GDN has a history of opening doors.
And it just opened another. It’s called LCP.

As we covered in our February, 2020 newsletter, last year was a very exciting year for the use of GDN in membrane protein structure determination. 2019 was the first year where the number of unique membrane protein structures determined using GDN was greater than the number of structures using Digitonin. As we hit the halfway mark of 2020, these trends are continuing, and in the past few months there have been a number of structures deposited using GDN, including:  
 
  • Human cholesterol transporter NPC1 (PDB: 6W5S) from the labs of Nieng Yan at Princeton University and Hongyuan Yang at the University of New South Wales(1).
  • Human pannexin 1 channel (PDB: 6WBF) from the labs of Juan Du and Wei Lu at the Van Andel Institute(2).
  • The glutamate transporter VGLUT2 (PDB: 6V4D) from the labs of Robert Edwards and Bob Stroud at UCSF(3).
  • Human calcium homeostasis modulator ion channels CALHM4 and CALHM6 (PDB: 6YTK/6YTV) from the lab of Raimund Dutzler at the University of Bern, Switzerland(4).
  • The mitochondrial respiratory supercomplex (PDB: 6T15) from the lab of Amandine Marechal at Birkbeck College(5).
  • The amino acid transporter b0,+AT-rBAT (PDB: 6LID) from the labs of Jing Huang and Qiang Zhou of Westlake University in China(6).

There is no doubt that GDN is becoming an invaluable tool for the Cryo-EM structure determination of membrane proteins. However, a common question we receive is if GDN is compatible with crystallization experiments. We are excited to highlight a recent publication from the lab of Martin Caffrey which shows the compatibility of GDN with the lipidic cubic phase (LCP) method(7). In this study, the cubic phase formed by the host lipid monoolein is not disrupted by concentrations of GDN up to 20 mM (2.3%) and over a wide range of temperatures. Typical concentrations of GDN used for protein purification and Cryo-EM structure determination are typically in the range of 0.17 mM – 0.5 mM (0.02% - 0.06%). These concentrations are well below the concentration where the cubic phase is destabilized, and membrane proteins that are stable and functional in GDN can proceed directly into LCP crystallization trials.

Tools for Cryo-EM and LCP crystallization from Anatrace and Molecular Dimensions:

 

 References:
  1. Qian H, Wu X, Du X, Yao X, Zhao X, Lee J, et al. Structural Basis of Low-pH-Dependent Lysosomal Cholesterol Egress by NPC1 and NPC2. Cell. 2020 Jun 11;
  2. Ruan Z, Orozco IJ, Du J, Lü W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating. Nature. 2020 Jun 3;
  3. Li F, Eriksen J, Finer-Moore J, Chang R, Nguyen P, Bowen A, et al. Ion transport and regulation in a synaptic vesicle glutamate transporter. Science. 2020 22;368(6493):893–7.
  4. Drożdżyk K, Sawicka M, Bahamonde-Santos M-I, Jonas Z, Deneka D, Albrecht C, et al. Cryo-EM structures and functional properties of CALHM channels of the human placenta. Elife. 2020 May 6;9.
  5. Hartley AM, Meunier B, Pinotsis N, Maréchal A. Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes. Proc Natl Acad Sci USA. 2020 Apr 28;117(17):9329–37.
  6. Yan R, Li Y, Shi Y, Zhou J, Lei J, Huang J, et al. Cryo-EM structure of the human heteromeric amino acid transporter b0,+AT-rBAT. Sci Adv. 2020 Apr;6(16):eaay6379.
  7. van Dalsen L, Weichert D, Caffrey M. In meso crystallogenesis. Compatibility of the lipid cubic phase with the synthetic digitonin analogue, glyco-diosgenin. J Appl Crystallogr. 2020 Apr 1;53(Pt 2):530–5.