A Solution of the Phase Problem in the Structure Determination of Biological Macromolecules

1981 ◽  
pp. 5-30 ◽  
Author(s):  
R. L. Mössbauer ◽  
F. Parak ◽  
W. Hoppe
Keyword(s):  
Structure Determination ◽  
Biological Macromolecules ◽  
Phase Problem

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

scholarly journals Structure calculation, refinement and validation usingCcpNmr Analysis

2015 ◽  
Vol 71 (1) ◽  
pp. 154-161 ◽  
Author(s):  
Simon P. Skinner ◽  
Benjamin T. Goult ◽  
Rasmus H. Fogh ◽  
Wayne Boucher ◽  
Tim J. Stevens ◽  
...  
Keyword(s):  
Structure Determination ◽  
Solution Structure ◽  
Residual Dipolar Couplings ◽  
Structure Calculation ◽  
Biological Macromolecules ◽  
Nmr Structure Determination ◽  
Determination Process ◽  
The Many ◽  
Nmr Restraints

CcpNmr Analysisprovides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes howCcpNmr Analysiscan seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral, hydrogen bonds and residual dipolar couplings (RDCs)], exporting these to and subsequently re-importing them from structure-calculation software (such as the programsCYANAorARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements toCcpNmr Analysisdescribed here, this process is now much more intuitive and efficient and less error-prone.

scholarly journals Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

2016 ◽  
Vol 113 (40) ◽  
pp. 11232-11236 ◽  
Author(s):  
Michael R. Sawaya ◽  
Jose Rodriguez ◽  
Duilio Cascio ◽  
Michael J. Collazo ◽  
Dan Shi ◽  
...  
Keyword(s):  
Ab Initio ◽  
Structure Determination ◽  
De Novo ◽  
Direct Methods ◽  
Biological Macromolecules ◽  
X Ray Diffraction ◽  
Em Method ◽  
Ab Initio Structure ◽  
Diffraction Patterns

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

scholarly journals The Use of Connected Masks for Reconstructing the Single Particle Image from X-Ray Diffraction Data. III. Maximum-Likelihood Based Strategies to Select Solution of the Phase Problem

2017 ◽  
Vol 12 (2) ◽  
pp. 521-535 ◽  
Author(s):  
Н.Л. Лунина ◽  
N.L. Lunina
Keyword(s):  
Bragg Diffraction ◽  
Biological Macromolecules ◽  
Phase Problem ◽  
X Ray Diffraction ◽  
Additional Advantage ◽  
X Ray ◽  
Experimental Limitation ◽  
New Criterion ◽  
Selection Of

The main experimental limitation of biological crystallography is associated with the need to prepare the object under study in the form of a single crystal. New powerful X-ray sources, namely free-electron X-ray lasers, makes it possible to raise the question of the determination of the structure of isolated biological macromolecules and their complexes in practice. An additional advantage of working with isolated particles is the possibility to obtain information about scattering in all directions, and not only in those limited by the Laue-Bragg diffraction conditions. This significantly facilitates the solution of the phase problem of X-ray diffraction analysis. This paper is devoted to two lines of development of the method for solving the phase problem, proposed earlier by the authors, which is based on the random scanning of the configuration space of potential solutions of the phase problem. The paper suggests a new criterion for the selection of "candidates" for solving the phase problem in the process of scanning. It involves the maximization of statistical likelihood, and its effectiveness is shown in test calculations. The second line concerns the choice of the optimal scanning strategy. It is shown that the gradual expansion of the set of experimental data used in the work allows obtaining solutions of a higher quality than those obtained with all available data included into the work simultaneously from the beginning.

scholarly journals BIODIFF: Diffractometer for large unit cells

2015 ◽  
Vol 1 ◽  
Author(s):  
Andresas Ostermann ◽  
Tobias Schrader
Keyword(s):  
Hydrogen Atom ◽  
Single Crystal ◽  
Structure Analysis ◽  
Structure Determination ◽  
Biological Macromolecules ◽  
Main Field ◽  
Large Unit ◽  
Neutron Structure ◽  
Unit Cells

The single crystal diffractometer BIODIFF, which is jointly operated by the Technische Universität München and JCNS, Forschungszentrum Jülich, is designed to handle crystals with large unit cells and is dedicated to the structure determination of biological macromolecules. The main field of application is the neutron structure analysis of proteins, especially the determination of hydrogen atom positions.

scholarly journals Programming conventional electron microscopes for solving ultrahigh-resolution structures of small and macro-molecules

2019 ◽  
Author(s):  
Heng zhou ◽  
Feng Luo ◽  
Zhipu Luo ◽  
Dan Li ◽  
Cong Liu ◽  
...  
Keyword(s):  
Organic Compounds ◽  
Structure Determination ◽  
Low Cost ◽  
Ccd Camera ◽  
Biological Macromolecules ◽  
Hydrogen Atoms ◽  
Ultrahigh Resolution ◽  
Electron Microscopes ◽  
Camera Synchronization

AbstractMicrocrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lacking of automated data collection method. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ~0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED, and demonstrates the capability of the low-end 120kV microscope with a CCD camera in solving ultra-high resolution structures of both organic compound and biological macromolecules.

Sign in / Sign up

Export Citation Format

Share Document