Ferritin and Transferrin Receptor 1: cryo-EM structural studies and protein engineering

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PhD course in Biochemistry

Tutor

Coordinator

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1. Introduction

1.1 Ferritin

Biochemical properties ≈ ≈

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β α

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1.3 Single-particle cryo-EM

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≈

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𝑑 = 𝜆

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3. Materials and methods

3.1 Cloning, expression and purification of

the proteins

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CD71

α

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ε

“Humanized” A.Fulgidus ferritin (HumAfFt)

′ ′

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∼

Lanthanide binding Ferritin (HFt-LBT Tb(III))

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3.2 Samples preparation for cryo-EM

acquisition

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    μ 

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μ

HFt-LBT Tb(III) μ

μ

3.3 Grids preparation for cryo-EM

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3.4 Cryo-EM data collections and analysis

Data collections

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μ

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Cryo-EM maps resolution estimations and sharpening

3.5 X-ray Crystallography for structure

determination

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HumAfFt

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apoHFt-LBT and HFt-LBT Tb(III)

μ

λ

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3.6 Samples composition assays

CD71/HFt SPR assay

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μ

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TfR1 silencing in HeLa cells

μ

HFt-LBT Tb(III) MALDI-TOF mass spectrometry μ

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HFt-LBT Tb(III) Fluorescence spectroscopy μ μ 18000 19400 20800 22200 23600 25000 M ass (m/z) 1102.4 0 10 20 30 40 50 60 70 80 90 100 % Int ens ity 4700 Linear Spec #1=>M C[BP = 22536.5, 1102] 22530.5098 22737.6582

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μ

3.7 Internalization assays of FITC-labelled

proteins in cells

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LC-MS measurements of FITC-labelled proteins

μ

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μ

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Flow Activated Cytometry Analysis (FACS)

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4 Results and discussion

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Cryo-EM data analysis

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2D Classifications.

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Structural analysis of the binding region ≈ β β β β 

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β β

β

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Characterization of mutants by SPR, FACS and confocal microscopy

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FACS, siRNA and confocal microscopy for HumAfFt cellular uptake

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μ −

μ μ

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σ

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5.Conclusions &

perspectives

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6. Appendix

6.1 CD71/HFt complex

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6.2 Humanized HumAfFt

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Cryo-EM map FSC curve

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6.3 Lanthanide Binding Ferritin

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