2021-07-21 REL PDBj PDBj PDBj 2019-08-07 2020-08-12 2020-08-12 2021-07-21 National Natural Science Foundation of China 31830020 China Ministry of Science and Technology (China) 2017YFA0504702 China Zhang J Zhang D Sun L Sun F Zhang J Zhang D Sun L Ji G Huang X Niu T Xu J Ma C Zhu Y Gao N Xu W Sun F J.Struct.Biol. US 213 107763 107763 2021 34174447 doi:10.1016/j.jsb.2021.107763 1095-8657 0803 JSBIEM Zhang J Zhang D Sun L Ji G Huang X Niu T Sun F Biorxiv US 2019 doi:10.1101/727149 EMD-0733 associated EM volume cryo-lamella of spinach leaf cryo-lamella of spinach leaf 0

A puncher was used to cut a circular slice of leaf at about 2 mm diameter. The slice was then fitting into carrier for high pressure freezing.

Spinacia oleracea tomography tissue 7.0

phosphate buffered saline (PBS)

NITROGEN

Leaf slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole composed sandwich was frozen.

OTHER

Leaf slice was put in the recess of the carrier and cryoprotectant 1-hexadecene was added to fill the surrounding area. Then a sapphire disk was loaded on top of the carrier before the whole composed sandwich was frozen.. The value given for _emd_high_pressure_freezing.instrument is HPF COMPACT 01. This is not in a list of allowed values {'BAL-TEC HPM 010', 'LEICA EM HPM100', 'OTHER', 'LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER'} so OTHER is written into the XML file.

1-hexadecene OTHER OTHER 30 0.08 3600 93 200 17

Then the carrier was transfer with the cryo-transfer shuttle into the SEM chamber by using Quorum PP3000T cryotransfer system under -180 degree. To improve sample conductivity and reduce curtaining artifacts, the samples were deposited with organometallic platinum using the in situ gas injection system (GIS) operated at 5 seconds gas injection time before milling. During the cryo-FIB milling process, the milling angle is nearly in parallel with the carrier, and the milling was performed parallel from both sides of the sample platform to produce lamella. Rough milling is produced with the accelerating voltage of the ion beam at 30 kV, and current at 0.79 nA-0.43 nA. The initial milling width is about 20 um and depth is about 20 um. To facilitate tomography data collection, ice at the notch above lamella was removed to get a trapezoid-shaped milling pattern. After rough milling, one side of the lamella is jagged from the main platform. When the thickness of lamella reaches about 1 um the ion current is reduced to 0.23 nA or 80 pA until thickness finally reaching 150 to 250 nm.. The value given for _emd_sectioning_focused_ion_beam.instrument is Helios NanoLab 600i. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 2.7 5.0 6.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GATAN K2 SUMMIT (4k x 4k) COUNTING 3838 3710 1-21 1.0 3.0 1 BACK PROJECTION IMOD 4.9.2 42