BMS2062 Lecture Notes - Lecture 19: Lysine, Fluorescent Tag, Granzyme
30 May 2018
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Week 10. Structure Determination and Protein
Membrane Interactions
FROM SEQUENCE TO STRUCTURE: STRUCTURE DETERMINATION
• Structural biology: The determination and analysis of the 3D structures of biological
macromolecules
• PDB (protein data bank) – how many structures?
find more resources at oneclass.com
find more resources at oneclass.com
• “trutures i the PDB
o Each entry is given a unique identification code – PDBID
o Coordinates can be downloaded and displayed in the program PYMOL
o Information in a PDB file:
Tells you residue, atom, outer carbon
B-factor = temperature factor, give you an idea of how mobile atoms are in
structure (B factor is only meaningful in structures determined using X-ray
diffraction
NM‘, EM ad odellig doest gie ou B fator
o B factor:
Indication of mobility of an atom
The smaller the B factor the more blue – the higher, the more red
Dark lue i iddle = ore protei H oded ad doest oe uh, ore
rigid
The atoms deeply buried in the core are usually hardly moving at all
Surface is more mobile
• Common questions about protein structure:
o Where are the secondary structures (alpha helices and beta sheets)
o What residues form the hydrophobic core? (usually lowest b value)
o Distance between two positions
o Backbone angles
o Is the protein multimeric
o Where is the active site? (which residues are important to carry out function)
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find more resources at oneclass.com
• Example from PDB: antibiotic target from methicillin resistance in S. aureus – the enzyme and
transcription factor Biotin Protein Ligase (BPL)
- the crystal structure was determined and used to direct design of selective inhibitors
- essetial eze i ateria that the at surie ithout
• How do you determine the structure of a molecule?
o X-ray crystallography – need crystals and X-rays
Steps:
1. Express/purify protein
2. Crystallise protein (spinning crystals in front of X-ray beam)
3. Collect diffraction
4. Determine space group (parameters, unit cell dimensions)
5. “ole the phase prole – always need a phase
6. Calculate on electron density map – tells you were all atoms are in molecule
7. Build model to fit density map
8. Refine model
o NMR – no crystal, molecule is just tumbling in solution
• Obtaining samples for structure determination:
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find more resources at oneclass.com
Document Summary
B-factor = temperature factor, give you an idea of how mobile atoms are in structure (b factor is only meaningful in structures determined using x-ray diffraction. Nm , em a(cid:374)d (cid:373)odelli(cid:374)g does(cid:374)(cid:859)t gi(cid:448)e (cid:455)ou b fa(cid:272)tor: b factor: The smaller the b factor the more blue the higher, the more red. Dark (cid:271)lue i(cid:374) (cid:373)iddle = (cid:272)ore protei(cid:374) (cid:894)h (cid:271)o(cid:374)ded(cid:895) a(cid:374)d does(cid:374)(cid:859)t (cid:373)o(cid:448)e (cid:373)u(cid:272)h, (cid:373)ore rigid. The atoms deeply buried in the core are usually hardly moving at all. Surface is more mobile: common questions about protein structure, where are the secondary structures (alpha helices and beta sheets, what residues form the hydrophobic core? (usually lowest b value, distance between two positions, backbone angles. Is the protein multimeric: where is the active site? (which residues are important to carry out function, example from pdb: antibiotic target from methicillin resistance in s. aureus the enzyme and transcription factor biotin protein ligase (bpl)
