Cryo-EM results are getting better and better.
We have the higher standards to match.

The past few years have been revolutionary for the field of single-particle electron cryo-microscopy (cryo-EM), with over 50% of the total deposited structures being determined since 2014. Currently, there are over 650 unique (<95% sequence identity) cryo-EM structures deposited in the PDB, 44 of which are membrane proteins. This sudden rise in the number of atomic level resolution cryo-EM structures is mainly due to the major technological advancements of direct electron detectors, and improved image processing algorithms(1, 2). Even with these remarkable developments, two major barriers remain for cryo-EM structures: the minimum size of the protein, and obtaining atomic level resolution (currently, only ~2% of structures are below 3.0 Å resolution). However, in June 2016, the size and resolution barriers were significantly lowered by the Subramaniam lab at the National Institutes of Health with the determination of the 93 kDa isocitrate dehydrogenase (PDB: 5K10) to 3.8 Å resolution, and the 334 kDa glutamate dehyrdrogenase (PDB: 5K12) to 1.8 Å resolution(3).

Here at Anatrace, we are often asked which detergents work best for cryo-EM studies of membrane proteins. The amphipol A8-35 has been emerging as a useful tool with a number of success stories(4, 5, 6, 7).  In an attempt to answer this question in more depth, we have compiled a list of the most recent cryo-EM structures of membrane proteins from the past two years and listed which detergents were utilized in their structure determination. The results from this analysis show that a large number of detergents are compatible with cryo-EM, including DDM, LMNG, and A8-35.

Recent membrane protein Cryo- EM Structures (sorted by PDB release date)

PDB

Name

Ref.

Resolution (Å)

MW (kDa)

Topology

Detergent

5LDW

Respiratory complex 1

8

4.27

892

α

Cymal 7

5LKH TcdA1 28 3.46 1416 α Lipid nanodiscs
0.01% Fluorinated OM added to improve vitrification.
5GKY RyR1 - Closed State 29 3.8 2312 α 0.015% Tween-20
0.5% CHAPS used for open state structure (PDB: 5GL1)
5SY1 STRA6 Receptor 30 3.9 186 α Amphipol A8-35

5KBS

GluA2-STZ

9

8.7

465

α

1 mM DDM, 0.01 mg/ml POPC:POPE:POPG (3:1:1)

5JZT

Aerolysin Pore

10

7.4

330

ß

0.02% LMNG

5KK2

GluA2-TARP

11

7.3

542

α

0.1% Digitonin

3JCU

PSII-LHCII

12

3.2

128

α

0.6% α-DDM

5IRZ

TRPV1

13

3.28

302

α

Lipid nanodiscs

5FXG

GluN1B-GluN2B

14

6.8

376

α

0.01% LMNG, 0.01 mg/ml POPC

5IPV

GluN1-GluN2B

15

9.25

372

α

1 mM DDM, 0.1 mM CHS

5GAQ

Lysenin Pore

16

3.1

315

ß

0.02% DDM

5HI9

TRPV2

17

4.4

351

α

0.064 mM DMNG

3JCF

Mg-channel CorA

18

3.8

208

α

0.5 mM DDM

5FMW

Poly-C9 MAC

19

6.7

1267

ß

N/A

3JBR

Cav1.1

20

4.2

395

α

0.1% Digitonin

5AN8

TRPV2

21

3.8

280

α

Amphipol A8-35

5FN2

γ-secretase

22

4.2

172

α

Amphipol A8-35

5A6E

Slo2.2 K+ channel

23

4.5

106

α

1.5 mM DDM, 0.05 mg/ml POPE:POPG (3:1)

5FIJ

ATP synthase

24

7.4

519

α

0.1% DDM

3JAV

IP3R1 channel

25

4.7

1255

α

0.4% CHAPS

3JAC

Piezo1 channel

26

4.8

7051

α

0.03% C12E10

3JAD

α1 glycine receptor

27

3.9

200

α

1 mM DDM



Special thanks to Mark Daniels (University of Virginia) for contributing to this month's newsletter.


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