and quality 2D crystals have been formed, cryo-EM grid preparation is tested with a screening cryo-EM. Cryo-EM grids under the
optimal conditions are then used for high-resolution data collection with a high-end cryo-EM and image processing.
Detailed reviews of 2D crystallization of membrane proteins
include [12–21]. Cryo-EM grid preparation of 2D crystals is based
on the back-injection method developed by Ku ¨hlbrandt and
Downing [22] and variations of this method with different sugars
have also described in detail, including electron diffraction data not
discussed here [6, 23–26]. The method was further modified to
increase sample and carbon film flatness [27] and to address charging effects of data collection at high tilt angles [28], which can
severely limit the resolution. Image processing approaches for highresolution data from 2D crystals build largely on [1, 2, 29, 30] and
references therein. Stahlberg and colleagues have automated data
collection of 2D crystals and developed the Focus software package
[31] to streamline the process of data collection and processing.
This chapter initially focuses on 2D crystallization of membrane proteins, as these are the protein samples the method has
most widely been applied to. However, the methods for screening
by negative stain TEM, cryo-EM data collection, and image processing are equally applicable to 2D crystals of soluble and membrane proteins.
2 Materials
Gloves are used for 2D crystallization trials and negative stain as
well as cryo-EM grid preparation. Caution and sufficient training is
required for work with chloroform and liquid nitrogen. Uranyl
acetate may require adherence to specific university regulations
for use and disposal.
2.1
Two-Dimensional
Crystallization Trials
1. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine
(DMPC,
Avanti Polar Lipids), in chloroform or powdered or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, Avanti Polar
Lipids), in chloroform (10 or 25 mg/mL) or powdered
(25 mg).
2. Round-bottom flask (5 or 10 mL).
3. Nitrogen gas.
4. Detergent (based on the detergent used for purification).
5. Water-bath sonicator.
6. 4–8 aliquots of 75–100 μL purified, detergent-solubilized
membrane protein.
7. 1 L detergent-free dialysis buffer (2 L for 8 crystallization
conditions).
2D Electron Crystallography of Membrane Proteins
229
optimal conditions are then used for high-resolution data collection with a high-end cryo-EM and image processing.
Detailed reviews of 2D crystallization of membrane proteins
include [12–21]. Cryo-EM grid preparation of 2D crystals is based
on the back-injection method developed by Ku ¨hlbrandt and
Downing [22] and variations of this method with different sugars
have also described in detail, including electron diffraction data not
discussed here [6, 23–26]. The method was further modified to
increase sample and carbon film flatness [27] and to address charging effects of data collection at high tilt angles [28], which can
severely limit the resolution. Image processing approaches for highresolution data from 2D crystals build largely on [1, 2, 29, 30] and
references therein. Stahlberg and colleagues have automated data
collection of 2D crystals and developed the Focus software package
[31] to streamline the process of data collection and processing.
This chapter initially focuses on 2D crystallization of membrane proteins, as these are the protein samples the method has
most widely been applied to. However, the methods for screening
by negative stain TEM, cryo-EM data collection, and image processing are equally applicable to 2D crystals of soluble and membrane proteins.
2 Materials
Gloves are used for 2D crystallization trials and negative stain as
well as cryo-EM grid preparation. Caution and sufficient training is
required for work with chloroform and liquid nitrogen. Uranyl
acetate may require adherence to specific university regulations
for use and disposal.
2.1
Two-Dimensional
Crystallization Trials
1. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine
(DMPC,
Avanti Polar Lipids), in chloroform or powdered or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, Avanti Polar
Lipids), in chloroform (10 or 25 mg/mL) or powdered
(25 mg).
2. Round-bottom flask (5 or 10 mL).
3. Nitrogen gas.
4. Detergent (based on the detergent used for purification).
5. Water-bath sonicator.
6. 4–8 aliquots of 75–100 μL purified, detergent-solubilized
membrane protein.
7. 1 L detergent-free dialysis buffer (2 L for 8 crystallization
conditions).
2D Electron Crystallography of Membrane Proteins
229