TY - JOUR
T1 - Transmission electron microscopy for the evaluation and optimization of crystal growth
AU - Stevenson, Hilary P.
AU - Lin, Guowu
AU - Barnes, Christopher O.
AU - Sutkeviciute, Ieva
AU - Krzysiak, Troy
AU - Weiss, Simon C.
AU - Reynolds, Shelley
AU - Wu, Ying
AU - Nagarajan, Veeranagu
AU - Makhov, Alexander M.
AU - Lawrence, Robert
AU - Lamm, Emily
AU - Clark, Lisa
AU - Gardella, Timothy J.
AU - Hogue, Brenda
AU - Ogata, Craig M.
AU - Ahn, Jinwoo
AU - Gronenborn, Angela M.
AU - Conway, James F.
AU - Vilardaga, Jean Pierre
AU - Cohen, Aina E.
AU - Calero, Guillermo
N1 - Funding Information:
HPS, COB and GL contributed to this work equally. HPS performed the electron microscopy with guidance from AMM, JFC and GC. GL, COB, SR, YW, SCW, EL, LJC, TK, RL and IS were responsible for crystallization and fragmentation. VN, SR and COB were responsible for UV-microscopy data analysis and crystal-fragment counting. COB, GL and AEC were responsible for X-ray data collection and analysis. CMO was responsible for crystal-rastering protocols and data analysis. JPV, TJG, BGH, AMG and JA provided reagents and data analysis. HPS, COB, GL and GC wrote the manuscript. All authors commented on and approved the manuscript. Portions of this research were carried out at the Linac Coherent Light Source (LCLS), a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. We thank Sebastien Boutet, Marc Messerschmidt, Daniel DePonte and Garth Williams of LCLS and Robert L. Shoeman and Sabine Botha of the Max Plank Institute for Medical Research for support during data collection at the coherent X-ray imaging (CXI) station. The CXI instrument was funded through the LCLS Ultrafast Science Instruments (LUSI) project funded by the US Department of Energy (DOE) Office of Basic Energy Sciences. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory is supported by the DOE Office of Science, Office of Basic Energy Sciences under contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research. The authors thank Sebastien Granier for his generous gift of the plasmid encoding BRIL-PTHR, Mark Gladwin for purified globin-X and Elena G. Kovaleva for crystals of the H200Q variant of homo-protecatechuate 2,3-dioxygenase. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDKK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH) under Award Nos. DK102495 (JPV), DK011794 (TJG), GM112686 (GC), DK102495 (GC) and P50GM082251 (AMG). COB acknowledges support from NIH F31-GM112497. HPS and GC acknowledge support from BioXFEL-STC1231306.
Funding Information:
The CXI instrument was funded through the LCLS Ultrafast Science Instruments (LUSI) project funded by the U.S. Department of Energy (DOE) Office of Basic Energy Sciences. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory is supported by the DOE Office of Science, Office of Basic Energy Sciences under contract DEAC02- 76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research. The authors thank Sebastien Granier for his generous gift of the plasmid encoding BRILPTHR, Mark Gladwin for purified globin-X and Elena G. Kovaleva for crystals of the H200Q variant of homoprotecatechuate 2,3-dioxygenase. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDKK) and the National Institute of General Medical Sciences (NIGMS) of the US National Institutes of Health (NIH) under Award Nos. DK102495 (JPV), DK011794 (TJG), GM112686 (GC), DK102495 (GC) and P50GM082251 (AMG). COB acknowledges support from NIH F31-GM112497. HPS and GC acknowledge support from BioXFEL-STC1231306.
Publisher Copyright:
© 2016 International Union of Crystallography.
PY - 2016
Y1 - 2016
N2 - The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.
AB - The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.
KW - Crystal optimization
KW - Crystal optimization
KW - Micro-electron diffraction
KW - Nanocrystallography
KW - Structural biology
KW - Transmission electron microscopy
KW - X-ray free-electron lasers
KW - XFELs
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U2 - 10.1107/S2059798316001546
DO - 10.1107/S2059798316001546
M3 - Article
C2 - 27139624
AN - SCOPUS:84974549964
SN - 0907-4449
VL - 72
SP - 603
EP - 615
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
IS - 5
ER -