A Workflow for Protein Structure Determination from Thin Crystal Lamella by Micro-Electron Diffraction

AbstractMicro-Electron Diffraction (MicroED) has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometres in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimised for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.

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Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

10.1101/2020.05.25.115246 ◽

2020 ◽

Cited By ~ 2

Author(s):

Michael W. Martynowycz ◽

Tamir Gonen

Keyword(s):

High Resolution ◽

Electron Diffraction ◽

Focused Ion Beam ◽

Ion Beam ◽

Proteinase K ◽

X Ray ◽

X Ray Crystallography ◽

High Resolution Structure ◽

Efficient Uptake ◽

Resolution Structure

AbstractA method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

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Serial Electron Diffraction Data Processing With diffractem and CrystFEL

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.624264 ◽

2021 ◽

Vol 8 ◽

Author(s):

Robert Bücker ◽

Pascal Hogan-Lamarre ◽

R. J. Dwayne Miller

Keyword(s):

Electron Diffraction ◽

Data Processing ◽

Three Dimensional ◽

Electron Diffraction Data ◽

New Techniques ◽

X Ray ◽

X Ray Crystallography ◽

Level Of Automation ◽

Rotation Method ◽

High Level

Serial electron diffraction (SerialED) is an emerging technique, which applies the snapshot data-collection mode of serial X-ray crystallography to three-dimensional electron diffraction (3D Electron Diffraction), forgoing the conventional rotation method. Similarly to serial X-ray crystallography, this approach leads to almost complete absence of radiation damage effects even for the most sensitive samples, and allows for a high level of automation. However, SerialED also necessitates new techniques of data processing, which combine existing pipelines for rotation electron diffraction and serial X-ray crystallography with some more particular solutions for challenges arising in SerialED specifically. Here, we introduce our analysis pipeline for SerialED data, and its implementation using the CrystFEL and diffractem program packages. Detailed examples are provided in extensive supplementary code.

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Crystal-bending and high-resolution protein-structure determination from electron-diffraction data

The Science of Nature ◽

10.1007/bf00440103 ◽

1977 ◽

Vol 64 (12) ◽

pp. 637-638 ◽

Cited By ~ 2

Author(s):

D. L. Dorset ◽

Sek -Wen Hui

Keyword(s):

Protein Structure ◽

High Resolution ◽

Electron Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Protein Structure Determination ◽

Electron Diffraction Data

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Single Crystal 3D Rotation Electron Diffraction from Nano-sized Crystals

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096338 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C366-C366

Author(s):

Xiaodong Zou

Keyword(s):

Electron Diffraction ◽

Data Collection ◽

Data Processing ◽

Single Crystal ◽

Diffraction Data ◽

Structure Determination ◽

Reciprocal Lattice ◽

Electron Diffraction Data ◽

Electron Crystallography ◽

Structure Solution

Electron crystallography is an important technique for structure analysis of nano-sized materials. Crystals too small or too complicated to be studied by X-ray diffraction can be investigated by electron crystallography. However, conventional TEM methods requires high TEM skills and strong crystallographic knowledge, which many synthetic materials scientists and chemists do not have. We recently developed the software-based Rotation Electron Diffraction (RED) method for automated collection and processing of 3D electron diffraction data. Complete single crystal 3D electron diffraction data can be collected from nano- and micron-sized crystals in less than one hour by combining electron beam tilt and goniometer tilt, which are controlled by the RED – data collection software.3 The unit cell, possible space groups and electron diffraction intensities can be obtained from the RED data using the RED data processing software. The figure below illustrates the data collection and data processing of a zeolite silicalite-1 by RED. 1427 ED frames were collected in less than 1 hour from a crystal of 800 x 400 x 200 nm in size. A 3D reciprocal lattice of silicalite-1 was reconstructed from the ED frames, from which the unit cell parameters and space group were determined (P21/n, a=20.02Å, b=20.25Å, c=13.35Å, alfa=90.130, beta=90.740, gamma=90.030. It was possible to cut the 3D reciprocal lattice perpendicular to any directions and study the reflection conditions. The reflection intensities could be extracted. The structure of the calcined silicalite-1 could be solved from the RED data by routine direct methods using SHELX-97. All 78 unique Si and O atoms could be located and refined to an accuracy better than 0.08 Å. The RED method has been applied for structure solution of a wide range of crystals and shown to be very powerful and efficient. Now a structure determination can be achieved within a few hours, from the data collection to structure solution. We will present several examples including unknown inorganic compounds, metal-organic frameworks and organic structures solved from the RED data. Different parameters that affect the RED data quality and thus the structure determination will be discussed. The methods are general and can be applied to any crystalline materials.

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Quasicrystal approximants solved by Rotation Electron Diffraction (RED)

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314088044 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1195-C1195 ◽

Cited By ~ 1

Author(s):

Sven Hovmöller ◽

Devinder SINGH ◽

Wei Wan ◽

Yifeng Yun ◽

Benjamin Grushko ◽

...

Keyword(s):

Electron Diffraction ◽

Data Processing ◽

Single Crystal ◽

Crystal Structures ◽

Reciprocal Lattice ◽

Electron Diffraction Data ◽

Data Set ◽

X Ray ◽

X Ray Crystallography ◽

3D Electron

We have developed single crystal electron diffraction for powder-sized samples, i.e. < 0.1μm in all dimensions. Complete 3D electron diffraction is collected by Rotation Electron Diffraction (RED) in about one hour. Data processing takes another hour. The crystal structures are solved by standard crystallographic techniques. X-ray crystallography requires crystals several micrometers big. For nanometer sized crystals, electron diffraction and electron microscopy (EM) are the only possibilities. Modern transmission EMs are equipped with the two things that are necessary for turning them into automatic single crystal diffractometers; they have CCD cameras and all lenses and the sample stage are computer-controlled. Two methods have been developed for collecting complete (except for a missing cone) 3D electron diffraction data; the Rotation Electron Diffraction (RED) [1] and Automated Electron Diffraction Tomography (ADT) by Kolb et al. [2]. Because of the very strong interaction between electrons and matter, an electron diffraction pattern with visible spots is obtained in one second from a submicron sized crystal in the EM. By collecting 1000-2000 electron diffraction patterns, a complete 3D data set is obtained. The geometry in RED is analogous to the rotation method in X-ray crystallography; the sample is rotated continuously along one rotation axis. The data processing results in a list of typically over 1000 reflections with h,k,l and Intensity. The unit cell is typically obtained correctly to within 1%. Space group determination is done as in X-ray crystallography from systematically absent reflections, but special care must be taken because occasionally multiple electron diffraction can give rise to very strong forbidden reflections. At +/-60° tilt with 0.1° steps, a complete data collection will be some 1200 frames. With one second exposures this takes about one hour. There is no need to align the crystal orientation. The reciprocal lattice can be rotated and displayed at any direction of view. Sections such as hk0, hk1, hk2, h0l and so on can easily be cut out and displayed. We have solved over 50 crystal structures by RED in one year. These include the most complex zeolites ever solved and quasicrystal approximants, such as the pseudo-decagonal approximants PD2 [3] and PD1 in AlCoNi. Observed and calculated sections of reciprocal space (cut at 1.0Å) are shown in Fig. 1. Notice the 10-fold symmetry of strong reflections.

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Structure determination from lipidic cubic phase embedded microcrystals by MicroED

10.1101/724575 ◽

2019 ◽

Cited By ~ 1

Author(s):

Lan Zhu ◽

Guanhong Bu ◽

Liang Jing ◽

Dan Shi ◽

Tamir Gonen ◽

...

Keyword(s):

High Resolution ◽

Electron Diffraction ◽

Structure Determination ◽

Model System ◽

Proteinase K ◽

Electron Diffraction Data ◽

Cubic Phase ◽

High Quality ◽

Small Crystals ◽

Lipidic Cubic Phase

AbstractThe lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP.

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Machining protein microcrystals for structure determination by electron diffraction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809978115 ◽

2018 ◽

Vol 115 (38) ◽

pp. 9569-9573 ◽

Cited By ~ 35

Author(s):

Helen M. E. Duyvesteyn ◽

Abhay Kotecha ◽

Helen M. Ginn ◽

Corey W. Hecksel ◽

Emma V. Beale ◽

...

Keyword(s):

Protein Structure ◽

Electron Diffraction ◽

Structure Determination ◽

Crystal Size ◽

Ion Beam ◽

Protein Structure Determination ◽

Atomic Resolution ◽

Nanometer Scale ◽

Hydrated Protein ◽

Thin Lamella

We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.

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Crystal Structure Determination of Glycerol-Containing Lipids from Electron Diffraction Data. Use of Analog Compounds Based upon Configurational Isomers of Cyclopentane-1, 2, 3-TRIOL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077761 ◽

1977 ◽

Vol 35 ◽

pp. 24-25

Author(s):

Douglas L. Dorset ◽

Anthony J. Hancock

Keyword(s):

Crystal Structure ◽

Electron Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Crystal Surface ◽

Coherent Scattering ◽

Crystal Structure Determination ◽

Electron Diffraction Data ◽

Polar Group ◽

Axis Orientation

Lipids containing long polymethylene chains were among the first compounds subjected to electron diffraction structure analysis. It was only recently realized, however, that various distortions of thin lipid microcrystal plates, e.g. bends, polar group and methyl end plane disorders, etc. (1-3), restrict coherent scattering to the methylene subcell alone, particularly if undistorted molecular layers have well-defined end planes. Thus, ab initio crystal structure determination on a given single uncharacterized natural lipid using electron diffraction data can only hope to identify the subcell packing and the chain axis orientation with respect to the crystal surface. In lipids based on glycerol, for example, conformations of long chains and polar groups about the C-C bonds of this moiety still would remain unknown.One possible means of surmounting this difficulty is to investigate structural analogs of the material of interest in conjunction with the natural compound itself. Suitable analogs to the glycerol lipids are compounds based on the three configurational isomers of cyclopentane-1,2,3-triol shown in Fig. 1, in which three rotameric forms of the natural glycerol derivatives are fixed by the ring structure (4-7).

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