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ABSTRACT Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous

membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and

determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure

protein sample. We also use the BaR methodology to elucidate structural information from _Escherichia coli_ K12 crude membrane and raw lysate. The findings demonstrate that it is possible to

solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly,

these results highlight the potential of cryo-EM for systems structural proteomics. Access through your institution Buy or subscribe This is a preview of subscription content, access via

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STRUCTURAL ANALYSIS OF BACTERIAL RIBOSOMES IN SITU Article Open access 28 January 2025 CRYOSTAR: LEVERAGING STRUCTURAL PRIORS AND CONSTRAINTS FOR CRYO-EM HETEROGENEOUS RECONSTRUCTION Article

29 October 2024 DATA AVAILABILITY Atomic coordinates and structure factors have been deposited with accession codes 6WTI (PDB) and EMD-21897 (EMDB) for cytochrome bo3; 6WU0 (PDB) and

EMD-21901 (EMDB) for BpHpnN; 6WTZ (PDB) and EMD-21900 (EMDB) for OmpF; 6WU6 (PDB) and EMD-21906 (EMDB) for SQR (3.60 Å); 7JZ3 (PDB) and EMD-22529 (EMDB) for OmpC; 7JZ2 (PDB) and EMD-22528

(EMDB) for SQR (2.50 Å); 7JZ6 (PDB) and EMD-22530 (EMDB) for KatG; and 7JZH (PDB) and EMD-22531 (EMDB) for GadB. Source data are provided with this paper. REFERENCES * Vinothkumar, K. R.

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Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank P. A. Klenotic for proofreading the manuscript. We are grateful to the Cryo-Electron Microscopy Core at the CWRU

School of Medicine and K. Li for access to the sample preparation and Cryo-EM instrumentation. We thank D. Wu and T. El-Baba for help with proteomics analysis. This work was supported by NIH

grant R01AI145069 (E.W.Y.) and MRC grant MR/N020413/1 (C.V.R.). This research was supported in part by the National Cryo-EM Facility of the National Cancer Institute at the Frederick

National Laboratory for Cancer Research under contract HSSN261200800001E. AUTHOR INFORMATION Author notes * These authors contributed equally: Chih-Chia Su, Meinan Lyu, Christopher E.

Morgan. AUTHORS AND AFFILIATIONS * Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA Chih-Chia Su, Meinan Lyu, Christopher E. Morgan & 

Edward W. Yu * Department of Chemistry, University of Oxford, Oxford, UK Jani Reddy Bolla & Carol V. Robinson Authors * Chih-Chia Su View author publications You can also search for this

author inPubMed Google Scholar * Meinan Lyu View author publications You can also search for this author inPubMed Google Scholar * Christopher E. Morgan View author publications You can

also search for this author inPubMed Google Scholar * Jani Reddy Bolla View author publications You can also search for this author inPubMed Google Scholar * Carol V. Robinson View author

publications You can also search for this author inPubMed Google Scholar * Edward W. Yu View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

C.-C.S., M.L., C.E.M. and E.W.Y. designed the cryo-EM experiments. J.R.B. and C.V.R. designed the nMS and proteomics experiments. C.-C.S., M.L. and C.E.M. made the BpHpnN, crude cell

membrane and raw cell lysate samples and performed the cryo-EM experiments. J.R.B. performed the nMS and proteomics experiments. E.W.Y. wrote the manuscript with input from all authors.

CORRESPONDING AUTHOR Correspondence to Edward W. Yu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION

Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. PUBLISHER’S NOTE Springer Nature

remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 Flowchart of the ‘Build and Retrieve’ (BaR)

iterative method. EXTENDED DATA FIG. 2 NATIVE MASS SPECTROMETRY (NMS) AND PROTEOMICS ANALYSIS SUGGEST THAT THE SAMPLE OF _B. PSEUDOMALLEI_ HPNN WAS CO-PURIFIED WITH SEVERAL OTHER PROTEINS.

(A) SDS–PAGE of the purified sample where the gel bands were sliced and subjected to tryptic digestion to identify the proteins. Proteomics analysis clearly indicated the presence of HpnN,

OmpF and different components of succinate dehydrogenase (SdhA and SdhB) and cytochrome bo3 oxidase (Subunit I, Subunit II and Subunit III). The protein bands were quantified and replicated

three times using ImageJ (imagej.net), showing the abundance of 60.8% for BpHpnN, 9.4% for SdhA + GlmS, 9.7% for subunit I, 6.4% for ArnC + subunit II+PfkA + GatD + TDH+ OmpF, 6.7% for SdhB

and 1.9% for subunit III. (B) Native mass spectra show several charge state distributions whose deconvoluted masses are shown in the Table. Among them, the 93,548 Da can be readily assigned

to monomeric HpnN and the 133,853 Da to dimeric glucosamine-6-phosphate synthase (GlmS). While the masses 65,233 Da and 75,031 Da can be assigned to SdhA bound to FAD and Subunit I bound to

heme b respectively based on the data from (a). Among the proteins identified in (a), PfkA and TDH are known to exist as tetramers. Therefore, the masses 139,385 Da and 149,545 Da can be

assigned to the PfkA and TDH tetramers. The collisionally induced dissociated products shown in (c) further supported this assignment. EXTENDED DATA FIG. 3 2D CLASSES OF THE CRYO-EM IMAGES.

The 2D classification indicates that there are at least five different proteins coexist in the nanodisc sample. EXTENDED DATA FIG. 4 CRYO-EM ANALYSIS OF THE _E. COLI_ CYTOCHROME BO3 COMPLEX.

(A) BaR processing flowchart. (B) Representative 2D classes. (C) Fourier Shell Correlation (FSC) curves. (D) Sharpened cryo-EM map of the cytochrome bo3 complex viewed in the membrane

plane. (E) Sharpened cryo-EM map of the cytochrome bo3 complex viewed from the cytoplasmic side. (F) Local EM density map of cytochrome bo3. EXTENDED DATA FIG. 5 CRYO-EM ANALYSIS OF THE _B.

PSEUDOMALLEI_ HPNN TRANSPORTER. (A) BaR processing flowchart. (B) Representative 2D classes. (C) Fourier Shell Correlation (FSC) curves. (D and E) Sharpened cryo-EM maps of the BpHpnN

transporter viewed in the membrane plane. (F) Local EM density map of BpHpnN. EXTENDED DATA FIG. 6 CRYO-EM ANALYSIS OF THE _E. COLI_ OMPF PORIN CHANNEL. (A) BaR processing flowchart. (B)

Representative 2D classes. (C) Fourier Shell Correlation (FSC) curves. (D) Sharpened cryo-EM map of the OmpF porin viewed in the membrane plane and from the periplasmic side. (E) Sharpened

cryo-EM map of the OmpF viewed from the exterior side. (F) Local EM density map of OmpF. EXTENDED DATA FIG. 7 CRYO-EM ANALYSIS OF THE _E. COLI_ SQR COMPLEX. (A) BaR processing flowchart. (B)

Representative 2D classes. (C) Fourier Shell Correlation (FSC) curves. (D) Sharpened cryo-EM map of the SQR complex viewed in the membrane plane. (E) Sharpened cryo-EM map of the SQR

complex viewed from the cytoplasmic side. (F) Local EM density map of SQR. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–6 and Supplementary Tables 1–5. REPORTING

SUMMARY SOURCE DATA SOURCE DATA EXTENDED DATA FIG. 2 Full scan image for Extended Data Figure 2a. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Su,

CC., Lyu, M., Morgan, C.E. _et al._ A ‘Build and Retrieve’ methodology to simultaneously solve cryo-EM structures of membrane proteins. _Nat Methods_ 18, 69–75 (2021).

https://doi.org/10.1038/s41592-020-01021-2 Download citation * Received: 08 June 2020 * Accepted: 16 November 2020 * Published: 06 January 2021 * Issue Date: January 2021 * DOI:

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