National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41GM136508
United States
Citation
Journal: Nat Commun / Year: 2023 Title: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals. Authors: Michael W Martynowycz / Anna Shiriaeva / Max T B Clabbers / William J Nicolas / Sara J Weaver / Johan Hattne / Tamir Gonen / Abstract: Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction ...Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
ProteinaseK / Endopeptidase K / Tritirachium alkaline proteinase
Mass: 28958.791 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) 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: 0.001 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1
Image scans
Sampling size: 28 µm / Width: 2048 / Height: 2048
EM diffraction
Camera length: 1202 mm / Tilt angle list: -30,30
EM diffraction shell
Resolution: 0.87→0.9 Å / Fourier space coverage: 37.64 % / Multiplicity: 2.1 / Num. of structure factors: 2783 / Phase residual: 30 °
EM diffraction stats
Fourier space coverage: 87.58 % / High resolution: 0.87 Å / Num. of intensities measured: 569407 / Num. of structure factors: 64974 / Phase error: 30 ° / Phase error rejection criteria: None / Rmerge: 0.236 / Rsym: 0.073
Reflection
Biso Wilson estimate: 12.65 Å2
-
Processing
Software
Name
Version
Classification
NB
phenix.refine
1.20.1_4487
refinement
PHENIX
1.20.1_4487
refinement
EM software
Name: AIMLESS / Category: crystallography merging
Image processing
Details: Binned by 2
EM 3D crystal entity
∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.05 Å / B: 67.05 Å / C: 107.02 Å / Space group name: 96 / Space group num: 96
CTF correction
Type: NONE
3D reconstruction
Resolution: 1.45 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL
Atomic model building
B value: 12.65 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
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