cryo-EM Study of Mm-Cpn and human D-crystallin Bo Chen - - PowerPoint PPT Presentation

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cryo-EM Study of Mm-Cpn and human D-crystallin Bo Chen Biochemistry and Molecular Biology Department Baylor College of Medicine NRAMM Workshop Nov. 15th, 2012 Protein Folding Machines Cells employs a cassette of chaperones to assist

SLIDE 1

cryo-EM Study of Mm-Cpn and human γD-crystallin

Bo Chen Biochemistry and Molecular Biology Department Baylor College of Medicine NRAMM Workshop

Nov. 15th, 2012

SLIDE 2

Protein Folding Machines

Cells employs a cassette of chaperones to assist protein folding

Hsp40, Hsp60, Hsp70, Hsp90, Hsp100

Hsp60 family is also called chaperonin.
Chaperonin family is divided into type I and type II.

Chaperonin Type I Type II Bacterial GroEL Mitochondria Hsp60 Chloroplast Hsp60 Archaea Thermosome Archaea Mm-Cpn Eukaryotic TRiC/CCT

SLIDE 3

Protein Folding Machines

Cells employs a cassette of chaperones to assist protein folding

Hsp40, Hsp60, Hsp70, Hsp90, Hsp100

Hsp60 family is also called chaperonin.
Chaperonin family is divided into type I and type II.

Chaperonin Type I Type II Bacterial GroEL-ES Mitochondria Hsp60 Chloroplast Hsp60 Archaea Thermosome Archaea Mm-Cpn Eukaryotic TRiC/CCT

SLIDE 4

Cryo-EM Structures of Mm-cpn

ATP-free (∆lid) Post ATP-hydrolysis Transition (∆lid) (Using ATP/AlFx) 8.0 Å 4.7 Å

Zhang et al Nature (2010

SLIDE 5

HγD-crystallin : substrate of Mm-Cpn

A 20 kDa protein with two domains
Is essential for maintaining lens transparency
Aggregation of misfolded HγD-crysallin is responsible for

the onset of cataract

Aggregation can be suppressed by Mm-Cpn
Refolding can occur with Mm-Cpn and ATP

SLIDE 6

Experimental Conditions

Specimens
Apo Mm-Cpn+GuHCl
Mm-Cpn + GuHCl denatured HγD-crystallin
Imaging of Mm-Cpn &HγD-crystallin Sample
3200FSC electron microscope
300 kV
Gatan 10k CCD camera
2.0 Å/pixel (detector magnification of 89,000x)
Defocus range: 2.0um ~ 3.5um
Data Processing
EMAN1

SLIDE 7

cryo-EM raw image of Cpn-hγD sample @ 3200FSC

500 Å

SLIDE 8

Reference free 2D class averages of Mm-Cpn & HγD- crystallin sample

Mm-Cpn only 10 20 30 40 50 60 70 80 90 100 End-on View Side View Percentage(%)

SLIDE 9

Multiple Reaction Products May Exist in Mm-Cpn & HγD-crystallin Sample

SLIDE 10

Unsupervised Multiple Model Refinement Process

Particle Sets (~100,000 particles, defocus range: 1.5- 3.5µµ)

C8- symmetry Imposed Model by EMAN1

Initial model Initial Model + Random Noise

Noise I-added Initial Model Noise II-added Initial model Noise III-added Initial model

C8-Symmetry C8-Symmetry C8-Symmetry Subset I (52%) Subset II (33%) Subset III (15%)

SLIDE 11

Subset I Model Resembles Apo State Mm-Cpn

Subset I (52%) Lidless Mm- Cpn (Zhang,

Nature, 2010)

p View

Bottom View Side View T

p View

Bottom View Side View

SLIDE 12

Refined Map of Subset II Shows One-ring Less Open and One-ring Open Conformation

Subset II (33%) Lidless Mm- Cpn (Zhang,

Nature, 2010)

p View

Bottom View Side View T

p View

Bottom View Side View

SLIDE 13

Symmetry-Free Reconstructions of 2 Subsets

p View of Apical

Domain Bottom View of Apical Domain Subset I (30,000 particles) Subset II (23,421 ptcls) T

p View of Apical

Domain Bottom View of Apical Domain

SLIDE 14

Conclusions

~50% of particles of Mm-cpn + denatured HγD-crystallin

(Subset I) correspond to Apo state Mm-Cpn.

~33% of complex in the Mm-Cpn + denatured HγD-crystallin

(Subset II) has one ring entirely open and another ring less

pen.
Symmetry free reconstruction shows additional density in the

apical region in one subunit of one ring with a break-down of 8 fold symmetry (subset II).

The observed protruding density may correspond to part of

the HγD-crystallin

SLIDE 15

Future Directions

Cryo-EM Biochemical gold labeling of HγD-crystallin

SLIDE 16

Acknowledgement

Joanita Jakana, BCM Junjie Zhang, BCM Steve Ludtke, BCM

Dr. Wah Chiu,

BCM

Dr. Jonathan King,

MIT Dan Goulet, MIT Kelly Knee, MIT

Current NCMI Members:

Joanita Jakana Jesus Galaz Montoya Boxue Ma Ming Cheng Htet Khant Caroline Fu All the NCMI personals

Center for Protein Folding Machinery and National Institutes

f Health Roadmap-supported Nanomedicine Development

Center.

Funding Resources

Oksana Sergeeva, MIT

Previous NCMI Members:

Junjie Zhang Yao Cong Ge (Jerry) Zhang Guang (Grant) T ang

SLIDE 17

Backup Slides

SLIDE 18

Unfolding/Misfolding of HγD-crystallin Causes Onset of Cataracts

Jonathan King lab homepage, http://web.mit.edu/king- lab/www/

SLIDE 19

Mm-Cpn Suppresses HγD-crystallin Aggregation

Knee et al, Protein Science, 2011

HγD-HγD-crystallin

alone Mm-Cpn+HγD- crystallin

Aggregation Level

SLIDE 20

Differences is Observed Between Subclass I & II 2D Side Views

2D class averages of subset I 2D class averages of subset II

SLIDE 21

Statistical Analysis of 2D Class Averages in Subset I and II

T B 2D class Averages

2D Class Averages Associated with Angular Projections of C8-symmetry Imposed Refinement Map Subset II Subset I

SLIDE 22

2D Class Averages Statistical Analysis Results

2D Class Averages Associated with Angular Projections of C8-symmetry Imposed Refinement Map Subset II Subset I

Apo Mm- Cpn control Apo Mm- Cpn control

2D Class Averages Associated with Angular Projections of C8- symmetry Imposed Refinement Map

SLIDE 23

5/10 Subclasses (49%) Particles Show Double-ring Open Conformation

Botto m Side

SLIDE 24

5/10 Subclasses (51%) Particles Show One-ring Open One-ring Closed Conformation

SLIDE 25

Continuous Density is Observed on the Apical Region of Top Ring in Subset II but not Subset I

Subset I Subset II