EM-FRAME · DNA-origami scaffolds for structure determination by single particle analysis
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2013-03-01 → 2015-02-28
- Финансиране от ЕС
- 221 606 €
- Участници
- 1
- Схема
- MC-IIF
Линиите свързват координатора с партньорите.
Накратко на български
Криоелектронната микроскопия помага за определяне на структурата на малки молекулярни комплекси, като например рибозомите при хората и дрождите. По-качествените изображения и новите методи за обработка позволяват по-точно да се разберат разположението и формата на тези частици.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
DNA-origami scaffolds for structure determination by single particle analysis
We aimed to improve cryo-EM structure determination by single-particle analysis, in particular for relatively small complexes (100-300 kDa). In that context, we highlighted three fundamental problems in cryo-EM structure determination that arise from the intrinsically low signal-to-noise ratios in the experimental images. Our proposed solutions to these problems were centered on the use of 3D DNA-origami objects (EM-frames). However, the advent of direct-electron detectors resulted in images of unprecedented quality. This had the unforeseen side effect that for a wide range of samples, the use of 3D DNA-origami frames was no longer necessary. We still addressed (and solved!) the 3 problems, but we did this in unanticipated ways. Problem 1): Selecting particles is hampered by very noisy backgrounds. Solution: better contrast of particles in the images from the new detectors greatly facilitated particle selection. Problem 2): Beam-induced motions affect the attainable resolution. Solution: we developed an image-processing procedure that uses movies from the new detectors to correct for sample motions. Using this procedure, we obtained near-atomic resolution reconstructions from only 30,000 particles (Bai et al. eLife 2013). Problem 3): The accuracy with which the orientations of individual particles can be determined is a limiting factor for structure solution. Solution: also in this case did the improved quality of the images lead to a huge improvement, such that we can now align particles in a wide size range. We then went on to demonstrate the potential of the developed procedures by solving near-atomic resolution cryo-EM structures for the mitochondrial ribosome from yeast (Amunts 2014, Science) and human (Brown 2015, Science), the yeast cytoplasmic ribosome in complex with initiation factor 5B (Fernandez, 2013, Science), the cytoplasmic ribosome from P. falciparum (Wong, 2014, eLIFE), human gamma-secretase (Lu, 2014, Nature, Bai 2015, Nature), the DARK apotosome (Pang, 2015, Gene & Development), and the rabbit ryanodine receptor RyR1 (Yan, 2015,Nature).
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Single-particle analysis of electron cryo-microscopy (cryo-EM) data can now provide near-atomic resolution information for large and high-symmetry protein complexes. However, application of the same method to small proteins is problematic, mainly because low signal-to-noise ratios hamper particle selection and orientation determination.The proposed research aims to tackle this problem by binding proteins to large 3D scaffolds made of DNA origami. The main idea is to use a hollow rectangular frame (called DNA alignment frame) with a dsDNA helix (called rotation axis) spanning the interior hole of the frame. By using a specific sequence, DNA-binding proteins may be bound to the rotation axis in a specific manner. The orientation of the bound protein may then be controlled by exploiting the helical character of the rotation axis in a series of experiments where the position of the binding site on the rotation axis is varied. Thus, experimental information about the orientation of the protein may be obtained, which will be used as probabilistic priors in a statistical 3D reconstruction approach. Application of this procedure to E. coli MutS and the human Trf2/Rap1 complex will serve as proof-of-principle and may lead to better understanding of their function.
Оригинален текст от CORDIS (на английски).
Участници
- MEDICAL RESEARCH COUNCIL · LONDONКоординаторОбединеното кралство
Връзки
Данни: CORDIS, © Европейски съюз