Salomon Garcia: Week 7: Difference between revisions
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**Contacsym program was used to assign van der waals contacts. | **Contacsym program was used to assign van der waals contacts. | ||
====Results and Discussion==== | |||
*Electron Density maps were good quality | |||
*R-values were higher due to constant refinement and adjustment | |||
====Results==== | |||
Revision as of 14:38, 8 March 2010
Preparation for Journal Club 2
- The Article for Journal Club 2; this is the link: Article for Journal Club 2
- Link to the powerpoint presentation: Journal Club 2 Powerpoint
Ten New words from the 2003 Stanfield Article:
- Twinning- The assemblage of two or more crystals, or parts of crystals, in reversed position with reference to each other in accordance with some definite law
- CDR- complementarity determining region (CDR) is a relatively short amino acid sequence found in the variable (V) domains of antigen receptors
- Fab proteins- fragment of immunoglobulin prepared by papain treatment.
- Deglycosylated-The removal of the sugar entity from a glycogen
- Peptide cyclization- Changing an open-chain hydrocarbon to a closed ring in a peptide bond
- TOM/FRODO- a computer program that builds graphic images of a particular model.
- Ramachandran plot- is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure.[1] It shows the possible conformations of ψ and φ angles for a polypeptide.
- Matthews coefficient- is used in machine learning as a measure of the quality of binary (two-class) classifications.
- ASW mice- a hybrid mice often used in experimentation
- BALB/c mice-is an albino, laboratory-bred strain of the House Mouse from which a number of common substrains are derived.
http:wikipedia.org
http:biology-online.org/dictionary
Outline
Introduction
- HIV has an outer surface of glyco proteins
- gp120 responsible for binding to CD4 receptors and coreceptors, CCR5 or CXCR4
- gp41 anchors gp120 to the viral membrane providing noncovalent association
Materials and Methods
- Mab 83.1 was made by immunization of an ASW mice with cyclic peptide RP70
- Antibody was produced in ascites fluid of a mice and purified with an immobilized protein A column
- Fab was made from immunoglobin by cleavage
- Fab was concentrated to 15.0 mg/ml for crystallization studies
- Fab Purification
- Fab was mixed with 16-mer peptide MP1 in a 6:1 mole ration
- Crystals were grown using sitting-drop vapor diffusion method with a reservoir solution of 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0.
- Crystals grow as clusters of thin plates
- Crystals used for this experiments grew over a 2-week period
- Fab Crystallization procedure
- Were cryocooled to liquid nitrogen temperatures in order to collect the data in a rapid manner.
- The crystals were protected by putting them in a solution containing the following 25% glycerol, 1.6 M Na/K phosphate, 5% isopropanol, pH 6.0
- HKL2000 was used to format all of the data that was obtained
- Structure determination
- Matthew coefficient was obtained by two Fab molecules
- Model was constructed from the constant region of Fab 58.2
- EPMR program was used to position the model in the cell
- EPMR also used to locate the first Fab molecule in the asymetric unit
- Model building and refinement
- TOM/FRODO was used to rebuild the mutated hybrid model and to correct the sequence and were subsequently refined with CNS version 1.1.
- Refinement was carried with tight NCS restraints in the beginning and progressively released towards the end of refinement
- Structural Analysis
- Kabat convention was used to number the molecules
- Light and heavy chains are labeled using “L” and “H”
- Peptide labeled “P” and was numbered according to HXB2 isolate sequence
- HBPLUS was used to evaluate the Hydrogen bonds
- Contacsym program was used to assign van der waals contacts.
- Kabat convention was used to number the molecules
Results and Discussion
- Electron Density maps were good quality
- R-values were higher due to constant refinement and adjustment