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Native-Source Structural Proteomics. Nathaniel Echols*, Monica Totir*, Andrew May#, Chloe Zubieta*, Alisa Moskaleva*, Tom Alber* * UC Berkeley # Fluidigm Corporation. Protein Structure Initiative Bottlenecks Workshop April 15 th , 2008. Native-source structural proteomics. - PowerPoint PPT Presentation
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Native-Source Structural Proteomics
Protein Structure Initiative Bottlenecks WorkshopApril 15th, 2008
Nathaniel Echols*, Monica Totir*, Andrew May#, Chloe Zubieta*,Alisa Moskaleva*, Tom Alber*
* UC Berkeley# Fluidigm Corporation
Native-source structural proteomics
• Native sources provide access to samples that may be
difficult to obtain by recombinant methods
• Project goal: obtain structures of complexes and
low-abundance proteins
1. Scale up purification (>100 g protein)
2. Scale down crystallization (picoliter
reactions)
• No cloning, no overexpression.
Experimental approach
• Use E. coli as a model system to develop the purification protocol necessary to go from grams of starting material to
100 μg fractions
• Screen the final samples at a concentration of >10 mg/ml in Fluidigm Topaz chips and identify the crystallizable fractions
• Identify samples by mass spectrometry
• Set the selected samples in diffraction capable chips or nanodrop crystallization trays for X-ray data collection
Proof-of-concept: the E. coli proteome• Small, well-studied proteome, but still some novelty:• 4243 predicted proteins (manageable number of molecular species)• 860 membrane proteins• 1000 proteins with > 90% sequence identity to known structures• 1250 with > 50% sequence identity• 2000 with > 30% sequence identity• Nearly 1400 uncharacterized non-membrane proteins
• Existing structures allow us to validate approach
• Easy to grow in massive quantities• Lysis and clarification are relatively simple
Proteome component sizes
Cellular protein content is dominated by large assemblies
Proteins/complexes bigger than500 kDa
Lyse at pH 7-8
Cross flow size fractionation – 500 kDa TFF
Sucrose gradients
Size exclusion chromatography
MonoQ
Proteins/complexes smaller than500 kDa
Scalable, gentle purification scheme
Purification schemeA new philosophy--keep everything--required new strategies
Superdex 200
Phenyl
MonoQ/MonoS
Capto Q
Phenyl
MonoQ/MonoS
SP Sepharose
Steps
Typical Anion Exchange chromatogram of the final samples
Blue Heparin Capto MMC
Proteins/complexes smaller than500 kDa
Superdex 200
Phenyl
MonoQ/MonoS
Purification scheme (continued)
Column size Approx. protein quantity1-2 L 50 g
300 mL 10 g
20-50 mL 1 g
1-8 mL 10-100 mg
Capto Q
Phenyl
MonoQ/MonoS
• 200 g of E. coli cells grown in M9 minimal medium and lysed
• Purification scheme:
• 272 fractions analyzed in 96-well Caliper electrophoresis robot and selected for crystallization
Caliper “gel”
The first large-scale prep
Microfluidic crystallization with the Fluidigm TOPAZ system (8.96 chips)
MS identification
Promising chip crystals
Purity checked by Caliper gel
Diffraction-capable chips96 well sitting drop
for further optimization
Sub-optimal chip crystals
MS identification
X-ray data collection
Crystallization pipeline
http://www.fluidigm.com/topaz.htm
Microfluidic crystallization• 272 samples set in Fluidigm TOPAZ 8.96 chips with
Index screen• Automated inspection and scoring required to find
crystals efficiently• 190/272 (70%) produced crystals or microcrystals in
chips (high redundancy in crystal forms)• 50 unique crystal forms by visual inspection• High-quality crystals possible even in very impure
samples
0
20
40
60
80
100
120
1.6 1.8 2 2.2 2.4 2.6
Resolution / Å
Purity / %
0
20
40
60
80
100
120
1.6 1.8 2 2.2 2.4 2.6
Resolution / Å
Purity / %
• 66 samples picked for optimization in nanodrop vapor diffusion trays (using Mosquito robot)• Protocol: sample 40%-100% precipitant concentration with different protein:well ratios (1:3, 1:1, 3:1)
• 50 of hits (76%) were reproducible by this method
Crystal optimization
ALS Beamline 8.3.1
Diffraction-capable microfluidic chips
Reagents Samples
10 nL sample chambers
“Hands-Free” data collection
Structure determination
• MS identification of unique crystals should be the first step
• 25 unique native datasets collected at ALS 8.3.1/12.3.1• 15 already published structures identified• 3 structures novel in E. coli, phased by MR
• Robotics and automation software used for data collection and processing whenever possible
Rapid structure identification by MR• Concept: identify protein from “anonymous” diffraction data (no mass spec info)
• Search set of every PDB structure homologous to an E. coli protein (~10,000 models)
• Molecular replacement rotation function run using each model
• Identical structures are usually high-scoring• Homologous proteins may still score better than average
• Potential solutions can be verified by full MR
Experimental phasing
• The largest bottleneck: much more manual labor required
• Cryoprotectants contain heavy monovalent ions (Br+, Rb-)
• Metal quick-soaks (0.5 - 5 mM):• Ethyl mercury phosphate/thimerosal• HgCl2 or PCMBS (p-Chloro-mercuric-
benzenesulphonate acid)
• SmCl3
• PtCl4, PtCl6
Cystathionine -synthase
Catalase HPII (also in truncated form)
5-keto-4-deoxyuronate isomerase Hsp31 chaperonePyruvate kinasePPIaseMolybdopterin biosynthesis prot. B
pSer aminotransferase
Arginosuccinate lyase Lysyl-tRNA synthetase
Methylglyoxal reductase(37%)
pGlucose isomerase(65%)
Dihydrodipicolinate synthase Citrate synthase
ycaC
ß-glucosidase (?) (bglA)(33%)
New:
Old:
(% identity to PDB)
Transhydrogenase domain I
(Structures labelled in red were identified by brute-force search.)
Current structures, new and old
Purity of crystallized samples
• Macro-to-micro strategy tested with E.coli
• Large-scale fractionation pipeline: • New approaches and equipment (TFF, larger columns,
Caliper CE robot) needed to scale up and keep everything• Currently 464 fractions isolated for crystallization
• Small-scale crystallization:• >50% of fractions crystallized in Topaz microfluidic
format• Many impure fractions yielded starting crystals• Optimization in sitting drops and new diffraction chips
was efficient
• Structure determination:• 25 data sets collected, 18 structures phased, all
oligomeric• 3 structures novel to E. coli• Brute-force molecular replacement was used in most cases
Summary
Future directions
• Continue improvements to purification methods
• Pathogenic organisms (e.g. Mycobacteria)• Plant/mammalian proteomes: diploid, much larger and more complex
• Smaller sets of related proteins:• Protease-resistant domains• Serum proteins• ATP-binding proteins• Metalloproteins• Large complexes
• Tom Alber, Monica Totir, Chloe Zubieta, Alisa Moskaleva
• Andy May (Fluidigm)• Scott Gradia, James Berger (UCB)• James Holton (ALS)• George Meigs, Jane Tanamatchi (ALS)
• ALS beamlines 8.3.1, 12.3.1• Tony Iavarone (QB3 MS facility)• Scripps Center for Mass Spectrometry
• W.M. Keck Foundation• Millipore Corporation• Funded in part by UC Discovery/Fluidigm Corporation and NIGMS grant GM71326-02
Acknowledgements
Second large-scale prep – a better purification scheme
• 1000 g of E.coli cells grown in M9 minimal medium and lysed
• 192 unique final samples to be screened in 8.96 chips and subsequently set upin diffraction-capable chips
Proteins/complexes smaller than500 kDa
SP Sepharose Blue Heparin Capto MMC
Lysate at pH 7
Cross flow size fractionation – 500 kDa TFF
Sucrose gradients
Size exclusion chromatography
MonoQ
Proteins/complexes bigger than500 kDa
Superdex 200
Phenyl
MonoQ/MonoS
Abundance ( # transcripts)
Apparently rare proteins accessible
# genesI will have to look this up. Or do we have smth like this?
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