Design of a molecular support for cryo-EM structure determination.
Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
Thomas G
TG
Martin
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.|Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
Tanmay A M
TA
Bharat
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.|German Cancer Consortium (DKTK), Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, 60438 Frankfurt am Main, Germany.
Andreas C
AC
Joerger
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
Xiao-Chen
XC
Bai
Physik Department, Walter Schottky Institute, Technische Universität München, 85748 Garching near Munich, Germany.
Florian
F
Praetorius
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
Alan R
AR
Fersht
Physik Department, Walter Schottky Institute, Technische Universität München, 85748 Garching near Munich, Germany scheres@mrc-lmb.cam.ac.uk dietz@tum.de.
Hendrik
H
Dietz
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom; scheres@mrc-lmb.cam.ac.uk dietz@tum.de.
Sjors H W
0000-0002-0462-6540
SH
Scheres
D020285:Cryoelectron Microscopy / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D046911:Macromolecular Substances / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*; D014867:Water
D046911:Macromolecular Substances; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D014867:Water; D004247:DNA
DNA-origami; cryo-EM; p53; single particle analysis; structural biology