National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM123159
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM124149
United States
National Science Foundation (NSF, United States)
STC-1231306
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM117126
United States
Citation
Journal: IUCrJ / Year: 2020 Title: Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals. Authors: Alexander M Wolff / Iris D Young / Raymond G Sierra / Aaron S Brewster / Michael W Martynowycz / Eriko Nango / Michihiro Sugahara / Takanori Nakane / Kazutaka Ito / Andrew Aquila / Asmit ...Authors: Alexander M Wolff / Iris D Young / Raymond G Sierra / Aaron S Brewster / Michael W Martynowycz / Eriko Nango / Michihiro Sugahara / Takanori Nakane / Kazutaka Ito / Andrew Aquila / Asmit Bhowmick / Justin T Biel / Sergio Carbajo / Aina E Cohen / Saul Cortez / Ana Gonzalez / Tomoya Hino / Dohyun Im / Jake D Koralek / Minoru Kubo / Tomas S Lazarou / Takashi Nomura / Shigeki Owada / Avi J Samelson / Tomoyuki Tanaka / Rie Tanaka / Erin M Thompson / Henry van den Bedem / Rahel A Woldeyes / Fumiaki Yumoto / Wei Zhao / Kensuke Tono / Sebastien Boutet / So Iwata / Tamir Gonen / Nicholas K Sauter / James S Fraser / Michael C Thompson / Abstract: Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal ...Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.
Details: 600 uL of protein at 60 mg/mL was combined with 400 uL of 50 percent PEG 3350 in a glass vial and stirred with an Octagon stir bar at 500 RPM Lipid mixture: NA / Temperature: 296 K
Buffer solution
pH: 7.5
Specimen
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: unspecified
Vitrification
Cryogen name: ETHANE
Crystal
Density Matthews: 2.75 Å3/Da / Density % sol: 55.34 %
-
Data collection
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
Microscopy
Model: FEI TALOS ARCTICA
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lens
Mode: DIFFRACTION
Image recording
Electron dose: 0.06 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of grids imaged: 1
EM diffraction
Camera length: 2055 mm
EM diffraction shell
Resolution (Å)
ID
EM diffraction stats-ID
Fourier space coverage (%)
Multiplicity
Num. of structure factors
Phase residual (°)
3.6045-30.4934
1
1
84
3.41
3581
14.45
2.8617-3.6045
2
1
87
3.64
3719
22.05
2.5002-2.8617
3
1
87
3.72
3746
32.3
EM diffraction stats
Fourier space coverage: 86 % / High resolution: 2.5 Å / Num. of intensities measured: 22370 / Num. of structure factors: 6236 / Phase error: 23.06 ° / Phase residual: 23.06 ° / Phase error rejection criteria: 0 / Rmerge: 0.217 / Rsym: 0.217
Reflection
Biso Wilson estimate: 35.52 Å2
-
Processing
Software
Name
Version
Classification
PHENIX
1.16_3549
refinement
PHASER
phasing
XDS
datascaling
XDS
datareduction
EM software
ID
Name
Version
Category
Details
6
PHENIX
dev2880
modelfitting
8
PHENIX
dev2880
molecularreplacement
PHASER
12
PHENIX
dev2880
3Dreconstruction
13
PHENIX
1.16-3549
modelrefinement
EM 3D crystal entity
∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 42.4 Å / B: 53.4 Å / C: 87.76 Å / Space group name: P212121 / Space group num: 19
CTF correction
Type: NONE
3D reconstruction
Resolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
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