BL3301 Introduction to protein structure determination
www.st-andrews.ac.uk/~glt2/BL3301
1915 Nobel prize for physics Nobel Prize science
Sir William Bragg Sir Lawrence Bragg
1962 Nobel Prizes “Nobel Prize for British Wife” Daily Mail 1964
Penicillin
Vitamin B12
Insulin Wilkins, Steinbeck, Kendrew, Perutz, Crick, Watson Dorothy Hodgkin
1 Nobel prize 1988 Nobel Prize 1982 Aaron Klug The first membrane protein structure Deisenhofer, Huber & Michel Protein-nucleic acid complexes EM & crystallography
Nobel Prize 1991 Nobel prize 2002 for the development of methods for Development of high field NMR identification and structure analyses of Richard Ernst biological macromolecules
John Fenn Koichi Tanaka Kurt Wüthrich Mass spectrometry Protein structure electrospray and laser desorption determination by NMR
Nobel prize 2003 Nobel Prize 2006 for discoveries concerning channels in membranes
Peter Agre Rod MacKinnon Water channels Ion channels Roger Kornberg
Cramer, Bushnell & Kornberg Science 292, 1863-1876 (2001)
Gnatt et al. Science 292, 1876-1882 (2001)
2 Structural Basis of Transcription: RNA Polymerase II at 2.8Å Resolution
Cramer, Bushnell & Kornberg Science 292, 1863-1876 (2001)
Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3Å
Gnatt et al. Science 292, 1876-1882 (2001)
Translation RNA→Protein
The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution
Ban et al. Science 2000, 289, 905-920.
The Structural Basis of Ribosome Activity in Peptide Bond Synthesis
Nissen et al. Science 2000, 289, 920-930.
50S subunit: 3000 nucleotides, 31 proteins, catalyses peptide bond formation
Structure of the 30S ribosomal subunit
Wimberly et al. Nature (2000) Nature, 407, 327-339
30S subunit: 1500 nucleotides, 20 proteins, Binds tRNA molecules
3 Aminoacyl site Translation Peptidyl site RNA→Protein Exit site Crystal Structure of the Ribosome at 5.5 Å Resolution Yusupov et al. Science 2001 292, 883-896.
Recognition of Cognate Transfer RNA by the 30S Ribosomal Subunit
Ogle et al. Science 2001 292, 897-902. E site P site A site
Anticodon Codon
1st position 1st position
2nd position 2nd position
3rd position 3rd position
4 LacC MRSA LpxD SUMO-SENP PrnA Lectin NanB NanJ
SUMO-SENP- pseudaminidase PSV131 RadA RanGAP PCNA sso1404 alba2 wza Psv165a
sirv21 Sso2273
Lm MK hydroxypropylpho MRSA681 sphonic acid Cathsili epoxidase
AlcC sirv48 Ranasmurfin PPAR-beta/delta sso0953
sirv30
PFK KDG kinase AtKAT Acsd x 3
Serine Palmitoyl PSV239 vc1805 mevalonate diphosphate transferase x 3 sso2462 pteridine reductase decarboxylase Human Qprtase DNA Glycosylase-1 x2
X-ray crystallography
• Advantages – Highest resolution (typically 0.2nm or 2Å) – Ideal for drug design – Effectively no size limit • Disadvantages – Need a crystal – Need milligrams of pure protein – Solid state, so molecule is “frozen”
X-ray data collection European Synchrotron Radiation Facility Grenoble, France
5 Nuclear magnetic resonance Diamond - near Oxford (NMR) • Advantages – No need for crystal - perform in solution – Can alter environment – Can monitor dynamics • Disadvantages – Limited to about 50kDa in size – Need milligrams of protein – Medium “resolution” compared to X-ray
NMR data collection
950MHz NMR Cryo-electron microscopy
• Advantages – No upper size limit – Need only nanograms of material • Disadvantages – Low resolution ( 1nm or 10Å at best) – Blobology – Lower size limit
6 Cryo-EM data collection
7