated procedure for the location of anomalous scatterers in macromolecular structures". Note: "currently the command line interface is limited to work with anomalous diffraction data" only.

Molscript 2.1.2

command: "molscript"
A program for displaying molecular 3D structures in both schematic and detailed representations. Molscript along with "Raster3D" can be used to generate high resolution images of 3D structures in common image formats such as jpg, tiff, and gif.

Mosflm/DPS 2.04

command: "dps"
An alternative for Denzo/HKL. It is a graphical interface of Mosflm for indexing and integration of X-ray diffraction images. Create a new directory to run DPS as a lot of temporary files will be copied and created.

Phaser

command: "phaser < input.inp > output.out &"
A new alpha (pre-beta) version program for MR, using the maximum likelihood methods for phasing macromolecular crystal structures.

PyMol 0.93

command: "pymol"
A new open-source program for molecular modeling. An alternative to XtalView.

Raster3D 2.7b

commands: "render", etc..
A set of tools for generating high quality raster images of proteins or other molecules. Raster3D can also be used to render pictures composed in other programs such as "Molscript" in glorious 3D with highlights, shadowing, etc.

 

Shake-and-Bake

command: "SnB"
SnB provides a graphical user interface for computing normalized structure-factor magnitudes, solving phases by the main SnB dual-space direct-methods, visualising and editing molecular structures. The SnB algorithm was entitled "The Top Ten Algorithms of the 20th Century" in a poster published by Computing in Science and Engineering in November, 1999.

SHARP/SUSHI

command: "sharp", or goto http://localhost:8080/ in web browser.  

A program using web browser interface for solving phases from MIR(AS), SIR(AS), SAD, and MAD data, as well as for density modification of electron maps.


SHELX 2004/1

commands: "shelxc", "shelxd", "shelxe", "shelxh", "shelxl", "shelxpro", "shelxs", "shelxwat"
"A set of programs for crystal structure determination from single-crystal diffraction data". shelxc--preparations of files for phasing; shelxd--structure solution for difficult problems and location of heavy atoms; shelxe--phases from shelxd heavy atom stides and density modification; shelxl--structure refinement (use shelxh for large structure); shexlpro--protein interface to shelx; shelxs--structure soltuion by Patterson and direct methods; shelxwat--automatic water divining for macromolecules. XPREP can be used for preparation of SHELX input files.

SOLVE/RESOLVE 2.06

commands: "solve", "solve-huge", "resolve", "resolve_huge"
SOLVE--Automated crystallographic structure solution for MIR, SAD, and MAD
RESOLVE--Statistical density modification, local pattern matching, automated model-building, and price-and-switch minimum bias phasing.

Turbo 2002 Release 1

commands: "turbo"
A general purpose molecular modelling environment for model building, viewing of 3D structures, as well as ligand fitting and protein stacking.

Xprep 6.15 (demo)

commands: "xprep", "bigxprep"
A standalone utility for processing MAD, SIR, SIRAS data; displaying reciprocal space to check completeness of data; displaying Harker sections of Patterson maps; and displaying self-rotation functions to search for non crystallographic symmetry and twinning.

XtalView 4.0

commands: "xfit", "xtalmgr"
Xfit is our lab most used program for fitting electron density maps and model building. Xtalmgr, the main menu of XtalView, can be used for creating new "crystal info" files for Xfit, as well as for solving structures by MIR and MAD.


Scripts:
 

Cleanpdb.com

command: "cleanpdb.com < input.pdb > output.pdb"
Clean PDB files to retain only the lines of "ATOM" and "HETATOM". Also remove any character within the empty fields of PDB that is generated from programs like SwissPDB.
 

Cns2fs

command: "cns2fsfour_linux" or "cns2fsfour_sgi" then "cns_input.map xfit_output.map"
Directly converts a CNS generated Map file into Xfit readable Map file. **NOTES: 1) Use the scripts for their corresponding computer platforms,ie. either Linux or SGI. 2) When generating the CNS map, look for the "extend of map" option and select "UNIT". 3) Recommend to creat the Xfit map and actually run Xfit from the same computer OS-platform or the map may not be read correctly. 4) To load the newly generate map from the script, RIGHT click on the "Load Map" tab in Xfit and pull down to select "Load FSFOUR Map".
 

Cns2xfit.awk

command: "cns2xfit.awk cns_input.coeff > output.phs"
This script convert CNS map coefficent file (.coeff) to Xfit phase file (.phs).
 

 

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This site was last updated 03/18/13