We have developed methods for the rapid detection and evaluation of the charge which builds up on biological specimens when they are examined by electron cryomicroscopy. Charging is assessed from images of protein crystals suspended in thin layers of glucose or vitreous ice. Effects of charging are manifested as an apparent increase in mass in areas illuminated by the electron beam and an effective increase in the strength of the objective lens. We assess the influence of a variety of factors related to the charging phenomena. A mechanism of charging is proposed in terms of the emission of secondary electrons upon irradiation of the specimen creating an electrostatic lens of variable strength at the specimen. This study has improved our understanding of specimen charging and demonstrated that charging can be substantially reduced by including part of the adjacent carbon film in the irradiated specimen area. This constitutes a viable strategy for high resolution imaging of specimens that are susceptible to charging. In order to deposit a layer of carbon film on the biological specimens already embedded in vitreous ice, we modified the Gatan coater by outfitting it with a new chamber that replaced ion-beam deposition with thermal evaporation of carbon rods. This chamber is larger in size to reduce thermal load on the specimens. It has an airlock to insert a cryo-specimen and a cryo-shield to protect the specimen from contamination. A separate variable transformer connected to a timer and a high-power relay allowed control of the amount of carbon evaporated onto the specimen surface. Frozen, hydrated specimens mounted in the Gatan 626 cryoholder at -168- C were inserted and coated in the modified coater with ~100 ? layer of carbon and transferred into a JEOL4000EX electron cryomicroscope operating at 400 kV. Ice-embedded catalase crystals prepared over holes were used as test specimens to estimate the effectiveness of coating cryospecimens with a thin carbon layer to minimize charging. The images were recorded, digitized and analyzed for their diffraction quality as shown in the IQ-plot. The plot shows reflections of IQ } 4 up to 3 ? resolution. Images obtained from carbon-coated catalase crystals tilted to 50- also show reflections of the same quality up to 4 ? resolution. These high resolution image data demonstrate the reduction of charging using our modified coater.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR002250-17
Application #
6568619
Study Section
Project Start
2001-12-01
Project End
2002-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
17
Fiscal Year
2002
Total Cost
$134,676
Indirect Cost
Name
Baylor College of Medicine
Department
Type
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Bucero, Marta Abril; Bajaj, Chandrajit; Mourrain, Bernard (2016) On the construction of general cubature formula by flat extensions. Linear Algebra Appl 502:104-125
Ebeida, Mohamed S; Rushdi, Ahmad A; Awad, Muhammad A et al. (2016) Disk Density Tuning of a Maximal Random Packing. Comput Graph Forum 35:259-269
Wensel, Theodore G; Zhang, Zhixian; Anastassov, Ivan A et al. (2016) Structural and molecular bases of rod photoreceptor morphogenesis and disease. Prog Retin Eye Res 55:32-51
Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I (2015) Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment. Eur J Transl Myol 25:4803
Rushdi, Ahmad A; Mitchell, Scott A; Bajaj, Chandrajit L et al. (2015) Robust All-quad Meshing of Domains with Connected Regions. Procedia Eng 124:96-108
Edwards, John; Daniel, Eric; Pascucci, Valerio et al. (2015) Approximating the Generalized Voronoi Diagram of Closely Spaced Objects. Comput Graph Forum 34:299-309
Wensel, Theodore G; Gilliam, Jared C (2015) Three-dimensional architecture of murine rod cilium revealed by cryo-EM. Methods Mol Biol 1271:267-92
Jeter, Cameron B; Patel, Saumil S; Morris, Jeffrey S et al. (2015) Oculomotor executive function abnormalities with increased tic severity in Tourette syndrome. J Child Psychol Psychiatry 56:193-202
Zhang, Qin; Cha, Deukhyun; Bajaj, Chandrajit (2015) Quality Partitioned Meshing of Multi-Material Objects. Procedia Eng 124:187-199
Baker, Mariah R; Fan, Guizhen; Serysheva, Irina I (2015) Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment. Eur J Transl Myol 25:35-48

Showing the most recent 10 out of 213 publications