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DESCRIPTION
To solve an unknown Protein Sequence
Citation preview
Submitted by
-Chainika Das (10110009)
-Jha Ashutosh (10110025)
Objective To identify the structure of the given protein
sequence:
>gi|1336632|gb|AAB18304.1| 2,3-dihydro-2,3-dihydroxybiphenyl-2, 3-dehydrogenase [Comamonas testosteroni]
MKLTGEVALITGGASGLGRALVDRFVAEGARVAVLDKSAERLRELEVAHGGNAVGVVGDVRSLQDQKRAAERCLAAFGKIDTLIPNAGIWDYSTALADLPEDKIDAAFDDIFHVNVKGYIHAVKACLPALVSSRGSVVFTISNAGFYPNGGGPLYTATKHAVVGLVRQMAFELAPHVRVNGVAPGGMNTDLRGPSSLGLSEQSISSVPLADMLKSVLPIGRMPALEEYTGAYVFFATRGDSLPATGALLNYDGGMGVRGFLTAAGGADLPEKLNINREGQE
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`Target MTZ
&
Sequence
Target
Details
TemplateSearch
Model
Preparation
Molecular Replacement
& Refinement
Check scores
and exit or
select the next
model
Overall Process
Steps to follow(when search
model is given)
Given mtz, seq & pdb
MOLREP & Refinement (obtain score,
contrast)
WINCOOT (mutation, translation ,
validation, save as Refmac)
Model preparation
Refinement (check for R factor and R
free)
If search model is not available
Pblast - database
Similarity known structure is obtained by
using already present sequence.
1. Blast
Choose the hit with sequence
identity more than 25%
2. Matthew’s Coefficient
3. Molrep
-Score
-contrast
4. Refmac
-R factor
-R free
5. Wincoot
-Mutations
-Rotamers
-Ramachandranplot
6. Pymol
Active site
Procedure
MOLREP
REFMAC
Wincoot
CCP4
Scope: covers data processing through to
refinement and validation
Molecular Replacement programs:
Molrep
Phaser
Create New
Directory
Matthews Coefficient
Water must be approx 50 %
Molecular Replacement Molrep is program for automated molecular replacement in
the case where a homologous structure has already been
identified.
The program will attempt to find the number of molecules
expected in the asymmetric unit as entered by the user.
A PDB file for the best solution is output.
Additional options
Self rotation function
Search for model in a map
Alignment only
Can perform individual steps in more difficult cases
MOLREP
3 inputs:
o Mtz file – experiment data
o Pdb file- search model
o Use Sequence
Output is .pdb (to be input in refmac) file
- positioned search model
-input for the next stage (restrained
refinement)
Steps
Log file
Steps
Score: product of correlation coefficient of intensity and maximum value of packing fraction.Contrast: Ratio of Top score to mean score. Should be >2.5
Steps
Refmac
The search model obtained from MOLREP
is passed to Refmac for refinement.
The change in the Rfree & R factor value
during refinement is used as a measure to
check how good the resulting model is.
Steps
Log file
Steps
1. Free-R: R-factor is calculated for a test-set of reflections that is never
included in refinement.
Difference between R and R-free is smaller for higher resolution and well-refined structures
Refinement is carried out in repetitive cycles till R-factor converges to a low value with appropriate geometry of the atomic model.
This value is 5% from the overall data.
2. R-factor:
It is a measure of how well the refined structure predicts the observed value
R =∑ |(Fobs)|-|(Fcalc)| ∑|(fobs)|
It should be 15-20% for macromolecules.
Wincoot
It is used for manipulation and
visualization of the Refmac5 pdb file.
With the help of overlapping electron
density the amino acids are identified and
fit in the structure.
The red and green regions are removed.
Steps
Steps
Steps
The red density indicates incorrect position
of proteins while the green density
indicates the scope of placing the
proteins.
On the overlap of blue and green density
side chains should be placed.
Steps
Steps
Validation
Ramachandran Plot
Rotamer analysis
Steps
If >80%, structure is correct.
6 outliers
Ramachandran Plot
Steps
Change residues Phi & Psi to remove outliers
Steps
Steps
Steps
Steps
Back To CCP4- Refmac
Steps
Input new
pdb file saved
from Coot
Steps
Steps
Rotamer Analysis
Red Bars Show Poor Rotamers
Steps
Poor rotamer is removed
Steps
Steps
Fill Incomplete areas, use Sequence file to know the missing sequence
Steps
Steps
Steps
Final R factor is less than the initial.
PyMOL
It is an open-source, user-sponsored,
molecular visualization system.
It is well suited to producing high quality
3D images of small molecules and
biological macromolecules such as
proteins.