skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold

Abstract

Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it as a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.

Authors:
 [1];  [1];  [2]
  1. Univ. of California, Los Angeles, CA (United States)
  2. Univ. of California, Los Angeles, CA (United States); California NanoSystems Inst., Los Angeles, CA (United States)
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1609012
Grant/Contract Number:  
FC02-02ER63421
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics

Citation Formats

Liu, Yuxi, Huynh, Duc T., and Yeates, Todd O. A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. United States: N. p., 2019. Web. doi:10.1038/s41467-019-09836-0.
Liu, Yuxi, Huynh, Duc T., & Yeates, Todd O. A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. United States. doi:10.1038/s41467-019-09836-0.
Liu, Yuxi, Huynh, Duc T., and Yeates, Todd O. Tue . "A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold". United States. doi:10.1038/s41467-019-09836-0. https://www.osti.gov/servlets/purl/1609012.
@article{osti_1609012,
title = {A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold},
author = {Liu, Yuxi and Huynh, Duc T. and Yeates, Todd O.},
abstractNote = {Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it as a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.},
doi = {10.1038/s41467-019-09836-0},
journal = {Nature Communications},
issn = {2041-1723},
number = 1,
volume = 10,
place = {United States},
year = {2019},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate
journal, June 2017

  • Khoshouei, Maryam; Radjainia, Mazdak; Baumeister, Wolfgang
  • Nature Communications, Vol. 8, Issue 1
  • DOI: 10.1038/ncomms16099

Intracellular Kinase Inhibitors Selected from Combinatorial Libraries of Designed Ankyrin Repeat Proteins
journal, April 2005

  • Amstutz, Patrick; Binz, H. Kaspar; Parizek, Petra
  • Journal of Biological Chemistry, Vol. 280, Issue 26
  • DOI: 10.1074/jbc.M501746200

Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005

  • Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
  • Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
  • DOI: 10.1038/nbt1172

Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein
journal, August 2016

  • Coscia, Francesca; Estrozi, Leandro F.; Hans, Fabienne
  • Scientific Reports, Vol. 6, Issue 1
  • DOI: 10.1038/srep30909

Routine single particle CryoEM sample and grid characterization by tomography
journal, May 2018


Accurate design of co-assembling multi-component protein nanomaterials
journal, May 2014

  • King, Neil P.; Bale, Jacob B.; Sheffler, William
  • Nature, Vol. 510, Issue 7503
  • DOI: 10.1038/nature13404

High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM
journal, March 2019


Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy
journal, January 2015


Atomic structure of the apoptosome: mechanism of cytochrome c - and dATP-mediated activation of Apaf-1
journal, November 2015


Design of a molecular support for cryo-EM structure determination
journal, November 2016

  • Martin, Thomas G.; Bharat, Tanmay A. M.; Joerger, Andreas C.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 47
  • DOI: 10.1073/pnas.1612720113

RELION: Implementation of a Bayesian approach to cryo-EM structure determination
journal, December 2012


Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy
journal, August 2015

  • Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei
  • Nature Methods, Vol. 12, Issue 10
  • DOI: 10.1038/nmeth.3541

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery
journal, June 2016


Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE
journal, September 2009

  • Veesler, David; Dreier, Birgit; Blangy, Stéphanie
  • Journal of Biological Chemistry, Vol. 284, Issue 44
  • DOI: 10.1074/jbc.M109.037812

Rigidly connected multispecific artificial binders with adjustable geometries
journal, September 2017


Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION
journal, June 2018


Volta potential phase plate for in-focus phase contrast transmission electron microscopy
journal, October 2014

  • Danev, R.; Buijsse, B.; Khoshouei, M.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 44
  • DOI: 10.1073/pnas.1418377111

CTFFIND4: Fast and accurate defocus estimation from electron micrographs
journal, November 2015


Chaperone-Assisted Crystallography with DARPins
journal, October 2008


A Suite of Engineered GFP Molecules for Oligomeric Scaffolding
journal, September 2015


Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins
journal, September 2003


Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry
journal, November 2014


MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy
journal, February 2017

  • Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul
  • Nature Methods, Vol. 14, Issue 4
  • DOI: 10.1038/nmeth.4193

The development of cryo-EM into a mainstream structural biology technique
journal, December 2015


Native display of complete foreign protein domains on the surface of hepatitis B virus capsids
journal, March 1999

  • Kratz, P. A.; Bottcher, B.; Nassal, M.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 5
  • DOI: 10.1073/pnas.96.5.1915

Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module
journal, December 2010

  • Kramer, Michaela A.; Wetzel, Svava K.; Plückthun, Andreas
  • Journal of Molecular Biology, Vol. 404, Issue 3
  • DOI: 10.1016/j.jmb.2010.09.023

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system
journal, March 2018

  • Liu, Yuxi; Gonen, Shane; Gonen, Tamir
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 13
  • DOI: 10.1073/pnas.1718825115

High-affinity binders selected from designed ankyrin repeat protein libraries
journal, April 2004

  • Binz, H. Kaspar; Amstutz, Patrick; Kohl, Andreas
  • Nature Biotechnology, Vol. 22, Issue 5, p. 575-582
  • DOI: 10.1038/nbt962

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination
journal, February 2017

  • Punjani, Ali; Rubinstein, John L.; Fleet, David J.
  • Nature Methods, Vol. 14, Issue 3
  • DOI: 10.1038/nmeth.4169

Quantifying the local resolution of cryo-EM density maps
journal, November 2013

  • Kucukelbir, Alp; Sigworth, Fred J.; Tagare, Hemant D.
  • Nature Methods, Vol. 11, Issue 1
  • DOI: 10.1038/nmeth.2727

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Sampling the conformational space of the catalytic subunit of human γ-secretase
journal, December 2015