ジャーナル: Elife / 年: 2020 タイトル: Fully automated, sequential focused ion beam milling for cryo-electron tomography. 著者: Tobias Zachs / Andreas Schertel / João Medeiros / Gregor L Weiss / Jannik Hugener / Joao Matos / Martin Pilhofer / 要旨: Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and ...Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
EMPIAR-10376 (タイトル: Yeast Tilt Series Collected on Lamella Generated by Fully Automated FIB Milling Data size: 2.6 Data #1: Yeast Tilt Series Collected on Lamella Generated by Fully Automated FIB Milling [tilt series])
A: 18718.72 Å / B: 23142.48 Å / C: 4222.68 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Integer*27
Å/pix. X/Y/Z
18.280000976563
18.28
18.28
M x/y/z
1024
1266
231
origin x/y/z
0.000
0.000
0.000
length x/y/z
18718.721
23142.480
4222.680
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
-208
209
116
NC/NR/NS
1024
1266
231
D min/max/mean
-579.000
300.000
1.603
-
添付データ
-
試料の構成要素
-
全体 : Yeast strain SK1
全体
名称: Yeast strain SK1
要素
細胞: Yeast strain SK1
-
超分子 #1: Yeast strain SK1
超分子
名称: Yeast strain SK1 / タイプ: cell / ID: 1 / 親要素: 0
由来(天然)
生物種: Saccharomyces cerevisiae (パン酵母) / 株: SK1
-
実験情報
-
構造解析
手法
クライオ電子顕微鏡法
解析
電子線トモグラフィー法
試料の集合状態
cell
-
試料調製
緩衝液
pH: 7
凍結
凍結剤: ETHANE-PROPANE
切片作成
集束イオンビーム - 装置: OTHER / 集束イオンビーム - イオン: OTHER / 集束イオンビーム - 電圧: 30 kV / 集束イオンビーム - 電流: 0.005 nA / 集束イオンビーム - 時間: 1545 sec. / 集束イオンビーム - 温度: 121 K / 集束イオンビーム - Initial thickness: 2000 nm / 集束イオンビーム - 最終 厚さ: 243 nm 集束イオンビーム - 詳細: The value given for _emd_sectioning_focused_ion_beam.instrument is Zeiss CrossBeam 550. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is ...集束イオンビーム - 詳細: The value given for _emd_sectioning_focused_ion_beam.instrument is Zeiss CrossBeam 550. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.
-
電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 平均電子線量: 1.96 e/Å2