Membrane protein session

Erice

16-6-06

Introduction Werner Kühlbrandt 5 min

Aquaporin 0 Tom Walz 30+5 min

Na+/H+ antiporter Carola Hunte 30+5 min

EM and x-ray Werner Kühlbrandt 30+5 min

Coffee break 30 min

Photosystem II Jan Kern 30+5 min

Solid-state NMR Hartmut Oschkinat 30+5 min

Programme

in pdb

PDB entries for membrane protein structures

0

50

100

150

200

250

1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Year

Number of structures

new totaldeposits

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all PDB entries

1976

2006

35,000

190 membrane protein entries

What makes it so hard ?

• Few membrane proteins are naturally abundant

• Most are difficult to express• Difficult to purify• Difficult to crystallize• Difficult to solve

Usual problems• Insufficient protein available• Protein unstable in detergent solution• Protein precipitates upon concentration• Detergent interferes with crystal order• Bound lipids may or may not be required• Crystals are often small, poorly ordered and

anisotropic • Crystals often cannot be frozen

Different types of membrane protein crystalsType 1 crystals Type 2 crystals

“Type 4” crystals“Type 3” crystals

Use of membrane protein crystals• 3D crystals for X-ray crystallography

– Protein in detergent solution– Initial crystals often poorly ordered– Antibody fragments as spacers– Lipidic cubic phases ?

• 2D crystals for electron microscopy– Protein in lipid bilayer– Physiological conditions– Good probability– Experimental phases

• Small crystals for solid-state NMR

Techniques for structure determination• X-ray crystallography (Carola Hunte, Jan Kern)

– atomic coordinates – single state (unless you are lucky)

• Electron crystallography – 2D crystals: 3 Å - 8 Å resolution with ab initio phases

(1.8 Å by electron diffraction: Tom Walz) – protein in membrane– different conformational states (Werner Kühlbrandt)

• Single-particle EM– 6 Å - 20 Å resolution– protein in solution or in membrane

• NMR– Solution: small -barrels– Solid-state (Hartmut Oschkinat)