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

Efficient Uptake ◽

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.

STRATEGY TO OBTAIN HIGH RESOLUTION STRUCTURE OF MEMBRANE PROTEINS BY X-RAY CRYSTALLOGRAPHY

Conformational Proteomics Of Macromolecular Architecture ◽

10.1142/9789812562647_0005 ◽

2004 ◽

pp. 111-132

Author(s):

Hiroshi Aoyama ◽

Eiki Yamashita ◽

Keisuke Sakurai ◽

Tomitake Tsukihara

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

X Ray ◽

X Ray Crystallography ◽

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

10.1101/2020.04.30.061895 ◽

2020 ◽

Cited By ~ 1

Author(s):

Emma V. Beale ◽

David G. Waterman ◽

Corey Hecksel ◽

Jason van Rooyen ◽

James B. Gilchrist ◽

...

Keyword(s):

Electron Diffraction ◽

Data Processing ◽

Structure Determination ◽

Focused Ion Beam ◽

Ion Beam ◽

Protein Structure Determination ◽

Proteinase K ◽

Protein Crystal ◽

Electron Diffraction Data ◽

X Ray Crystallography

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.

Defect Structure of Fe1-x0

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100055588 ◽

1982 ◽

Vol 40 ◽

pp. 548-549

Author(s):

C. Lebreton ◽

L. W. Hobbs

Keyword(s):

High Resolution ◽

Electron Diffraction ◽

Phase Boundary ◽

Defect Structure ◽

Short Range ◽

Ion Beam ◽

Floating Zone ◽

Long Range Ordering ◽

Short-range and long-range ordering of defects in wüstite (Fe1-xO) have been studied using electron diffraction and high-resolution structure imaging in TEM. Single crystals of high purity were grown by a floating zone technique, sectioned and equilibrated in CO/CO2 atmospheres between 1175 K and 1625 K to obtain a series of compositions across the wüstite phase field from Fe0.9 5O (Fe/FeO phase boundary) to Fe0.8 7O (Fe/Fe3O4, phase boundary). Equilibrated specimens were quenched, in some cases subsequently annealed between 475 K and 575 K, and reduced to electron transparency by mechanical polishing and ion-beam thinning.

Insight into the binding affinity of thiourea in the calcium binding pocket of proteinase K, through high resolution X-ray crystallography

Bioorganic Chemistry ◽

10.1016/j.bioorg.2019.103443 ◽

2020 ◽

Vol 94 ◽

pp. 103443

Author(s):

Malik Shoaib Ahmad ◽

Zeeshan Akbar ◽

M. Iqbal Choudhary

Keyword(s):

High Resolution ◽

Binding Affinity ◽

Calcium Binding ◽

Binding Pocket ◽

Proteinase K ◽

X Ray ◽

X Ray Crystallography ◽

Insight Into

The molecular structure of silyl cyanide, determined in the gas phase by electron diffraction, microwave spectroscopy and high resolution infra-red spectroscopy, in solution in a nematic phase by NMR spectroscopy, and in the crystalline phase at 100 K by X-ray crystallography

Journal of Molecular Structure ◽

10.1016/0022-2860(89)80139-5 ◽

1989 ◽

Vol 193 ◽

pp. 279-294 ◽

Cited By ~ 20

Author(s):

Peter D. Blair ◽

Alexander J. Blake ◽

Russell W. Cockman ◽

Stephen Cradock ◽

E.A.V. Ebsworth ◽

...

Keyword(s):

Molecular Structure ◽

Nmr Spectroscopy ◽

High Resolution ◽

Electron Diffraction ◽

Gas Phase ◽

Nematic Phase ◽

Microwave Spectroscopy ◽

X Ray ◽

X Ray Crystallography ◽

Infra Red

MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020010117 ◽

2020 ◽

Vol 117 (51) ◽

pp. 32380-32385

Author(s):

Michael W. Martynowycz ◽

Farha Khan ◽

Johan Hattne ◽

Jeff Abramson ◽

Tamir Gonen

Keyword(s):

Membrane Protein ◽

Focused Ion Beam ◽

Ion Beam ◽

Anion Channel ◽

Voltage Dependent Anion Channel ◽

X Ray ◽

X Ray Crystallography ◽

Voltage Dependent ◽

Viscous Media ◽

Scanning Electron

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Study of Diffuse Scattering by Means of High Resolution Structure Images

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092347 ◽

1976 ◽

Vol 34 ◽

pp. 476-477

Author(s):

Sumio Iijima ◽

G. R. Anstis

Keyword(s):

High Resolution ◽

Neutron Diffraction ◽

Diffuse Scattering ◽

Reciprocal Space ◽

The Other ◽

X Ray ◽

Actual Model ◽

Made In ◽

Disorders in crystals with relatively simple structures which gave diffuse scattering have been extensively studied by X-ray or neutron diffraction methods. All these investigations were based on traditional diffraction methods and observations were made in reciprocal space (note observable diffraction intensities can be considered only in terms of interatomic vectors) and therefore the results obtained there leaves considerable ambiguity, particularly when we try to derive an actual model of the disordered crystals. A solution of this problem will be given only by knowing all atom positions in an assembly of atoms and for this case the observable diffracted intensity is given bywhere (xi,yi) and (xj,yj) represent position vectors of the i th and j th atoms with scattering factors fi and fj from an arbitrary origin. On the other hand, a crystal containing imperfections can be defined by

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609243114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2247-2252 ◽

Cited By ~ 41

Author(s):

Cornelius Gati ◽

Dominik Oberthuer ◽

Oleksandr Yefanov ◽

Richard D. Bunker ◽

Francesco Stellato ◽

...

Keyword(s):

High Resolution ◽

Single Molecule ◽

Free Electron ◽

Structural Changes ◽

Protein Crystals ◽

Experimental Conditions ◽

X Ray ◽

X Ray Crystallography ◽

Unit Cells ◽

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.