Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data

2011 ◽  
Vol 68 (1) ◽  
pp. 62-68 ◽  
Author(s):  
Michael R. Sawaya ◽  
Brad L. Pentelute ◽  
Stephen B. H. Kent ◽  
Todd O. Yeates
2014 ◽  
Vol 70 (a1) ◽  
pp. C337-C337
Author(s):  
Carina Lobley ◽  
Juan Sanchez-Weatherby ◽  
James Sandy ◽  
Marco Mazzorana ◽  
Tobias Krojer ◽  
...  

A typical protein crystal contains 30-60% solvent. For a naked crystal, this solvent is distributed between solvent shells, where water and solvent molecules make specific interactions with the crystalline protein, and solvent channels filled with disordered solvent molecules. This internal solvent map of the crystal can be modified by placing the crystal in a dehydrating environment. This may in turn induce changes to the crystal lattice and affect mosaicity, resolution and quality of diffraction data. A dehydrating environment can be generated around a crystal in several ways with various degrees of precision and complexity. In this study we have used the HC1 device (Maatel) to mount crystals an air stream of known relative humidity – a precise yet hassle-free approach to altering crystal hydration. We set out to analyse a range of different crystals to establish usable protocols that will allow one to explore to crystal hydration space, either by preparing samples before synchrotron beamtime or by undertaking the experiments during beamtime. Our results, considered in the light of the literature surrounding crystal dehydration, provide guidance for when dehydration can help diffraction.


2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Qing-di Cheng ◽  
Hsiang-Yu Chung ◽  
Robin Schubert ◽  
Shih-Hsuan Chia ◽  
Sven Falke ◽  
...  

Abstract There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.


1999 ◽  
Vol 6 (5) ◽  
pp. 995-1006 ◽  
Author(s):  
Y. P. Nieh ◽  
J. Raftery ◽  
S. Weisgerber ◽  
J. Habash ◽  
F. Schotte ◽  
...  

2014 ◽  
Vol 70 (a1) ◽  
pp. C334-C334
Author(s):  
Yoshiaki Kawano ◽  
Takaaki Hikima ◽  
Kunio Hirata ◽  
Seiki Baba ◽  
Hironori Murakami ◽  
...  

The absorption of X-rays which pass through the protein crystal is possibly the largest source of systematic errors in macromolecular crystallography. Therefore we are developing protein crystal processing system using Pulsed UV Laser Soft Ablation (PULSA) technique [1] to reduce the systematic error as well as background scattering from cryoprotectant agents. For high-quality diffraction data collection from organic material, crystals are usually processed to spherical shape in order to keep X-ray path length in crystal constant. This dramatically reduces systematic errors caused by `absorption of X-rays'. Although shaping crystal was thought to be effective for protein crystallography, there was no usual technique to achieve this because protein crystals are exceedingly fragile against mechanical stress. We are developing protein crystal processing system using PULSA technique. In this system, short pulsed UV-laser (maximum power: 1.0 μJ/pulse, wavelength: 193.4 nm, duration: less than 1.3 nsec) is raised by NSL-193L (Nikon Corporation) and focused on 4 μmφ (FWHM). The focused laser is controlled by galvanomic mirror system and irradiates a sample. Combining this mirror system with four-axis goniometer enables to process crystal to arbitrary shape that is easily defined on GUI. Several protein crystals have been successfully processed into spherical, column and square pole shape, etc. In the case of crystal processed into column shape (diameter is 50 μm), in addition to reducing absorption effects, signal-noise ratio of diffraction data can be increased by removing cryoprotectant agent surrounding the crystal. This work was supported by "Platform for Drug Discovery, Informatics, and Structural Life Science" from MEXT, Japan.


Author(s):  
W. I. F. David ◽  
K. Shankland

Advances made over the past decade in structure determination from powder diffraction data are reviewed with particular emphasis on algorithmic developments and the successes and limitations of the technique. While global optimization methods have been successful in the solution of molecular crystal structures, new methods are required to make the solution of inorganic crystal structures more routine. The use of complementary techniques such as NMR to assist structure solution is discussed and the potential for the combined use of X-ray and neutron diffraction data for structure verification is explored. Structures that have proved difficult to solve from powder diffraction data are reviewed and the limitations of structure determination from powder diffraction data are discussed. Furthermore, the prospects of solving small protein crystal structures over the next decade are assessed.


2013 ◽  
Vol 69 (6) ◽  
pp. 1062-1072 ◽  
Author(s):  
Jeffrey J. Lovelace ◽  
Peter D. Simone ◽  
Václav Petříček ◽  
Gloria E. O. Borgstahl

1999 ◽  
Vol 32 (6) ◽  
pp. 1084-1089 ◽  
Author(s):  
R. B. Von Dreele

By combining high-resolution X-ray powder diffraction data and stereochemical restraints, Rietveld refinement of protein crystal structures has been shown to be feasible. A refinement of the 1261-atom protein metmyoglobin was achieved by combining 5338 stereochemical restraints with a 4648-step (dmin= 3.3 Å) powder diffraction pattern to give the residualsRwp= 2.32%,Rp= 1.66%,R(F2) = 3.10%. The resulting tertiary structure of the protein is essentially identical to that obtained from previous single-crystal studies.


Crystals ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 501 ◽  
Author(s):  
Li ◽  
Yan ◽  
Liu ◽  
Wu ◽  
Liu ◽  
...  

We present a systematic quality comparison of protein crystals obtained with and without cross-linked protein crystal (CLPC) seeds. Four proteins were used to conduct the experiments, and the results showed that crystals obtained in the presence of CLPC seeds exhibited a better morphology. In addition, the X-ray diffraction data showed that the CLPC seeds method is a powerful tool to obtain high-quality protein crystals. Therefore, we recommend the use of CLPC seeds in preparing high-quality diffracting protein crystals.


2013 ◽  
Vol 69 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
Igor Nederlof ◽  
Eric van Genderen ◽  
Yao-Wang Li ◽  
Jan Pieter Abrahams

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e− Å−2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLMandSCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.


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