Single-particle cryo-EM
protocol for preparing MP/APol samples (Protocol
12.1), 579–583
random conical tilt reconstruction method, 536,
557, 563
specimen preparation, 535
Single-particle cryo-EM studies of MP/APol complexes
overview, 566
Single-particle EM, 535
achieving near-atomic resolution, 539
application to MPs, 540
assessment of resolution achieved, 536, 537
the basics, 535
correcting for sample drift, 538
direct-detection devices, 538
flow-chart of an experiment, 540
of frozen specimens, 536
impact of technological advances, 541
of negatively-stained specimens, 536
principle, 539
the resolution revolution, 538, 541
use of APols, 541–578
Single-particle EM studies of MP/APol complexes
comparative studies using various surfactants, 556
cryo-EM studies, 562–573
early studies, 549–554
film thickness in APol- vs. detergent-based cryo-EM
samples, 550, 551
flow-chart of an experiment, 579
NS studies, 554–561
orientation of MP/APol vs. MP/detergent particles, 575
overview, 544
particle distribution in APol- vs. detergent-based cryoEM samples, 550, 551
STEM studies, 549–554
siRNA
MD simulation of delivery by APols, 264
use of APols for delivery, 238
Size exclusion chromatography (SEC), 417
of MP/APol complexes, 444–446
protocol for analyzing MP/APol complexes (Protocol
9.2), 444–446
Sligar, S.G., 105, 107
SLS, see Static light scattering
Small multidrug-resistance protein (Smr)
in bicelles, 100
Small-angle neutron scattering (SANS), 426–438, 537
effect of deuterating the sample, 428
effect of deuterating the solvent, 428
information on the internal dynamics
of the particles, 430
measuring D t , 430
Stuhrmann plot, 433, 434
Small-angle X-ray and neutron scattering (SAXS
and SANS)
deviations from linearity in Guinier plots, 429
effect of aggregation, 431
Guinier approximation, 429
Guinier plot, 429, 431
information on the shape of particles, 429
interparticle effects, 431
interpretation of scattering curves, 429
pair distribution function, 430, 431
principle, 428
Small-angle X-ray scattering (SAXS), 426–438, 537
SMFS, see Single-molecule force spectroscopy (SMFS)
SNAREs
vesicle-bound, interaction with ND-trapped VAMP-2,
116, 117
Sodium dodecylsulfate (SDS), 64
chemical structure, 61
CMC, 61
Sokalan CP9
¼ DIBMA, 164
Solid-state NMR (ssNMR), 454
possible uses of APols, 490
Solution NMR (sNMR), 454
Solution NMR studies of APol-trapped MPs, 461–488
A8-35 trapped melanocortin receptors, 488
accessibility of MP surface in OmpX/A8-35 vs.
OmpX/di-C 6 -PC complexes, 471, 472
accessibility of MP surface to a water-soluble
paramagnetic agent, 471, 472
advantages and drawbacks with respect to other
systems, 488
biologically oriented studies, 482–488
comparative studies in diC 6 PC and various
APols, 465
comparison of 2D NMR spectra of tOmpA/SAPol
complexes at 30, 50 and 70
C, 476
comparison of NMR spectra of OmpX in NAPol vs.
A8-35, 478
conformation of LTB 4 bound to the BLT2 leukotriene
receptor, 483–487
dipolar broadening in HAPol vs. DAPol, 469, 470
dynamics of the TM β-barrel of OmpX, 483–485
improved stability of GLUT4 in A8-35 vs. LMNG, 475
intermolecular contacts mapped by heteronuclear
dipole-to-dipole cross-relaxation, 470
mapping intermolecular contacts, 469, 470
methodologically oriented studies, 463–473
MOMP/A8-35 complexes, 488
15
N1
H CRINEPT spectra of BLT2 in the presence or
absence of LTB 4 , 487
overview, 461, 462
pH-insensitive APols, 473–479
relative advantages and drawbacks of A8-35, SAPols
and NAPols, 479
2D NMR spectrum of CRFR2β in NVoy, 479
use of deuterated APols, 479–482
use of NAPols, 477, 478
validation, 463–473
Solution NMR studies of MPs
APols as helpers for sample preparation, 456
in bicelles, 457
complete assignments, 458
effect of detergent choice and temperature setting, 460
in NDs, 458
overview, 455–461
producing the protein, 455
706
Index
protocol for preparing MP/APol samples (Protocol
12.1), 579–583
random conical tilt reconstruction method, 536,
557, 563
specimen preparation, 535
Single-particle cryo-EM studies of MP/APol complexes
overview, 566
Single-particle EM, 535
achieving near-atomic resolution, 539
application to MPs, 540
assessment of resolution achieved, 536, 537
the basics, 535
correcting for sample drift, 538
direct-detection devices, 538
flow-chart of an experiment, 540
of frozen specimens, 536
impact of technological advances, 541
of negatively-stained specimens, 536
principle, 539
the resolution revolution, 538, 541
use of APols, 541–578
Single-particle EM studies of MP/APol complexes
comparative studies using various surfactants, 556
cryo-EM studies, 562–573
early studies, 549–554
film thickness in APol- vs. detergent-based cryo-EM
samples, 550, 551
flow-chart of an experiment, 579
NS studies, 554–561
orientation of MP/APol vs. MP/detergent particles, 575
overview, 544
particle distribution in APol- vs. detergent-based cryoEM samples, 550, 551
STEM studies, 549–554
siRNA
MD simulation of delivery by APols, 264
use of APols for delivery, 238
Size exclusion chromatography (SEC), 417
of MP/APol complexes, 444–446
protocol for analyzing MP/APol complexes (Protocol
9.2), 444–446
Sligar, S.G., 105, 107
SLS, see Static light scattering
Small multidrug-resistance protein (Smr)
in bicelles, 100
Small-angle neutron scattering (SANS), 426–438, 537
effect of deuterating the sample, 428
effect of deuterating the solvent, 428
information on the internal dynamics
of the particles, 430
measuring D t , 430
Stuhrmann plot, 433, 434
Small-angle X-ray and neutron scattering (SAXS
and SANS)
deviations from linearity in Guinier plots, 429
effect of aggregation, 431
Guinier approximation, 429
Guinier plot, 429, 431
information on the shape of particles, 429
interparticle effects, 431
interpretation of scattering curves, 429
pair distribution function, 430, 431
principle, 428
Small-angle X-ray scattering (SAXS), 426–438, 537
SMFS, see Single-molecule force spectroscopy (SMFS)
SNAREs
vesicle-bound, interaction with ND-trapped VAMP-2,
116, 117
Sodium dodecylsulfate (SDS), 64
chemical structure, 61
CMC, 61
Sokalan CP9
¼ DIBMA, 164
Solid-state NMR (ssNMR), 454
possible uses of APols, 490
Solution NMR (sNMR), 454
Solution NMR studies of APol-trapped MPs, 461–488
A8-35 trapped melanocortin receptors, 488
accessibility of MP surface in OmpX/A8-35 vs.
OmpX/di-C 6 -PC complexes, 471, 472
accessibility of MP surface to a water-soluble
paramagnetic agent, 471, 472
advantages and drawbacks with respect to other
systems, 488
biologically oriented studies, 482–488
comparative studies in diC 6 PC and various
APols, 465
comparison of 2D NMR spectra of tOmpA/SAPol
complexes at 30, 50 and 70
C, 476
comparison of NMR spectra of OmpX in NAPol vs.
A8-35, 478
conformation of LTB 4 bound to the BLT2 leukotriene
receptor, 483–487
dipolar broadening in HAPol vs. DAPol, 469, 470
dynamics of the TM β-barrel of OmpX, 483–485
improved stability of GLUT4 in A8-35 vs. LMNG, 475
intermolecular contacts mapped by heteronuclear
dipole-to-dipole cross-relaxation, 470
mapping intermolecular contacts, 469, 470
methodologically oriented studies, 463–473
MOMP/A8-35 complexes, 488
15
N1
H CRINEPT spectra of BLT2 in the presence or
absence of LTB 4 , 487
overview, 461, 462
pH-insensitive APols, 473–479
relative advantages and drawbacks of A8-35, SAPols
and NAPols, 479
2D NMR spectrum of CRFR2β in NVoy, 479
use of deuterated APols, 479–482
use of NAPols, 477, 478
validation, 463–473
Solution NMR studies of MPs
APols as helpers for sample preparation, 456
in bicelles, 457
complete assignments, 458
effect of detergent choice and temperature setting, 460
in NDs, 458
overview, 455–461
producing the protein, 455
706
Index