scholarly journals Atomic resolution structure of serine protease proteinase K at ambient temperature

2017 ◽  
Author(s):  
Tetsuya Masuda ◽  
Mamoru Suzuki ◽  
Shigeyuki Inoue ◽  
Changyong Song ◽  
Takanori Nakane ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Active Sites ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Water Molecules ◽  
Specific Substrate ◽  
Hydrogen Atoms ◽  
X Ray Crystallography ◽  
Chain Conformations ◽  
Resolution Structure

AbstractAtomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

scholarly journals Atomic resolution structure of serine protease proteinase K at ambient temperature

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Tetsuya Masuda ◽  
Mamoru Suzuki ◽  
Shigeyuki Inoue ◽  
Changyong Song ◽  
Takanori Nakane ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Serine Protease ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Resolution Structure

scholarly journals Resolving Individual-Atom of Protein Complex using Commonly Available 300-kV Cryo-electron Microscopes

2020 ◽  
Author(s):  
Kaiming Zhang ◽  
Grigore D. Pintilie ◽  
Shanshan Li ◽  
Michael F. Schmid ◽  
Wah Chiu
Keyword(s):  
Structure Determination ◽  
Single Particle ◽  
Heavy Atom ◽  
Atomic Resolution ◽  
Water Molecules ◽  
Hydrogen Atoms ◽  
Cryo Electron Microscopy ◽  
Resolution Model ◽  
Electron Microscopes ◽  
Resolution Structure

AbstractBreakthroughs in single-particle cryo-electron microscopy (cryo-EM) technology have made near-atomic resolution structure determination possible. Here, we report a ∼1.35-Å structure of apoferritin reconstructed from images recorded on a Gatan K3 or a Thermo Fisher Falcon 4 detector in a commonly available 300-kV Titan Krios microscope (G3i) equipped with or without a Gatan post-column energy filter. Our results demonstrate that the atomic-resolution structure determination can be achieved by single-particle cryo-EM with a fraction of a day of automated data collection. These structures resolve unambiguously each heavy atom (C, N, O, and S) in the amino acid side chains with an indication of hydrogen atoms’ presence and position, as well as the unambiguous existence of multiple rotameric configurations for some residues. We also develop a statistical and chemical based protocol to assess the positions of the water molecules directly from the cryo-EM map. In addition, we have introduced a B’ factor equivalent to the conventional B factor traditionally used by crystallography to annotate the atomic resolution model for determined structures. Our findings will be of immense interest among protein and medicinal scientists engaging in both basic and translational research.

scholarly journals Atomic-resolution structure of cytoskeletal bactofilin by solid-state NMR

2015 ◽  
Vol 1 (11) ◽  
pp. e1501087 ◽  
Author(s):  
Chaowei Shi ◽  
Pascal Fricke ◽  
Lin Lin ◽  
Veniamin Chevelkov ◽  
Melanie Wegstroth ◽  
...  
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Caulobacter Crescentus ◽  
Heavy Atom ◽  
Cytoskeletal Proteins ◽  
Atomic Resolution ◽  
Hydrophobic Core ◽  
X Ray Crystallography ◽  
Wide Range ◽  
Resolution Structure

Bactofilins are a recently discovered class of cytoskeletal proteins of which no atomic-resolution structure has been reported thus far. The bacterial cytoskeleton plays an essential role in a wide range of processes, including morphogenesis, cell division, and motility. Among the cytoskeletal proteins, the bactofilins are bacteria-specific and do not have a eukaryotic counterpart. The bactofilin BacA of the speciesCaulobacter crescentusis not amenable to study by x-ray crystallography or solution nuclear magnetic resonance (NMR) because of its inherent noncrystallinity and insolubility. We present the atomic structure of BacA calculated from solid-state NMR–derived distance restraints. We show that the core domain of BacA forms a right-handed β helix with six windings and a triangular hydrophobic core. The BacA structure was determined to 1.0 Å precision (heavy-atom root mean square deviation) on the basis of unambiguous restraints derived from four-dimensional (4D) HN-HN and 2D C-C NMR spectra.

Structures of human cytosolic and mitochondrial nucleotidases: implications for structure-based design of selective inhibitors

2014 ◽  
Vol 70 (2) ◽  
pp. 461-470 ◽  
Author(s):  
Petr Pachl ◽  
Milan Fábry ◽  
Ivan Rosenberg ◽  
Ondřej Šimák ◽  
Pavlína Řezáčová ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Specific Inhibitor ◽  
Detailed Comparison ◽  
Atomic Resolution ◽  
Phosphate Anion ◽  
Protonation State ◽  
Interatomic Distances ◽  
Enzyme Active Site ◽  
Resolution Structure

The human 5′(3′)-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.

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

2020 ◽  
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.

scholarly journals Analysis of global and site-specific radiation damage in cryo-EM

2018 ◽  
Author(s):  
Johan Hattne ◽  
Dan Shi ◽  
Calina Glynn ◽  
Chih-Te Zee ◽  
Marcus Gallagher-Jones ◽  
...  
Keyword(s):  
Electron Beam ◽  
Radiation Damage ◽  
Disulfide Bonds ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Electron Radiation ◽  
Site Specific ◽  
X Ray ◽  
X Ray Crystallography ◽  
Crystal Electron

SummaryMicro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

Stereospecific Association of C-20 Epimers of 3β-Hydroxy-16-oxo-24-nor-17-azachol-5-eno-23-nitryle

1997 ◽  
Vol 52 (6) ◽  
pp. 749-756
Author(s):  
Zofia Plesnar ◽  
Stanisław Malanowski ◽  
Zenon Lotowski ◽  
Jacek W. Morzycki ◽  
Jadwiga Frelek ◽  
...  
Keyword(s):  
Proton Nuclear Magnetic Resonance ◽  
Side Chain ◽  
Water Molecules ◽  
Carbonyl Groups ◽  
Nmr Data ◽  
Molecular Modelling Studies ◽  
Modelling Studies ◽  
Lactam Carbonyl ◽  
Self Association ◽  
Chain Conformations

Abstract The cryoscopic measurements show that title compounds are strongly associated in CHCl3 solutions. The association of the 20 R epimer is distinctly less pronounced than that of the 20 S epipmer. Self-association of the 20 S epimer leads to the formation of very large com­plexes. The 20 R epimer forms associates via water molecules. The dissimilarity may be ex­plained in terms of different accessibility of the lactam carbonyl groups in the two epimers for the association. It is proposed that the association process is controlled by the configura­tion at the carbon atom C(20) and conformation around the C(20)-C(22) bond. Populations of side chain conformations of both epimers were determined by means of proton nuclear magnetic resonance. It was found for the 20 R epimer that the t and the -g rotamers are almost equally populated, and the rotamer +g is excluded. For the 20 S epimer the +g rotamer predominates over the t one, and the -g rotamer is excluded. The NMR data are fully consistent with the results of the molecular modelling studies.

scholarly journals CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nathanael A. Caveney ◽  
Sean D. Workman ◽  
Rui Yan ◽  
Claire E. Atkinson ◽  
Zhiheng Yu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acid Anhydride ◽  
Atomic Resolution ◽  
Inherent Difficulty ◽  
Penicillin Binding Proteins ◽  
Antibiotic Development ◽  
Class A ◽  
Membrane Mimetic ◽  
Antibacterial Target ◽  
Resolution Structure

AbstractThe pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

scholarly journals MicroED Structures from Micrometer Thick Protein Crystals

2017 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Calina Glynn ◽  
Jennifer Miao ◽  
M. Jason de la Cruz ◽  
Johan Hattne ◽  
...  
Keyword(s):  
Theoretical Calculations ◽  
Protein Structures ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Protein Crystals ◽  
Molecular Replacement ◽  
Varying Thickness ◽  
Order Of Magnitude ◽  
Human Prion Protein ◽  
Do So

AbstractTheoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

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