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OutlineOutline Introduction
What is homology modeling
Theory Basic principles and procedure
InsightII and homology module How to start it
Building your first model from sequence
Dihydrofolate Reductase Improving your model
Relaxation
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IntroductionIntroduction
Goal
Create a 3-D structure of a protein Why
When the 3-D structure is not available from
X-ray or NMR studies What do you need to know
Protein sequence of interest (target sequence)
Protein structure of at least one homologousprotein (3-D template)
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IntroductionIntroduction
What is a homologous protein
A protein that have a high sequence similarityto the target protein sequence
Basic assumption of Homology Modeling Proteins sharing high sequence similarity
should have similar protein fold
. . . . (if they dont misfold)
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IntroductionIntroduction There are exceptions !!
Protein can be designed with a fewmutations/insertions/deletions to completelyunfold, misfold and even fold to another
secondary structure
Proteins may adopt completely different folds
depending on environmental variables such aspH, temperature, salt conc. etc. which is notaddress by homology modeling
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IntroductionIntroduction
Why are we still doing it ??
If someone ask you, Given a class of proteinsthat folds in a certain way, what do you think
the structure of this protein, whose structureis not yet solved experimentally, should be ??
The bottom line . . . . . What is your best guess ?
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In perspectiveIn perspective
Human has 30K to 35K Genes
Number of proteins expressed in human alone canbe 10 X number of Genes
Total number of proteins expressed by all organisms
.. MANY
Number of protein structures at PDB as of May 05is 31K
There is a good chance that the protein you arelooking for is not solved !!!!
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Some Basic TheorySome Basic Theory
When is homology modeling applicable
When the sequence similarity is greater than 40 %
When there is more than one instance that supportour initial assumption. E.g. If you can find a proteinfrom different organisms with slightly different
protein sequence sharing a very similar proteinstructure (fold).
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ProcedureProcedure
Assign coordinates from SCR
Align Target Sequence to SCR sequence
Extract Sequence
Struct/Seq Alignment
Get homologous protein structure
Load Target Sequence
Generate loops
Repair the ends
Optimize structure
Determine SCR
Similar Sequence
Find homologous proteins
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Getting a Protein StructureGetting a Protein Structure
Go to protein data bank at
http://www.rcsb.org
Find a series of proteins structure thatare homologous to your protein.
Download the coordinate file (in PDBformat) to your working directory
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InsightII:InsightII:
One stop Graphical User InterfaceOne stop Graphical User Interfacefor life sciencesfor life sciences
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InsightII features cover todayInsightII features cover today
Molecular Visualization
Structure comparison Sequence alignment
Structure building
Molecular Modeling Energy Calculation
Minimization
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How to run InsightIIHow to run InsightII
Available on SGI and Linux workstations
module load insightII
start_insightII
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InsightIIInsightIIMSI logo
Viewer toolbar
Module toolbar
Frequently
Used buttons
Frequently used buttons
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LoadingLoading
moleculemolecule
Note: Molecule/GetNote: Molecule/Get
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DisplayingDisplaying
StructureStructure
Note: Molecule/DisplayNote: Molecule/Display
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HomologyHomology
Here I am
Homology toolbar
Note: Accelrys Logo/homologyNote: Accelrys Logo/homology
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ExtractingExtracting
SequenceSequence
Sequence Viewing
Window
Note: Sequences/ExtractNote: Sequences/Extract
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Manipulating Sequence Manually with the mouseManipulating Sequence Manually with the mouse
Toggle on Seq on Sequence Viewing Window
Moving Entire Sequence
Middle click on a residue and drag to move the entire sequence Creating Gap
Right click on a residue and drag to the right to create gap to theright of residue
Left click on a residue and drag to the left to create gap to the leftof residue
Moving entire Gap Right click on a gap and drag to the left to move entire gap to left Left click on a gap and drag to the right to move entire gap to right
Splitting Gap Right click on a gap and drag to the right to split gap to right Left click on a gap and drag to the left to split gap to left
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MultipleMultiple
SequenceSequenceAlignmentAlignment
Note: Alignment/Multiple SequenceNote: Alignment/Multiple Sequence
SuperimposeStructure usingSCR
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AlignedAlignedStructureStructure
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Aligned SequenceAligned Sequence
Structurally ConservedRegions (SCR)
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GetGet
SequenceSequence
Note: Sequences/GetNote: Sequences/Get
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PairwisePairwise
SequenceSequenceAlignmentAlignment
Note: Alignment/Note: Alignment/Pairwise_SequencePairwise_Sequence
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Aligned SequenceAligned Sequence
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SequenceSequenceIdentityIdentity
Note: Alignment/Note: Alignment/Percent_IdentityPercent_Identity
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Manipulating Boxes Manually with the mouseManipulating Boxes Manually with the mouse
Toggle on Box on Sequence Viewing Window
Creating Boxes
Left click on one residue of a sequence and drag to anotherresidue of another sequence to create a box Altering Boxes
Right click on left or right side of box and drag to shrink orexpand box
Moving Boxes Middle click on edge of box to move box
Freezing/Unfreezing Boxes Middle click then left click to freeze box
Repeat to unfreeze box Deleting Boxes
Middle click, hold down control on keyboard, then left clickto delete box
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Assigning SCR manuallyAssigning SCR manually
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AssigningAssigning
CoordinatesCoordinatesfrom Alignedfrom AlignedSequenceSequence
Note: Sequences/Note: Sequences/Assign_CoordsAssign_Coords
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GeneratingGenerating
LoopsLoops
Note: Loops/GenerateNote: Loops/Generate
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DisplayingDisplaying
LoopsLoops
Note: Loops/DisplayNote: Loops/Display
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AssignAssign
CoordinatesCoordinatesto Loopsto Loops
Note: Loops/DisplayNote: Loops/Display
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SettingSetting
VariableVariableto Localto LocalPDBPDB
Note: Session/Note: Session/Env_VarEnv_Var//usr/local/db/pdb/current/entriesusr/local/db/pdb/current/entries
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SearchingSearching
LoopsLoops
Note: Loops/SearchNote: Loops/Search
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End RepairEnd Repair
Note: Refine/Note: Refine/EndRepairEndRepair
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DisplayingDisplaying
TraceTrace
Note: Molecule/DisplayNote: Molecule/Display
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SCR andSCR and
LoopLoopRegionsRegions
Note: Molecule/DisplayNote: Molecule/Display
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RelaxRelaxSetupSetup
Note: Refine/RelaxNote: Refine/Relax
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RelaxRelax
Note: Refine/RelaxNote: Refine/Relax
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Compare withCompare with
ExperimentalExperimentalStructureStructure(1CD2)(1CD2)
Note: Loops/DisplayNote: Loops/Display
R i
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RunningRunning
FastaFasta
Note: Databases/InputNote: Databases/Input
R iR i
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RunningRunning
FastaFasta
Note: Databases/OutputNote: Databases/Output
R iR i
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RunningRunning
FastaFasta
Note: Databases/RunNote: Databases/Run
F tF t
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FastaFasta
PrintoutPrintout
F tF st
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FastaFasta
PrintoutPrintout
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Learning more about InsightIILearning more about InsightIIand its modules on your ownand its modules on your own
Pilot tutorials
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InsightII DocumentationInsightII Documentation
http://www.accelrys.com/doc/life/index.html
Username: msi
Passwd: msi-doc
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To get helpTo get help
By mail [email protected]
Web www.msi.umn.edu
Phone 612 626 0802
Appointment TBA
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Contact InformationContact Information
Yuk Sham
Computational Biology/Biochemistry Consultant
Phone: (612) 624 7427 (Walter Library)
Phone: (612) 624 0783 (VWL)Email: [email protected]