National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41GM136508
United States
Citation
Journal: J Struct Biol / Year: 2022 Title: Electron-counting MicroED data with the K2 and K3 direct electron detectors. Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Brent L Nannenga / Tamir Gonen / Abstract: Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances ...Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
ProteinaseK / Endopeptidase K / Tritirachium alkaline proteinase
Mass: 28958.791 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Parengyodontium album (fungus) / Gene: PROK / Production host: Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recording
Average exposure time: 0.5 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1
Image scans
Sampling size: 5 µm / Width: 5760 / Height: 4092
EM diffraction
Camera length: 745 mm
EM diffraction shell
Resolution: 2.7→3.4 Å / Fourier space coverage: 83 % / Multiplicity: 6 / Num. of structure factors: 2499 / Phase residual: 33 °
EM diffraction stats
Fourier space coverage: 83 % / High resolution: 2.7 Å / Num. of intensities measured: 30326 / Num. of structure factors: 5453 / Phase error: 28 ° / Phase error rejection criteria: None / Rmerge: 64 / Rsym: 30
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Processing
Software
Name: REFMAC / Version: 5.8.0267 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: 2020-24-08 Description: (un)restrained refinement or idealisation of macromolecular structures
EM software
ID
Name
Category
11
AIMLESS
crystallographymerging
12
REFMAC
3Dreconstruction
EM 3D crystal entity
∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.01 Å / B: 67.01 Å / C: 106.56 Å / Space group name: P43212 / Space group num: 96
CTF correction
Type: NONE
3D reconstruction
Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model building
B value: 15.89 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum liklihood
Refinement
Resolution: 1.7→43.333 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.919 / SU B: 9.248 / SU ML: 0.12 / Cross valid method: THROUGHOUT / ESU R: 0.208 / ESU R Free: 0.132 Details: Hydrogens have been added in their riding positions
Rfactor
Num. reflection
% reflection
Rfree
0.2542
1286
4.779 %
Rwork
0.1757
25625
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all
0.179
-
-
obs
-
26911
98.14 %
Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT