Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM

Ricardo Righetto, Nikhil Biyani, Julia Kowal, Mohamed Chami, Henning Stahlberg

Preprint posted on 6 December 2018

Article now published in Nature Communications at

2D crystals revisited – High resolution structures of the MloK1 ion channel are solved from “imperfect” 2D crystals by treating them as a single particle cryoEM sample

Selected by David Wright


Membrane proteins, despite making up over half of small molecule drug targets (1), make up less than 2% of the Protein Data Bank (PDB). Historically, X-ray crystallography was the most popular technique for the solution of membrane protein structures, however cryogenic electron microscopy (CryoEM) is now the go-to technique for large, flexible membrane proteins. There are many reasons why cryoEM is particularly suited for membrane proteins, but one of the most important is that cryoEM copes particularly well with the multitude of conformations commonly found in a membrane protein sample. MloK1 is a homotetrameric prokaryotic ion channel, which is used in this preprint as a test case for membrane proteins more generally.

The very first membrane protein structures were generated from 2D crystals (2), formed from the proteins aligning side-by-side as if in a native bilayer. Due to the fact that there is only one protein molecule in the z-axis, the resolution in this plane is often very poor. With the resolution revolution (3) due to improved hardware and software, single particle cryoEM has emerged as a high resolution technique to rival X-ray crystallography. In single particle cryoEM of membrane proteins the sample is typically solubilised in detergent, which might lead to a non-native structure.  On the other hand, 2D crystals are grown in a lipidic environment, so might result in a more native-like structure. This preprint uses novel software for single particle reconstruction of 2D crystals deemed “too imperfect” for traditional 2D electron crystallography.


The first point worthy of note is that the overall resolution of the final model was 4 Å, which is higher than the previously available MloK1 structure (4). Further to this, the authors calculate the Brag reflections of these data to be 10 Å or worse, suggesting that this hybrid approach actually improves the resolution of the model generated from these data. It might be expected that the “single particle” selected in analysis was one tetramer; however it seems that there are at least 9 tetramers in each single particle. Even though this ion channel is homotetrameric, it might be possible for the structures to be asymmetric; however, using localized reconstruction, “no significant deviations from 4-fold symmetry” were detected.

The most interesting result, in my opinion, is that eight different structures are generated from one crystal system. If this were crystallography, we would expect a single “averaged” structure, much like the 4 Å structure described above. By generating 3D classes, however, the different conformations can be identified, resulting in structures to 4.4 – 5.6 Å resolution. The largest RMSD between states was less than 1 Å, suggesting that there were no vast global differences between states (this is illustrated in figure 4 included below of the preprint and can be visualised in supplementary movies 1-3). It was also noted that there was no correlation between structure and position on the crystal e.g. that state A was not always found on the edge of the crystal (see supplementary figure 8).

Conclusions and comments

This work resulted in a structure with a higher resolution than that previously seen with MloK1. 2D crystallography generally leads to a structure with good resolution in the x and y planes (as there are thousands of molecules here) but very poor resolution in the z plane (as only one molecule is present). This results in what is often known as the “missing cone” of data. As these crystals are not perfectly planar, the “single particles” are tilted relative to each other, which offers slightly different views i.e. not all from directly above. There is still significant missing data, but this technique, for imperfect crystals, is clearly an improvement on typical 2D crystallography. To the authors’ knowledge, this is the first example of 3D classification within a 2D dataset, and I think it will certainly not be the last. This technique could be very interesting for use in 2D arrays of membrane proteins that are sometimes natively observed, for example in mitochondria and chloroplasts.

Why I chose this preprint

This is a really exciting article where the authors rescue poorly ordered crystals and generate a really nice set of structures. It is really impressive that the imperfections in the crystal that led to non-planarity have been exploited to minimise the “missing cone” that usually affects resolution in the z-axis. Hopefully this work will be applicable to other projects that have stalled with poorly-diffracting 2D crystals. This workflow has also been incorporated into the FOCUS package for other users’ future benefit.

Outstanding questions

  1. How easy it is to make 2D crystals in reality? How much sample is required compared to standard single particle cryoEM?
  2. If purified protein is available, is there any benefit to forming 2D crystals over single particle cryoEM?
  3. How many examples of native 2D crystals are there and would this approach be able to handle crystals with multiple, potentially heterogeneous components?


  1. Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, et al. A comprehensive map of molecular drug targets. Nature Reviews Drug Discovery. 2016 12/02/online;16:19.
  2. Henderson R, Unwin PN. Three-dimensional model of purple membrane obtained by electron microscopy. Nature. 1975 Sep 4;257(5521):28-32. PubMed PMID: 1161000. Epub 1975/09/04. eng.
  3. Kühlbrandt W. The Resolution Revolution. Science. 2014;343(6178):1443.
  4. Kowal J, Biyani N, Chami M, Scherer S, Rzepiela AJ, Baumgartner P, et al. High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer. Structure. 2018 2018/01/02/;26(1):20-7.e3.


Tags: ion channel, lipid

Posted on: 17 December 2018


Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the biochemistry category:

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.


List by Marcus Oliveira

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link:


List by Nándor Lipták


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Also in the molecular biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023


List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23


List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra