Journal: J Struct Biol / Year: 2019 Title: Using focus ion beam to prepare crystal lamella for electron diffraction. Authors: Heng Zhou / Zhipu Luo / Xueming Li / Abstract: Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the ...Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Vitrification
Cryogen name: ETHANE
-
Data collection
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
Microscopy
Model: FEI TECNAI F20
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: DIFFRACTION
Image recording
Electron dose: 0.057 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
EM diffraction
Camera length: 1200 mm
EM diffraction shell
Resolution: 1.7→1.76 Å / Fourier space coverage: 79.1 % / Multiplicity: 9.7 / Num. of structure factors: 1002 / Phase residual: 1 °
EM diffraction stats
Fourier space coverage: 89 % / High resolution: 1.7 Å / Num. of intensities measured: 121300 / Num. of structure factors: 11548 / Phase error: 0 ° / Phase residual: 1 ° / Phase error rejection criteria: 60 / Rmerge: 0.281 / Rsym: 0.281
Detector
Date: Feb 2, 2018
-
Processing
Software
Name
Version
Classification
NB
REFMAC
5.8.0222
refinement
PDB_EXTRACT
3.25
dataextraction
EM 3D crystal entity
∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 78.99 Å / B: 78.99 Å / C: 37.89 Å / Space group name: P43212 / Space group num: 96
CTF correction
Type: NONE
3D reconstruction
Resolution: 1.73 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model building
Protocol: FLEXIBLE FIT / Space: RECIPROCAL
Refinement
Resolution: 1.73→14.67 Å / Cor.coef. Fo:Fc: 0.94 / Cor.coef. Fo:Fc free: 0.924 / SU B: 3.69 / SU ML: 0.113 / Cross valid method: THROUGHOUT / ESU R: 0.16 / ESU R Free: 0.147 Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.2575
550
4.8 %
RANDOM
Rwork
0.2203
-
-
-
obs
0.2221
10947
88.68 %
-
Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
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