(Hong.Zhou@UCLA.edu) Direct Electron D-12 DDD camera Campbell MG, - - PowerPoint PPT Presentation

▶

Dec 21, 2022 385 likes •555 views

Deriving atomic models from cryoEM images recorded on a DE12 DDD camera Xing Zhang 1 , Jingchen Sun 2 , Xuekui Yu 3 , Z. H. Zhou 1,3 1 California NanoSystems Institute, University of California, Los Angeles (UCLA), Los Angeles, CA 90095-7151, USA 2

SLIDE 1

Deriving atomic models from cryoEM images recorded on a DE12 DDD camera

Xing Zhang1, Jingchen Sun2, Xuekui Yu3, Z. H. Zhou1,3

1 California NanoSystems Institute, University of California, Los Angeles (UCLA), Los

Angeles, CA 90095-7151, USA

2 Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular

Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, China

3 Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los

Angeles, CA 90095-7364, USA

(Hong.Zhou@UCLA.edu)

SLIDE 2

Direct Electron D-12 DDD camera

Campbell MG, Cheng A, Brilot AF, Moeller A, Lyumkis D, Veesler D, Pan J, Harrison SC, Potter CS, Carragher B, Grigorieff N, Structure, Nov, 2012 Brilot AF, Chen JZ, Cheng A, Pan J, Harrison SC, Potter CS, Carragher B, Henderson R, Grigorieff N Journal of Structural Biology, 177 (3): 630-7. March 2012. Bammes BE, Rochat RH, Jakana J, Chen DH, Chiu W Journal of Structural Biology, 177 (3): 589-601. March 2012. Milazzo AC, Cheng A, Moeller A, Lyumkis D, Jacovetty E, Polukas J, Ellisman MH, Xuong NH, Carragher B, Potter CS Journal of Structural Biology, 176 (3): 404-8. December 2011. Milazzo AC, Moldovan G, Lanman J, Jin L, Bouwer JC, Klienfelder S, Peltier ST, Ellisman MH, Kirkland AI, Xuong NH Ultramicroscopy, 110 (7): 744-7. June 2010.

Best resolution of final 3D reconstruction is the 4.4Å structure by movie.

SLIDE 3

Confirming Thon Ring of Carbon at ¾ Nyquist at cryoEM condition

1/3 Å-1

25e-/Å2

Earlier work at low mag/low resolution: contrast at ¾ Nyquist Bammes BE, Rochat RH, Jakana J, Chen DH, Chiu W Journal of Structural Biology, 177 (3): 589-601. March 2012.

SLIDE 4

Imaging Cytoplasmic Polyhedrosis Virus (CPV) on DDD camera at 3.5-Å

SLIDE 5

Holey Quantifoil grids “baked” by 100kV electrons
Imaged with Leginon – For all images, including the

carbon images, mag=53,600, and pixelsize=1.12Å/pix, dose=25e/Å2

2653 DDD pictures with defocus from -0.5 to -3µm

were used for processing

42082 particles were automatically selected and 33660

(80%) were used for the final reconstruction

Same program (IMIRS with G3D) was used for

alignment and reconstruction of DDD and film data

The resolution of the capsid is estimated as 3.5Å based
n density feature and FSC (Rosenthal and Henderson,

i.e., 0.143) after 7 cycles of alignment/refinement

Data Statistics

SLIDE 6

Close-up view of a small region

SLIDE 7

Segmented CSP-A monomer

SLIDE 8

Close-up view of helices

SLIDE 9

Close-up view of a -turn

SLIDE 10

Close-up view of a loop

SLIDE 11

Comparison between DED and film structures at 3.5 Å

DED film

SLIDE 12

Comparison between DED and film structures at 3.5 Å

DED film

SLIDE 13

DED film

Comparison between DED and film structures at 3.5 Å

SLIDE 14

Summary

DDD (D-12) cryoEM images of CPV and

3D reconstruction at 3.5Å resolution

Single-frame images of DDD sufficiently

good quality – no drift-correction necessary for the Titan Krios

Direct electron recording has come of

age for atomic modeling

SLIDE 15

Acknowledgement

Direct Electrons: Liang Jin, Robert Bilhorn and Donghua Chen The Leginon team (NRAMM): Anchi Cheng, Jim Pulokas FEI company UCLA: Peng Ge