Molecular dynamics-based model refinement and validation for sub-5 Å cryo-electron microscopy maps

Abstract
Data availability
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Abstract

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5-Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a convergence radius of ~25Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2Å and 3.4Å resolution. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the respective macromolecules studied, captured employing local root mean square fluctuations. The MDFF tools are made available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

Data availability

The following previously published data sets were used

(2003) NIZN[FE4S4] AND NINI[FE4S4] CLUSTERS IN CLOSED AND OPEN ALPHA SUBUNITS OF ACETYL-COA SYNTHASE/CARBON MONOXIDE DEHYDROGENASE
Publicly available at the Protien Data Bank (accession no. 1OAO).

http://www.rcsb.org/pdb/explore/explore.do?structureId=1OAO
1. Liao M
2. Cao E
3. Julius D
4. Cheng Y
(2013) Structure of TRPV1 ion channel determined by single particle electron cryo-microscopy
Publicly available at the Protien Data Bank (accession no. 3J5P).

http://www.rcsb.org/pdb/explore/explore.do?structureId=3J5P
1. Liao M
2. Cao E
3. Julius D
4. Cheng Y
(2015) Structure of the capsaicin receptor, TRPV1, determined by single particle electron cryo-microscopy
Publicly available at the EMDataBank (accesion no. EMD-5778).

http://emsearch.rutgers.edu/atlas/5778_summary.html
1. Bartesaghi A
2. Merk A
3. Banerjee S
4. Matthies D
5. Wu X
6. Milne J
7. Subramaniam S
(2015) 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
Publicly available at the Protien Data Bank (accession no. 5A1A).

http://dx.doi.org/10.2210/pdb5a1a/pdb
1. Bartesaghi A
2. Merk A
3. Banerjee S
4. Matthies D
5. Wu X
6. Milne J
7. Subramaniam S
(2015) 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
Publicly available at the EMDataBank (accesion no. EMD-2984).

http://emsearch.rutgers.edu/atlas/2984_summary.html
1. Bai X
2. Yan C
3. Yang G
4. Lu P
5. Ma D
6. Sun L
7. Zhou R
8. Scheres SHW
9. Shi Y
(2015) Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolution.
Publicly available at the Protien Data Bank (accession no. 5A63).

http://www.rcsb.org/pdb/explore/explore.do?structureId=5A63
1. Bai XC
2. Yan CY
3. Yang GH
4. Lu PL
5. Ma D
6. Sun LF
7. Zhou R
8. Scheres SHW
9. Shi YG
(2015) Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolution
Publicly available at the EMDataBank (accesion no. EMD-3061).

http://emsearch.rutgers.edu/atlas/3061_summary.html
1. Lu P
2. Bai XC
3. Ma D
4. Xie T
5. Yan C
6. Sun L
7. Yang G
8. Zhao Y
9. Zhou R
10. Scheres SHW
11. Shi Y
(2014) Structure of a extracellular domain
Publicly available at the Protien Data Bank (accession no. 4UPC).

http://www.rcsb.org/pdb/explore/explore.do?structureId=4UPC
1. Lu PL
2. Bai XC
3. Ma D
4. Xie T
5. Yan CY
6. Sun LF
7. Yang GH
8. Zhao YY
9. Zhou R
10. Scheres SHW
11. Shi YG
(2015) Three-dimensional structure of human gamma-secretase at 4.5 angstrom resolution
Publicly available at the EMDataBank (accesion no. EMD-2677).

http://emsearch.rutgers.edu/atlas/2677_summary.html
1. Li X
2. Mooney P
3. Zheng S
4. Booth C
5. Braunfeld MB
6. Gubbens S
7. Agard DA
8. Cheng Y
(2015) Thermoplasma acidophilum 20S proteasome
Publicly available at the Protien Data Bank (accession no. 3J9I).

http://www.rcsb.org/pdb/explore/explore.do?structureId=3J9I
1. Li X
2. Mooney P
3. Zheng S
4. Booth C
5. Braunfeld MB
6. Gubbens S
7. Agard DA
8. Cheng Y
(2015) 3D reconstruction of archaeal 20S proteasome
Publicly available at the EMDataBank (accesion no. EMD-5623).

http://emsearch.rutgers.edu/atlas/5623_summary.html
(2014) Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
Publicly available at the Protien Data Bank (accession no. 3J7H).

http://www.rcsb.org/pdb/explore/explore.do?structureId=3J7H
(2015) Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
Publicly available at the EMDataBank (accesion no. EMD-5995).

http://emsearch.rutgers.edu/atlas/5995_summary.html

Article and author information

Author details

Abhishek Singharoy

Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana Champaign, Urbana, United States

For correspondence
singharo@illinois.edu

Competing interests
The authors declare that no competing interests exist.
Ivan Teo

Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States

Competing interests
The authors declare that no competing interests exist.
Ryan McGreevy

Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, United States

Competing interests
The authors declare that no competing interests exist.
John E Stone

Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States

Competing interests
The authors declare that no competing interests exist.
Jianhua Zhao

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

Competing interests
The authors declare that no competing interests exist.
Klaus Schulten

Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States

For correspondence
kschulte@ks.uiuc.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7192-9632

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