PT  - JOURNAL ARTICLE
AU  - Ricardo D. Righetto
AU  - Nikhil Biyani
AU  - Julia Kowal
AU  - Mohamed Chami
AU  - Henning Stahlberg
TI  - Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM
AID  - 10.1101/488403
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 488403
4099  - http://biorxiv.org/content/early/2018/12/06/488403.short
4100  - http://biorxiv.org/content/early/2018/12/06/488403.full
AB  - Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 Å were obtained from images of 2D crystals that do not diffract beyond 10 Å. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.Abbreviations2Dtwo-dimensional3Dthree-dimensionalcAMPcyclic adenosine monophosphateCCcross-correlationCNBDcyclic nucleotide binding domainCTFcontrast transfer functioncryo-EMcryo-electron microscopyDEDdirect electron detectorFSCFourier shell correlationFTFourier transformGUIgraphical user interfaceSNRsignal-to-noise ratioSPAsingle particle analysisTEMtransmission electron microscopyTMDtransmembrane domainsVSDvoltage sensor domain