10.3.3 Biologically Oriented Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
10.3.3.1 Dynamics of the Transmembrane β-Barrel of OmpX . . . . . . . . 483
10.3.3.2 Conformation of LTB 4 Bound to the BLT2 Leukotriene
Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
10.3.3.3 Preliminary Studies of Chlamydia trachomatis MOMP
and Two Melanocortin Receptors . . . . . . . . . . . . . . . . . . . . . . 488
10.4 Challenges and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
11 Amphipols and Membrane Protein Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . 497
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
11.2 Context: Classical and Less Classical Approaches to Crystallizing Membrane
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
11.2.1 Factors Contributing to the Steady Growth of the Number
of MP Structures Solved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
11.2.2 The Formation of Membrane Protein Crystals . . . . . . . . . . . . . . . . . . . . 500
11.2.2.1 Crystals Formed in Detergent Solutions . . . . . . . . . . . . . . . . . 503
11.2.2.2 Forming Crystals in Lipid Three-Dimensional Phases . . . . . . . 506
11.3 Using Amphipols for Crystallizing Membrane Proteins . . . . . . . . . . . . . . . . . . . . 508
11.3.1 Crystallization of Cytochrome bc 1 /A8-35 Complexes in Aqueous
Solutions: Failures and Partial Successes and the Possible Reasons
Underlying Them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
11.3.2 Using Amphipols as a Vehicle to Crystallize Membrane Proteins
in Meso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
11.3.3 Challenges and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
11.4.1 Pre-crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
11.4.2 Crystallization of Ternary Protein/Amphipol/Detergent Complexes . . . . . 524
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
12 The Use of Amphipols for Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
12.2 Context: Electron Microscopy Studies of Membrane Proteins . . . . . . . . . . . . . . . . 534
12.2.1 The Various Approaches to Studying Membrane Proteins
by Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
12.2.2 Single-Particle Electron Microscopy Studies of Biological
Molecules: The Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
12.2.3 Single-Particle Cryo-EM Studies of Biological Molecules:
The Resolution Revolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
12.2.4 Single-Particle Electron Microscopy Studies of Membrane Proteins . . . . . 540
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies . . . . . 541
12.3.1 The Early Years: 1998–2008 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
12.3.2 Recent Studies of Negatively Stained MP/APol Complexes
(2011–Present) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
12.3.2.1 Conclusions from Negative-Stain Studies . . . . . . . . . . . . . . . . 561
12.3.3 Cryo-EM Studies of MP/APol Complexes (2011–Present) . . . . . . . . . . . 562
Contents
xxiii
10.3.3.1 Dynamics of the Transmembrane β-Barrel of OmpX . . . . . . . . 483
10.3.3.2 Conformation of LTB 4 Bound to the BLT2 Leukotriene
Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
10.3.3.3 Preliminary Studies of Chlamydia trachomatis MOMP
and Two Melanocortin Receptors . . . . . . . . . . . . . . . . . . . . . . 488
10.4 Challenges and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
11 Amphipols and Membrane Protein Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . 497
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
11.2 Context: Classical and Less Classical Approaches to Crystallizing Membrane
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
11.2.1 Factors Contributing to the Steady Growth of the Number
of MP Structures Solved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
11.2.2 The Formation of Membrane Protein Crystals . . . . . . . . . . . . . . . . . . . . 500
11.2.2.1 Crystals Formed in Detergent Solutions . . . . . . . . . . . . . . . . . 503
11.2.2.2 Forming Crystals in Lipid Three-Dimensional Phases . . . . . . . 506
11.3 Using Amphipols for Crystallizing Membrane Proteins . . . . . . . . . . . . . . . . . . . . 508
11.3.1 Crystallization of Cytochrome bc 1 /A8-35 Complexes in Aqueous
Solutions: Failures and Partial Successes and the Possible Reasons
Underlying Them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
11.3.2 Using Amphipols as a Vehicle to Crystallize Membrane Proteins
in Meso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
11.3.3 Challenges and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
11.4 Protocol: Crystallizing bc 1 /A8-35/Detergent Complexes from Aqueous
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
11.4.1 Pre-crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
11.4.2 Crystallization of Ternary Protein/Amphipol/Detergent Complexes . . . . . 524
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
12 The Use of Amphipols for Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
12.2 Context: Electron Microscopy Studies of Membrane Proteins . . . . . . . . . . . . . . . . 534
12.2.1 The Various Approaches to Studying Membrane Proteins
by Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
12.2.2 Single-Particle Electron Microscopy Studies of Biological
Molecules: The Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
12.2.3 Single-Particle Cryo-EM Studies of Biological Molecules:
The Resolution Revolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
12.2.4 Single-Particle Electron Microscopy Studies of Membrane Proteins . . . . . 540
12.3 The Use of Amphipols for Membrane Protein Electron Microscopy Studies . . . . . 541
12.3.1 The Early Years: 1998–2008 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
12.3.2 Recent Studies of Negatively Stained MP/APol Complexes
(2011–Present) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
12.3.2.1 Conclusions from Negative-Stain Studies . . . . . . . . . . . . . . . . 561
12.3.3 Cryo-EM Studies of MP/APol Complexes (2011–Present) . . . . . . . . . . . 562
Contents
xxiii