Dataset Information

Single particle reconstruction of 52 kDa apo-state streptavidin at 3.3 Angstrom resolution

ABSTRACT:

SUBMITTER: Hong-Wei Wang

PROVIDER: EMPIAR-10269 | biostudies-other |

REPOSITORIES: biostudies-other

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Publications

Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution.

Fan Xiao X Wang Jia J Zhang Xing X Yang Zi Z Zhang Jin-Can JC Zhao Lingyun L Peng Hai-Lin HL Lei Jianlin J Wang Hong-Wei HW

Nature communications 20190603 1

The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of ...[more]

PMID: 31160591

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Project description:Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ?0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 Å localization precision for 40 nm AuNPs and 1.9 Å localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 ?s time resolution, localization precisions at 5.4 Å for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors.

Dataset Information

Single particle reconstruction of 52 kDa apo-state streptavidin at 3.3 Angstrom resolution

Publications

Single particle cryo-EM reconstruction of 52 kDa streptavidin at 3.2 Angstrom resolution.

Similar Datasets

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