National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
P41GM103832
United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
R01GM079429
United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
S10OD021600
United States
Citation
Journal: Nat Commun / Year: 2019 Title: Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution. Authors: Kaiming Zhang / Shanshan Li / Kalli Kappel / Grigore Pintilie / Zhaoming Su / Tung-Chung Mou / Michael F Schmid / Rhiju Das / Wah Chiu / Abstract: Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain ...Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.
Average exposure time: 6 sec. / Electron dose: 7.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7200
EM imaging optics
Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scans
Movie frames/image: 30 / Used frames/image: 1-30
-
Processing
EM software
ID
Name
Version
Category
2
EPU
imageacquisition
4
CTFFIND
4
CTFcorrection
7
UCSF Chimera
1.13.1
modelfitting
9
RELION
2.1
initialEulerassignment
10
cryoSPARC
2
finalEulerassignment
11
cryoSPARC
2
classification
12
cryoSPARC
2
3Dreconstruction
13
PHENIX
1.14
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 2102569
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 796923 / Symmetry type: POINT
Atomic model building
Protocol: BACKBONE TRACE / Space: REAL
+
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