Cryo-EM Structure of the Human Mitochondrial Translocase … · The Tim9/10a/10b hexamer, similar to a 118 hub, was located at the center of TIM22 complex, and was encircled by the

1

Cryo-EM Structure of the Human Mitochondrial Translocase 1

TIM22 Complex 2

Liangbo Qi1, 5

, Qiang Wang1, 5

, Zeyuan Guan1, 5

, Yan Wu1, Jianbo Cao

2, Xing Zhang

3, 3

Chuangye Yan4, #

, and Ping Yin1, 6, #

4

5

1National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene 6

Research, Huazhong Agricultural University, Wuhan 430070, China. 7

2Public Laboratory of Electron Microscopy, Huazhong Agricultural University, Wuhan, 8

China 9

3Department of Biophysics, and Department of Pathology of Sir Run Run Shaw Hospital, 10

Zhejiang University School of Medicine, Center of Cryo Electron Microscopy, Zhejiang 11

University, Hangzhou, China. 12

4Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center 13

for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China 14

5These authors contributed equally to this work. 15

6Lead contact 16

17

#To whom correspondence should be addressed. E-mail: [email protected] to Ping 18

Yin or [email protected] to Chuangye Yan 19

20

.CC-BY-NC-ND 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted December 9, 2019. ; https://doi.org/10.1101/869289doi: bioRxiv preprint

mailto:[email protected]

https://doi.org/10.1101/869289

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

Abstract 21

Mitochondria play vital functions in cellular metabolism, homeostasis, and apoptosis1-3

. 22

Most of the mitochondrial proteins are synthesized as precursors in the cytosol and 23

imported into mitochondria for folding or maturation4,5

. The translocase TIM22 24

complex is responsible for the import of multiple hydrophobic carrier proteins that are 25

then folded in the inner membrane of mitochondria6-8

. In mammalian cells, the TIM22 26

complex consists of at least six components, Tim22, Tim29, AGK, and three Tim 27

chaperones (Tim9, Tim10a and Tim10b)9-14

. Here, we report the cryo-EM structure of 28

the human translocase TIM22 complex at an overall resolution of 3.7 angstrom. The 29

core subunit, Tim22, contains four transmembrane helices, forming a partial pore that is 30

open to the lipid bilayer. Tim29 is a single transmembrane protein that provides an 31

N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space (IMS) 32

domain to connect AGK and two TIM chaperone hexamers to maintain complex 33

integrity. One TIM hexamer comprises Tim9 and Tim10a in a 3:3 molar ratio, and the 34

other consists of two Tim9 units, three Tim10a units, and one Tim10b unit. The latter 35

hexamer faces the intramembrane region of Tim22, likely providing the dock to load the 36

precursors to the partial pore of Tim22. Our structure serves as a molecular basis for 37

the mechanistic understanding of TIM22 complex function. 38

39

40



https://doi.org/10.1101/869289


3

Mitochondria are essential eukaryotic cellular organelles with multiple vital functions, 41

such as energetics, metabolism, and cellular signaling1,2

. These functions are performed by 42

more than 1,000 proteins3,5

. However, only a small set of these proteins are synthesized in the 43

mitochondria; most of the mitochondria functioning proteins (~99%) are encoded by nuclear 44

genes, and imported into the correct mitochondrial compartment by specific preprotein 45

translocase complexes3,5,15,16

. These complexes include the translocase of outer membrane 46

(the TOM complex)17-20

, the carrier translocase of inner membrane complex (the TIM22 47

complex)21-24

, the presequence translocase of the inner membrane (the TIM23 complex)4,19,25

, 48

the sorting and assembly machinery (the SAM complex)26,27

, and the mitochondrial import 49

complex (the MIM complex)28,29

, etc. These translocation machineries are crucial for 50

mitochondrial biogenesis, dynamics, and function3,4,30,31

. Their dysregulations are connected 51

to dozens of rare mitochondrial diseases and disorders, suggesting an involvement in disease 52

pathogenesis3,31-33

. 53

54

The carrier pathway is one major protein import pathway that is responsible for the 55

translocation and insertion of carrier proteins into the mitochondrial inner membrane4,5,34-38

. 56

The carrier precursors pass through the pore of the TOM complex, and are then transferred by 57

a hexameric small TIM chaperon complex (the Tim9/Tim10a complex is a main form) to the 58

TIM22 complex4,5

. Consequently, the TIM22 complex mediates the insertion and lateral 59

release of precursors into the inner membrane in a membrane potential-dependent manner39

. 60

In addition, the TIM22 complex also transports other multiple-transmembrane-segments 61

containing inner-membrane proteins, including members of the Tim17, Tim22, and Tim23 62



https://doi.org/10.1101/869289


4

family40,41

. 63

64

In human, the TIM22 complex consists of at least six components: a hypothetic 65

channel-forming protein Tim229,21,24

; three chaperone components (Tim9, Tim10a and 66

Tim10b)10,42

; newly identified Tim2911,12

and acylglycerol kinase (AGK)13,14

(Fig. 1a). Tim22 67

and small chaperones are well conserved from yeast to mammals9, but Tim29 and AGK are 68

specific in metazoan11-14

. AGK participates in lipid biosynthesis43

, and mutations in AGK gene 69

have been associated with Sengers syndrome44-48

. Mutations in the TIMM8A gene (also 70

known as DDP1 encoding a small chaperone homologue TIM8A) cause deafness dystonia 71

syndrome49,50

. Recently, mutations in the TIM22 gene have been identified to cause 72

early-onset mitochondrial myopathy51

. 73

74

Despite advances in our understanding of the function and pathophysiology of the 75

TIM22 complex, structural characterization has been sparse. The limited structural 76

information of the TIM22 complex restricted to the crystal structures of Tim9/Tim10a 77

hexameric chaperone and homologues, and a nuclear magnetic resonance (NMR) analysis of 78

carrier precursor associated Tim9/Tim10a52-55

. Electron microscopy has been also applied to 79

structural studies of translocase complexes. The negatively stained images and the recent 80

cryoelectron microscopy (cryo-EM) structure of the TOM core complex at approximately 6.8 81

Å revealed pore-forming architecture56-59

. In contrast, only the low-resolution electron 82

micrographs of the yeast TIM22 complex revealed a two-pore like shape, which was 83

supposed to display a smaller pore diameter than the pores of the TOM core complex39

. 84



https://doi.org/10.1101/869289


5

85

Here, we report the structure of the human TIM22 complex at resolutions of 3.7 Å for 86

the overall structure and 3.5 Å for the inter-membrane region, determined using 87

single-particle cryo-EM. The structure reveals the assembly and detailed structural 88

information of the complex, and provides an important framework for understanding the 89

function and mechanism of the TIM22 complex in carrier protein maturation. 90

91

We co-expressed all six components of the TIM22 complex in human embryonic kidney 92

(HEK) 293F cells. After Flag-tag affinity purification followed by gel filtration, the resulting 93

TIM22 complex displayed good solution (Fig. 1b). The apparent molecular weight was 94

approximately 440 kDa assessed by blue native PAGE (Extended Data Fig. 1), in line to 95

previous findings. Analysis of the purified complex by mass spectrometry (MS) confirmed the 96

presence of all the components of the TIM22 complex. Details of grid preparation, cryo-EM 97

data acquisition, and structural determination of the TIM22 complex can be found in the 98

Methods. Following the initial 2D classification, 3D classification and refinement of the 99

cryo-EM particle images yielded a final 3D EM reconstruction map at an overall 3.7 Å 100

resolution and 3.5 Å resolution for the inter-membrane region of out of 482,959 selected 101

particles (Fig. 1c, Extended Data Fig. 2 and Table 1), according to the gold-standard Fourier 102

shell correlation (FSC) 0.143 criterion (Extended Data Fig. 3). Atomic models were built into 103

the map for Tim22, Tim9, Tim10a, Tim10b, Tim29 and AGK (Fig. 1d and Extended Data 104

Table 2; examples of local densities are shown in Extended Data Fig. 4). The densities for the 105

Tim29 N-terminal helix were of lower resolutions, and poly-Ala was assigned to this region. 106



https://doi.org/10.1101/869289


6

107

The overall structure of the TIM22 complex was approximately 100 Å in height and 160 108

Å in the longest dimension of width (Fig. 1e). In the structure, there was one Tim22, one 109

Tim29, one AGK, and two hexamer chaperones, Tim9/10a and Tim9/10a/10b, whose 110

stoichiometries were 3:3 and 2:3:1, respectively (Extended Data Fig. 5). Most of the 111

structures were located at the intermembrane space (IMS), including the N-terminus of Tim22 112

and the large extended C-terminus portion of Tim29 (Extended Data Fig. 5a). Four 113

transmembrane segments (TMs) of Tim22 together with a single TM of Tim29 constitute the 114

transmembrane element at the center of the TIM22 complex. Only one N-terminal helix of 115

Tim29 protrudes from the core transmembrane region and was oriented nearly parallel to the 116

plane of the membrane at the matrix side (Fig. 1e). The Tim9/10a/10b hexamer, similar to a 117

hub, was located at the center of TIM22 complex, and was encircled by the N-terminus of 118

Tim22, the middle portion of Tim29, AGK, and the Tim9/10a chaperone. Interestingly, the 119

Tim9/10a/10b hexamer was not perpendicular to the membrane, but rather approximately 45° 120

tilted (Fig. 1e). The Tim9/10a chaperone and AGK were located at the nearly-opposite side of 121

the Tim9/10a/10b hexamer, and AGK was anchored to the membrane via its portion of the 122

N-terminal helix and an additional membrane anchor helix loop helix (Fig. 1e). 123

124

Tim22 contains two helices (1 and 2) connected by an extended loop 1, and 4 125

transmembrane segments (TM 1-4) (Fig. 2a and Extended Data Fig. 5a). The 23 N-terminal 126

residues and the matrix loop (residues 94-118; connection between TM1 and TM2) had no 127

EM density, most likely due to their intrinsic flexibility. Two helices (1 and 2) protrude 128



https://doi.org/10.1101/869289


7

toward the intermembrane space and interact with the Tim9/10a/10b hexamer. Helix 1 and 129

loop 1, similar to a hook, sinuously wind around the groove between the inner helices and the 130

outer helices of Tim9 and Tim10a. Helix 1 was nestled in a greasy pocket formed by Tim9 131

and Tim10a (Fig. 2b), and likely functioned as a plug to obstruct the hydrophobic carrier 132

precursor from wedging within the hexamer chaperone from this side (Extended Data Fig. 6a). 133

The recently identified disease-related mutation (Val33Leu) of Tim22 was located in the helix 134

151

. 135

136

Specific interactions comprise van der Waals contacts and one hydrogen bond. 137

Hydrophobic residues, Leu28, Leu29, Leu32, and Val33 interacted with residues Leu9, Leu13, 138

Met17, and Leu69 from helices of Tim9, and residues Phe14, Phe17, and Leu18 from the 139

inner helix of Tim10a. The aromatic ring of Tyr25 from Tim22 stacked against Phe14 and 140

Phe17 from Tim10a. Asn75 from Tim10a, was found to form a hydrogen bond with the main 141

chain of Arg40 from Tim22 (Fig. 2c). Reinforcing these interactions, one residue from helix 142

2 and four residues from the last turn of TM2 of Tim22 contacted residues of the outer helix 143

of Tim9. Glu67 from Tim22 interacted with Lys45 from Tim9 to form a salt bridge. The side 144

chains of Glu144 and Ser150 from Tim22 formed hydrogen bonds with Ser44 and the main 145

chain of Gly46 from Tim9. In addition, the main chains of Ser145 and Tyr146 from Tim22 146

accepted hydrogen bonds from Arg53 of Tim9 (Fig. 2c). 147

148

Notably, only one Tim22 was observed in the complex. The four TMs of Tim22 were 149

unlike a closed pore channel but constituted a lateral hydrophobic cave that was exposed to 150



https://doi.org/10.1101/869289


8

the lipid bilayer (Fig. 2a), which is reminiscent of insertase60

. A disulfide bond formed 151

between Cys69 and Cys141 appeared to stabilize the conformations of TM1 and TM2 152

(Extended Data Fig. 6b, c), which was consistent with biochemical studies. Tim22 153

homologues from yeast to human share more than 40% similarity, and most of the interacting 154

residues and two cysteines are conserved (Extended Data Fig. 6c), suggesting that the Tim22 155

homologues exhibit similar folds9. 156

157

Tim29 was recently identified as a metazoan-specific subunit of the human TIM22 158

complex, which is required for the stability of the complex. Our structure corroborated this 159

observation, since. Tim29 exhibited an extended conformation comprising of a long 160

N-terminal helix 1 in the matrix, a single TM, an intermembrane space domain (IMS 161

Domain) and a C-terminal chaperone Tim9/10a recruiting motif (CRM) (Fig. 3a and Extended 162

Data Fig. 5a). One phospholipid, which was most likely to be phosphatidylethanolamine, was 163

observed at the IMS (Extended Data Fig. 7a, b). The helix 1 was oriented nearly parallel to 164

the plane of the membrane in the matrix and perpendicular to the TM of Tim29 (Fig. 3a, b), 165

which appeared to interact with TM3 of Tim22 and stabilize it. However, the lack of EM 166

density for the side chains of helix 1 prevented an unambiguous assessment of the details of 167

these molecular interactions. Interestingly, the single TM of Tim29 was positioned far from 168

the TMs of Tim22, of some extent, suggesting this TM was unable to strictly associate with 169

TMs of Tim22 (Fig. 3b). This arrangement suggests the TM is flexible for regulation of 170

precursor insertion. 171

172



https://doi.org/10.1101/869289


9

The IMS domain of Tim29 interacts with the Tim9/10a/10b hexamer mainly through the 173

helix4, as illustrated by three polar residues Glu107, Gln111, and Arg119 hydrogen-bonding 174

with His37, Ser35, Arg31 from Tim10b, respectively (Fig. 3b, d). Additionally, the main 175

chains of Ser143 from Tim29 and Lys32 from Tim10a form a hydrogen bond (Fig. 3d). These 176

interactions robustly fix the orientation of the Tim9/10a/10b hexamer, in part due to the 177

presence of only one Tim10b in the heterohexamer (Fig. 3b and Extended Data Fig. 5a). 178

179

The CRM of Tim29 makes contact with the Tim9/10a chaperone via extensive 180

electrostatic interactions (Fig. 3b). The negatively charged residues of Asp183, Asp186, 181

Glu210 and Asp214 interact with several positively charged residues, Arg39 from Tim9, 182

including Arg53, Lys 45, Lys32, and Arg31 from Tim10a, respectively (Fig. 3c). Interestingly, 183

the external C-terminal helix, 9, has no interaction with the Tim9/10a chaperone. Given that 184

this portion is rich in negatively charged residues (Extended Data Fig. 7c, d), it might recruit 185

the other Tim9/Tim10a chaperone or interact with the TOM complex, which has been shown 186

to occur biochemically12

. 187

188

The Tim9/10a chaperone and the Tim9/10a/10b chaperone share almost identical 189

symmetric hexamer structures as that of the reported free Tim9/10a hexamer52

(Fig. 4 and 190

Extended Data Fig. 8a). Each individual subunit displays a helix-loop-helix fold and is 191

stabilized by two intramolecular disulfide bonds from a highly conserved “twin CX3C motif” 192

(Fig. 4b). The six inner helices (helix 1) collectively form a donut-shape with a 14~16 Å 193

diameter central hole. The outer helices (helix 2) act as tentacles that radiate from the central 194



https://doi.org/10.1101/869289


10

region (Fig. 4a. b). Superimposing Tim9, Tim10a, and Tim10b revealed that the less 195

conserved connecting loops (C loops) exhibit distinct conformations (Fig. 4c), in line to the 196

observation that the C loop of Tim10b is primarily responsible for interaction with Tim29 (Fig. 197

3d). Furthermore, the C-terminus of the outer helix of Tim10b tilts towards the inner helix 198

(Fig. 4b), mainly due to its Pro72 residue (Fig. 4d). Thus, the major conformational difference 199

between these two chaperones appears to be the outer helix of Tim10b, while the diameter of 200

the Tim9/10a hole appears to be subtly narrowed (Extended Data Fig. 8b). A circular groove 201

was observed between the inner and outer helices, which was previously hypothesized to hold 202

unstructured carrier precursors52,55

. The twist outer helix of Tim10b disrupts the continuous 203

groove (Extended Data Fig. 8c, d), which might be involved in precursors unloading from the 204

Tim9/10a chaperone. The Tim9/10a chaperone and the Tim9/10a/10b hexamer were 205

selectively bound to the CRM and IMS domain of Tim29 as determined by Tim10b (Fig. 3c, 206

d). Tim10b interacted with the IMS domain of Tim29 and the DGK domain of AGK 207

(described below). These specific interactions prevented the binding of the Tim9/10a 208

chaperone in this position (Fig 3b). 209

210

AGK is a mitochondrial lipid kinase that converts monoacylglycerol (MAG) and 211

diacylglycerol (DAG) to lysophosphatidic acid (lyso-PA) and phosphatidic acid (PA), 212

respectively43

. Similar to the DAG kinase homologue, DgkB from Staphylococcus aureus 213

(SaDgkB), the AGK structure exhibits a typical two-domain fold61

(Fig. 5a and Extended 214

Data Fig. 5b). Notably, a predicted TM (helix 1) preceding domain 1 is partially embedded 215

in the membrane (Fig. 5a), and is consistent with previous findings13,14

. A protruded helix 9 216



https://doi.org/10.1101/869289


11

and an ensuing loop of domain 2 were found to be anchored to the membrane via Trp225, 217

Tyr226, L227, L230, Phe237 and Phe238 (Fig. 5a), generating a positively-charged cavity 218

between AGK and the membrane to potentially facilitate the carrier precursor insertion (Fig. 219

5b). These membrane-anchoring structural features were found to be AGK-specific (Fig. 5c). 220

AGK interacts with both Tim29 and the Tim9/10a/10b hexamer, corroborating previous 221

studies that suggested AGK is a bona fide subunit of the TIM22 complex. Arg40 from helix 222

1 offers hydrogen bonds to the main chains of Tyr151 and Gln153 from Tim29 (Fig. 5d). 223

Gln52, Ala58, and Asp94 were found to hydrogen-bond with Lys45 from Tim10a, Arg62 from 224

Tim10b, and Arg39 from Tim9, respectively (Fig. 5e). 225

226

The structure of the TIM22 complex presented here not only offers the first molecular 227

level view of the interactions that facilitate the mammalian TIM22 complex assembly, but 228

also provides a framework for a mechanistic understanding of the import of carrier proteins 229

through the TIM22 complex. Typically, the carrier protein contains six TMs that are 230

assembled in three repeats, each comprising two TMs connected by a short matrix helix62-65

. 231

Both the amino and carboxy termini are oriented towards the intermembrane space63,64

. And, 232

the precursor protein is wrapped into the hydrophobic groove of the Tim9/10a chaperone55

. 233

Based on our structural analysis, we proposed a “bending-in” insertion model, wherein Tim22 234

and AGK generate a space-limited environment to recruit the two transmembrane helices of 235

the carrier proteins at once, and then bend them into the membrane (Extended Data Fig. 9). 236

After insertion of one repeat into the membrane, the two TM segments intermediate might be 237

transiently stabilized by the Tim22 until the next repeat inserts. This scenario the structures of 238



https://doi.org/10.1101/869289


12

the TIM22 complex with the carrier precursor during an insertion cycle are necessitated to 239

fully understand the carrier translocation mechanism. 240

241

242

243

References 244

1 Harbauer, A. B., Zahedi, R. P., Sickmann, A., Pfanner, N. & Meisinger, C. The protein 245

import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease. 246

Cell Metab 19, 357-372 (2014). 247

2 Kasahara, A. & Scorrano, L. Mitochondria: from cell death executioners to regulators 248

of cell differentiation. Trends Cell Biol 24, 761-770 (2014). 249

3 Pfanner, N., Warscheid, B. & Wiedemann, N. Mitochondrial proteins: from 250

biogenesis to functional networks. Nat Rev Mol Cell Biol 20, 267-284 (2019). 251

4 Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing 252

mitochondrial proteins: machineries and mechanisms. Cell 138, 628-644 (2009). 253

5 Wiedemann, N. & Pfanner, N. Mitochondrial Machineries for Protein Import and 254

Assembly. Annu Rev Biochem 86, 685-714 (2017). 255

6 Rehling, P., Brandner, K. & Pfanner, N. Mitochondrial import and the twin-pore 256

translocase. Nat Rev Mol Cell Biol 5, 519-530 (2004). 257

7 Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Annu 258

Rev Biochem 76, 723-749 (2007). 259

8 Bohnert, M., Pfanner, N. & van der Laan, M. Mitochondrial machineries for insertion 260

of membrane proteins. Curr Opin Struct Biol 33, 92-102 (2015). 261

9 Bauer, M. F. et al. The mitochondrial TIM22 preprotein translocase is highly 262

conserved throughout the eukaryotic kingdom. FEBS Letters 464, 41-47 (1999). 263

10 Muhlenbein, N., Hofmann, S., Rothbauer, U. & Bauer, M. F. Organization and 264

function of the small Tim complexes acting along the import pathway of metabolite 265

carriers into mammalian mitochondria. J Biol Chem 279, 13540-13546 (2004). 266

11 Callegari, S. et al. TIM29 is a subunit of the human carrier translocase required for 267



https://doi.org/10.1101/869289


13

protein transport. FEBS Lett 590, 4147-4158 (2016). 268

12 Kang, Y. et al. Tim29 is a novel subunit of the human TIM22 translocase and is 269

involved in complex assembly and stability. Elife 5, e17463 (2016). 270

13 Kang, Y. et al. Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a 271

Subunit of the Human TIM22 Protein Import Complex. Mol Cell 67, 457-470 (2017). 272

14 Vukotic, M. et al. Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of 273

the TIM22 Protein Translocase in Mitochondria. Mol Cell 67, 471-483 (2017). 274

15 Schleiff, E. & Becker, T. Common ground for protein translocation: access control for 275

mitochondria and chloroplasts. Nat Rev Mol Cell Biol 12, 48-59 (2011). 276

16 Schulz, C., Schendzielorz, A. & Rehling, P. Unlocking the presequence import 277

pathway. Trends Cell Biol 25, 265-275 (2015). 278

17 Kiebler, M. et al. Identification of a mitochondrial receptor complex required for 279

recognition and membrane insertion of precursor proteins. Nature 348, 610-616 280

(1990). 281

18 Hill, K. et al. Tom40 forms the hydrophilic channel of the mitochondrial import pore 282

for preproteins. Nature 395, 516-521 (1998). 283

19 Shiota, T. et al. Molecular architecture of the active mitochondrial protein gate. 284

Science 349, 1544-1548 (2015). 285

20 Mokranjac, D. & Neupert, W. Cell biology: Architecture of a protein entry gate. 286

Nature 528, 201-202 (2015). 287

21 Sirrenberg, C., Bauer, M. F., Guiard, B., Neupert, W. & Brunner, M. Import of carrier 288

proteins into the mitochondrial inner membrane mediated by Tim22. Nature 384, 289

582-585 (1996). 290

22 Koehler, C. M. et al. Import of mitochondrial carriers mediated by essential proteins 291

of the intermembrane space. Science 279, 369-673 (1998). 292

23 Sirrenberg, C. et al. Carrier protein import into mitochondria mediated by the 293

intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5. Nature 391, 912-915 (1998). 294

24 Kovermann, P. et al. Tim22, the essential core of the mitochondrial protein insertion 295

complex, forms a voltage-activated and signal-gated channel. Mol Cell 9, 363-373 296

(2002). 297

25 Chacinska, A. et al. Mitochondrial presequence translocase: switching between TOM 298

tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817-829 299

(2005). 300



https://doi.org/10.1101/869289


14

26 Wiedemann, N. et al. Machinery for protein sorting and assembly in the 301

mitochondrial outer membrane. Nature 424, 565-571 (2003). 302

27 Paschen, S. A. et al. Evolutionary conservation of biogenesis of beta-barrel 303

membrane proteins. Nature 426, 862-866 (2003). 304

28 Chacinska, A. et al. Essential role of Mia40 in import and assembly of mitochondrial 305

intermembrane space proteins. EMBO J 23, 3735-3746 (2004). 306

29 Naoe, M. et al. Identification of Tim40 that mediates protein sorting to the 307

mitochondrial intermembrane space. J Biol Chem 279, 47815-47821 (2004). 308

30 Opalinska, M. & Meisinger, C. Metabolic control via the mitochondrial protein 309

import machinery. Curr Opin Cell Biol 33, 42-48 (2015). 310

31 Kang, Y., Fielden, L. F. & Stojanovski, D. Mitochondrial protein transport in health 311

and disease. Semin Cell Dev Biol 76, 142-153 (2018). 312

32 Sokol, A. M., Sztolsztener, M. E., Wasilewski, M., Heinz, E. & Chacinska, A. 313

Mitochondrial protein translocases for survival and wellbeing. FEBS Lett 588, 314

2484-2495 (2014). 315

33 Martensson, C. U. & Becker, T. Acylglycerol Kinase: Mitochondrial Protein 316

Transport Meets Lipid Biosynthesis. Trends Cell Biol 27, 700-702 (2017). 317

34 Mokranjac, D. & Neupert, W. Thirty years of protein translocation into mitochondria: 318

unexpectedly complex and still puzzling. Biochimica et biophysica acta 1793, 33-41 319

(2009). 320

35 Wagner, K., Mick, D. U. & Rehling, P. Protein transport machineries for precursor 321

translocation across the inner mitochondrial membrane. Biochimica et biophysica 322

acta 1793, 52-59 (2009). 323

36 Webb, C. T. & Lithgow, T. Mitochondrial biogenesis: sorting mechanisms cooperate 324

in ABC transporter assembly. Current biology : CB 20, R564-567 (2010). 325

37 Marom, M., Azem, A. & Mokranjac, D. Understanding the molecular mechanism of 326

protein translocation across the mitochondrial inner membrane: still a long way to go. 327

Biochimica et biophysica acta 1808, 990-1001 (2011). 328

38 Becker, T., Bottinger, L. & Pfanner, N. Mitochondrial protein import: from transport 329

pathways to an integrated network. Trends Biochem Sci 37, 85-91 (2012). 330

39 Rehling, P. et al. Protein insertion into the mitochondrial inner membrane by a 331

twin-pore translocase. Science 299, 1747-1751 (2003). 332

40 Beverly, K. N., Sawaya, M. R., Schmid, E. & Koehler, C. M. The Tim8-Tim13 333



https://doi.org/10.1101/869289


15

complex has multiple substrate binding sites and binds cooperatively to Tim23. J Mol 334

Biol 382, 1144-1156 (2008). 335

41 Curran, S. P., Leuenberger, D., Schmidt, E. & Koehler, C. M. The role of the 336

Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic 337

inner membrane proteins. J Cell Biol 158, 1017-1027 (2002). 338

42 Kerscher, O., Sepuri, N. B. & Jensen, R. E. Tim18p is a new component of the 339

Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol Biol Cell 11, 340

103-116 (2000). 341

43 Bektas, M. et al. A novel acylglycerol kinase that produces lysophosphatidic acid 342

modulates cross talk with EGFR in prostate cancer cells. J Cell Biol 169, 801-811 343

(2005). 344

44 Aldahmesh, M. A., Khan, A. O., Mohamed, J. Y., Alghamdi, M. H. & Alkuraya, F. S. 345

Identification of a truncation mutation of acylglycerol kinase (AGK) gene in a novel 346

autosomal recessive cataract locus. Hum Mutat 33, 960-962 (2012). 347

45 Haghighi, A. et al. Sengers syndrome: six novel AGK mutations in seven new 348

families and review of the phenotypic and mutational spectrum of 29 patients. 349

Orphanet J Rare Dis 9, 199 (2014). 350

46 Siriwardena, K. et al. Mitochondrial citrate synthase crystals: novel finding in 351

Sengers syndrome caused by acylglycerol kinase (AGK) mutations. Mol Genet Metab 352

108, 40-50 (2013). 353

47 Mayr, J. A. et al. Lack of the mitochondrial protein acylglycerol kinase causes 354

Sengers syndrome. Am J Hum Genet 90, 314-320 (2012). 355

48 Calvo, S. E. et al. Molecular diagnosis of infantile mitochondrial disease with 356

targeted next-generation sequencing. Sci Transl Med 4, 118ra110 (2012). 357

49 Koehler, C. M. et al. Human deafness dystonia syndrome is a mitochondrial disease. 358

Proc Natl Acad Sci U S A 96, 2141-2146 (1999). 359

50 Roesch, K., Curran, S. P., Tranebjaerg, L. & Koehler, C. M. Human deafness dystonia 360

syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex. 361

Hum Mol Genet 11, 477-486 (2002). 362

51 Pacheu-Grau, D. et al. Mutations of the mitochondrial carrier translocase channel 363

subunit TIM22 cause early-onset mitochondrial myopathy. Hum Mol Genet 27, 364

4135-4144 (2018). 365

52 Webb, C. T., Gorman, M. A., Lazarou, M., Ryan, M. T. & Gulbis, J. M. Crystal 366

structure of the mitochondrial chaperone TIM9.10 reveals a six-bladed 367

alpha-propeller. Mol Cell 21, 123-133 (2006). 368



https://doi.org/10.1101/869289


16

53 Baker, M. J. et al. Structural and functional requirements for activity of the 369

Tim9-Tim10 complex in mitochondrial protein import. Mol Biol Cell 20, 769-779 370

(2009). 371

54 Endo, T., Yamano, K. & Kawano, S. Structural insight into the mitochondrial protein 372

import system. Biochimica et biophysica acta 1808, 955-970 (2011). 373

55 Weinhaupl, K. et al. Structural Basis of Membrane Protein Chaperoning through the 374

Mitochondrial Intermembrane Space. Cell 175, 1365-1379 (2018). 375

56 Ahting, U. et al. The TOM core complex: the general protein import pore of the outer 376

membrane of mitochondria. J Cell Biol 147, 959-968 (1999). 377

57 Kunkele, K. P. et al. The preprotein translocation channel of the outer membrane of 378

mitochondria. Cell 93, 1009-1019 (1998). 379

58 Model, K., Meisinger, C. & Kuhlbrandt, W. Cryo-electron microscopy structure of a 380

yeast mitochondrial preprotein translocase. J Mol Biol 383, 1049-1057 (2008). 381

59 Bausewein, T. et al. Cryo-EM Structure of the TOM Core Complex from Neurospora 382

crassa. Cell 170, 693-700 (2017). 383

60 Kumazaki, K. et al. Structural basis of Sec-independent membrane protein insertion 384

by YidC. Nature 509, 516-520 (2014). 385

61 Miller, D. J., Jerga, A., Rock, C. O. & White, S. W. Analysis of the Staphylococcus 386

aureus DgkB structure reveals a common catalytic mechanism for the soluble 387

diacylglycerol kinases. Structure 16, 1036-1046 (2008). 388

62 Endres, M., Neupert, W. & Brunner, M. Transport of the ADP/ATP carrier of 389

mitochondria from the TOM complex to the TIM22.54 complex. EMBO J 18, 390

3214-3221 (1999). 391

63 Nelson, D. R., Felix, C. M. & Swanson, J. M. Highly conserved charge-pair networks 392

in the mitochondrial carrier family. J Mol Biol 277, 285-308 (1998). 393

64 Nury, H. et al. Relations between structure and function of the mitochondrial 394

ADP/ATP carrier. Annu Rev Biochem 75, 713-741 (2006). 395

65 Pebay-Peyroula, E. et al. Structure of mitochondrial ADP/ATP carrier in complex 396

with carboxyatractyloside. Nature 426, 39-44 (2003). 397

398

Data availability 399



https://doi.org/10.1101/869289


17

Cryo-EM map for the human TIM22 complex is available on the Electron Microscopy Data 400

Bank under accession number EMD-9958. Coordinate of the atomic structure have been 401

deposited in the Protein Data Bank under accession number 6KC9. All other data are 402

available from the corresponding authors upon reasonable request. 403



https://doi.org/10.1101/869289


18

Main Figures 404

405

406

Fig. 1 | Cryo-EM structure of the human TIM22 complex. a, The schematic diagram for 407

each subunit of the TIM22 complex. b, A representative gel filtration chromatography of the 408

TIM22 complex. The peak fractions were pooled for cryo-EM study. c, A representative 409

electron micrograph of TIM22 complex. The typical particles are marked by yellow circles. d, 410

Cryo-EM density map of the TIM22 complex. Tim9, cyan; Tim10a, slate; Tim10b, magenta; 411

Tim22, orange; Tim29, yellow; AGK, chartreuse; the disc-shape micelle, gray. e, The 412

opposing side views of the TIM22 complex. The inner membrane (IM) is indicated by two 413

lines between the intermembrane space (IMS) and the matrix. All structure figures were 414

prepared using PyMol. 415

416

417

418

419

420

421

422

423

424

425



https://doi.org/10.1101/869289


19

426

427

428

Fig. 2 | Structure of the Tim22 subunit. a, Two side views of Tim22. The transmembrane 429

(TM) segments are labeled. b, Tim22 contacts the Tim9/10a/10b chaperone mainly via the 430

N-terminal helix in the intermembrane space and the helices bundle at the inner membrane. 431

The two interfaces are highlighted by red and green box. c, Key residues mediating the 432

interactions between Tim22 subunit and Tim9/10a/10b chaperone are shown as sticks. 433

434

435

436

437

438

439

440



https://doi.org/10.1101/869289


20

441

442

Fig. 3 | Structure of the Tim29 subunit. a, Tim29 subunit forms extensive contacts with 443

both Tim9/10a and Tim9/10a/10b chaperones via the C-terminal segments. The interacted 444

regions are indicated by pink and red box, respectively. b, The overall structure of the Tim29 445

subunit. The C-terminus of Tim29 is divided into two segments: IMS (intermembrane space) 446

domain and CRM (C-terminal recruiting motif). The secondary structural elements are labeled. 447

c, d, Key residues mediating the interactions between Tim29 subunit and Tim9/10a (c) or 448

Tim9/10a/10b (d) are shown as sticks. 449

450

451

452

453

454

455



https://doi.org/10.1101/869289


21

456

457

Fig. 4 | Structure of the Tim9/10a/10b hexamer. a, Two perpendicular views of 458

Tim9/10a/10b hexamer. The Tim9/10a/10b hexamer forms a pore. b, Structural 459

superimposition of Tim9, Tim10a and Tim10b. Disulfide bonds formed from the signature 460

cysteines of twin CX3C motif are indicated. The C (center) loop is highlighted by green box. c, 461

Structural alignment of the C-loop of Tim9, Tim10a, and Tim10b. d, Sequence alignment of 462

Tim9, Tim10a and Tim10b. The conserved residues are colored red. Residues in the C loop 463

involved in the interaction with the Tim29 subunit are colored green. Cysteines in the twin 464

CX3C motif forming the disulfide bonds are shown with yellow numbers. Proline72 is colored 465

purple. The inner and outer helices are drawn above the sequences. 466

467

468

469

470

471

472

473

474

475



https://doi.org/10.1101/869289


22

476

477

Fig. 5 | Structure of the AGK subunit. a, Two opposing views of AGK, which is composed 478

by domain 1 and domain 2. The first -helix (light pink) is embedded into the membrane. 479

Key residues bound to the membrane are shown as orange sticks. b, A positively charged 480

cavity of AGK is formed in between the first -helix and the anchor. c, Structural alignment 481

of AGK (Homo sapiens) and DgkB (Staphylococcus aureus, PDB code: 2VQ7). AGK 482

exhibits additional membrane anchor (a dashed circle) and the transmembrane d, e, Key 483

residues involved in the interactions between AGK and Tim29 (d) or Tim9/10a/10b chaperone 484

(e) are shown as sticks. 485

486



https://doi.org/10.1101/869289


23

Methods 487

Transient expression of the human TIM22 complex 488

The codon-optimized full-length cDNAs for subunits of the human TIM22 complex were 489

synthesized by General Biosystems Company (Tim22, Uniport: Q9Y584; Tim29, Uniport: 490

Q9BSF4; Tim9, Uniport: Q9Y5J7; Tim10a, Uniport: P62072, Tim10b, Uniport: Q9Y5J6; 491

AGK, Uniport: Q53H12). TIM22, TIM29 and AGK were subcloned into the plasmid A; TIM9, 492

TIM10A and TIM10B were subcloned into the plasmid B using the pMlink vector1. Tim22 493

contains a N-terminal triple Flag tag. Expi293FTM

(Invitrogen) cells were cultured in SMM 494

293TI medium (Sino Biological Inc.) at 37 °C under 5% CO2 in a shaker and diluted into 2.0 495

× 106 cells ml

-1 with fresh medium when the cell density reached 3.5 × 10

6 ~ 4.0 × 10

6 cells 496

ml-1

for further transfection. For 1 liter cell culture, 1.4 mg plasmid A and 0.6 mg plasmid B 497

were pre-incubated with 4 mg linear polyethylenimines (PEIs) (Polysciences) in 50 ml fresh 498

medium for 20 min. The transfection was initiated by adding the mixture into the diluted cell 499

culture. Transfected cells were cultured for 48 hours before harvesting. 500

501

Mitochondria Preparation 502

The cells were harvested by centrifugation at 800 g for 20 min and washed with PBS, then 503

resuspended in the buffer A containing 10 mM Tris-HCl pH 7.5, 70 mM sucrose, 210 mM 504

mannitol, 1 mM EDTA, 1mg ml-1

BSA and 1mM PMSF2. The cells were disrupted using 505

dounce homogenizer (sigma) for 80 cycles on ice and the homogenate was centrifuged at 506

3,000 g for 10 min. The supernatant was further centrifuged at 20,000 g for 20 min to obtain 507

the crude mitochondrial pellet. The pellet was resuspended in buffer B containing10 mM Tris 508



https://www.uniprot.org/uniprot/Q9Y584

https://www.uniprot.org/uniprot/Q9BSF4

https://www.uniprot.org/uniprot/Q9Y5J7

https://www.uniprot.org/uniprot/P62072

https://www.uniprot.org/uniprot/Q9Y5J6

https://www.uniprot.org/uniprot/Q53H12

https://doi.org/10.1101/869289


24

pH 7.5, 250 mM sucrose, 60 mM KCl and 0.1 mM EDTA and centrifuged at 60,000 g at 4 °C 509

for 20 min in a discontinuous Percoll density gradient3. The clear mitochondria layer was 510

obtained carefully and diluted with buffer C containing 10 mM Tris-HCl pH 7.5, 70 mM 511

sucrose, 210 mM mannitol. The highly pure mitochondria were collected by centrifugation at 512

20,000 g for 30 min and stored in buffer C at -80 °C before use. 513

514

Purification of the TIM22 complex 515

The TIM22 complex were extracted from pure mitochondria by 1% LMNG (Anatrace), 0.25% 516

soybean lipids (Sigma) and 0.1 % CHS (Anatrace) in lysis buffer containing 25 mM HEPES 517

pH 7.4, 100 mM KOAc, 10 mM Mg(OAc)2, 0.1 mM EDTA, 10% glycerol, 1 mM PMSF, 4 518

µg ml-1

pepstatin A, 4 µg ml-1

aprotinin and 5 µg ml-1

leupeptin at 4 °C for 1.5 h4. The 519

extraction was centrifuged at 100,000 g for 30 min to remove the unsoluble component. The 520

supernatant was incubated with anti-Flag G1 affinity resin (Genscript) at 4 °C for 2 h and then 521

washed with 30 bed volumes of lysis buffer added 0.05% GDN (Anatrace). The protein was 522

eluted with lysis buffer added 0.05% GDN and 300 g ml-1

Flag peptide (Genscript). The 523

protein solution was concentrated with a 100-kDa cut-off centricon (Milipore) and further 524

purified by Superose-6 increase 10/300 column (GE Healthcare) using a buffer containing 50 525

mM Imidazole pH 6.0, 150 mM NaCl, 2 mM MgCl2 and 0.05% GDN5. The peak fractions 526

were pooled and concentrated to 7 mg ml-1

for further cryo-EM study. 527

528

Blue native-PAGE analysis 529

Blue native PAGE technique was used to determine native TIM22 complex mass as described 530



https://doi.org/10.1101/869289


25

previously6. Chromatographically purified TIM22 complex sample was mixed with 10 × 531

loading buffer (0.1% (w/v) Ponceau S, 50% (w/v) glycerol) and subjected to 4%-16% blue 532

native PAGE mini gel (Invitrogen) for electrophoresis at 4 °C. The TIM22 complex was 533

transferred onto a PVDF membrane and detected by immunoblotting using Tim22 antibody 534

(cat number: 14927-1-AP, Proteintech). The size of the TIM22 complex was determined 535

based on the mobility pattern of a native protein molecular weight standard (cat number: 536

LC0725, Invitrogen). 537

538

Mass spectrometry analysis 539

The TIM22 complex proteins were separated by 1D SDS-PAGE, the gel bands of interest 540

were excised from the gel, reduced with 5 mM of dithiotreitol, and alkylated with 11 mM 541

iodoacetamide. In gel digestion was then carried out with sequencing grade modified trypsin 542

in 50 mM ammonium bicarbonate at 37 °C overnight. The peptides were extracted twice with 543

0.1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min. Extracts were then 544

centrifuged in a speedvac to reduce the volume. Tryptic peptides were redissolved in 20 l 0.1% 545

TFA and analyzed by LC-MS/MS. 546

547

Cryo-EM Data Acquisition 548

For cryo-EM sample preparation, 3.5 l aliquots of the recombinant human TIM22 complex 549

(7 mg ml-1

) were dropped onto glow discharged holey carbon grids (Quantifoil Au R1.2/1.3, 550

300 mesh), blotted with a Vitrobot Mark IV (ThemoFisher Scientific) using 5 s blotting time 551

with 100% humidity at 8 °C, and plunged into liquid ethane cooled by liquid nitrogen. The 552



https://doi.org/10.1101/869289


26

sample was imaged on an FEI Titan Krios transmission electron microscope at 300 kV with a 553

magnification of 29,000 ×. Images were recorded by a Gatan K2 Summit direct electron 554

detector using the counting mode. Defocus values varied from -1.8 to -2.5 m. Each image 555

was dose fractionated to 32 frames with a total electron dose of 60 e- Å

-2 and a total exposure 556

time of 8.0 s. SerialEM7 was used for fully automated data collection. All stacks were motion 557

corrected using MotionCor28 with a binning factor of 1, resulting in a pixel size of 1.014 Å 558

and dose weighting was performed concurrently. The defocus values were estimated using 559

Gctf9. 560

561

Preliminary data processing 562

1,951 micrographs were used for initial data analysis. A small sets of particles for the TIM22 563

complex were auto-picked using reference-free autopicking method with 564

Laplacian-of-Gaussian in RELION3.010

. Good 2D averages were generated after 2D 565

classification and 552,779 particles were auto-picked with selected good 2D averages as 566

template. One round of 2D classification was performed, remaining 305,689 good particles. 567

Initial model was generated with stochastic gradient descent method in RELION10

using 568

about 1000 particles. Global 3D classification was performed with this initial model as 569

reference, generating one good class with 36.8% of input particles and three bad classes. 570

571

Data processing of the TIM22 complex 572

For the complete data set of the TIM22 complex, 2,401,464 particles were auto-picked from 573

14,105 micrographs. A guided multi-reference global classification procedure was applied to 574



https://doi.org/10.1101/869289


27

the full dataset. One good and five bad references from preliminary data processing were used 575

as the initial references (Round 1). These six references were low-pass filtered to 40 Å. To 576

avoid the problem of discarding good particles, we simultaneously performed three parallel 577

multi-reference 3D global classifications. After the global classification, particles that belong 578

to the good classes from 24/27/30 iterations of the three parallel runs were separately used as 579

the input for follow-up local classification. Six 3D models from global classification were 580

used as references for the multi-reference local classification. Good particles from these 3X3 581

parallel runs were merged, and the duplicated particles were removed as described 582

previously11

. 1,107,389 particles (46.1% of the original input) remained for following 583

processing. 584

A second round (Round 2) of multi-reference local 3D classification was performed with 585

five references (from global classification, scale to pixel size 2.028 Å) using re-extracted and 586

re-centered 2x binned particles (pixel size: 2.028 Å). Particles from the good classes 587

(representing 45.4/47.8/50.2% of the input particles) were combined to yield 621,049 588

particles (representing 25.9% of the total original particles). 589

A third round (Round 3) local classification was performed using unbinned particles (pixel 590

size: 1.014 Å), focusing on the inter-membrane region. The largest class of 482,959 particles 591

(representing 77.8% of the input particles or 20.1% of the total original particles) yielded an 592

average resolution of 3.73 Å after auto-refinement. With application of a soft mask on the 593

inter-membrane region, these particles gave an average resolution of 3.53 Å for this region 594

after continuous auto-refinement. 595

In the 3.73Å overall and 3.53Å intermembrane region maps of the TIM22 complex, the 596



https://doi.org/10.1101/869289


28

local resolution reaches 3.2-3.5 Å in the core regions. The angular distributions of the 597

particles used for the final reconstruction of the TIM22 complexes are reasonable, and the 598

refinement of the atomic coordinates did not suffer from severe over-fitting. The resulting EM 599

density maps display distinguishing features for the amino acid side chains in most regions. 600

Reported resolutions were calculated on the basis of the FSC 0.143 criterion, and the FSC 601

curves were corrected for the effects of a soft mask on the FSC curve using high-resolution 602

noise substitution12

. Prior to visualization, all density maps were corrected for the modulation 603

transfer function (MTF) of the detector, and then sharpened by applying a negative B-factor 604

that was estimated using automated procedures13

. Local resolution variations were estimated 605

using RELION10

. 606

607

Model building and refinement 608

We combined homology modeling and de-novo model building to generate the atomic 609

models. 610

Identification and docking of the two chaperones Tim9/10a and Tim9/10a/10b were 611

facilitated by the crystal structure of the human mitochondrial Tim9/10a hexametric complex 612

(PDB code: 2BSK). Tim10b was manually identified by comparing the sequence variance of 613

these three proteins and the cryo-EM density, and then manually build by COOT14

. 614

Identification of AGK protein was facilitated by the homology structure of a diacylglycerol 615

kinase DgkB from Staphylococcus aureus (PDB code: 2QV7). The atomic model of AGK 616

were generated by CHAINSAW15

and the backbone was manually adjusted using COOT14

. 617

After that, automated model rebuilding was performed with RosettaCM using the adjusted 618



https://doi.org/10.1101/869289


29

model as the template and the experimental cryo-EM density as a guide16-18

. Then the 619

hydrogen atoms of the generated model were removed and model building was further 620

performed manually using COOT14

, The single trans-membrane helix and a membrane 621

anchoring motif was de-novo built using COOT14

. 622

The intermembrane space (IMS) domain of Tim29 was located under the Tim9/10a/10b 623

hexamer. The local resolution of IMS domain in cryo-EM map reaches around 3.2 Å, 624

allowing us de-novo building of these regions. The transmembrane helix and a C-terminal 625

fragment which recruits the Tim9/10a chaperone was also de-novo built by COOT14

. A 626

N-terminal helix of Tim29 was identified to be located in the matrix, adhering to the 627

membrane and stabilizing the four transmembrane helices Tim22. Due to limited resolution 628

(5~7 Å), only poly-ALA helix was built for the N-helix. 629

Tim22 is supposed to have four transmembrane helices19

. The cryo-EM density of the 630

membrane region is relatively lower than the soluble region, ranging from 5 Å to 3.2 Å. 631

Luckily, the local resolution of the loop between TM2 and TM3 is high enough, around 3.5 Å, 632

which allow us identify three bulky residues (Y146, R147, and W152) in the linker. The 633

density of the loop connecting TM3 and TM4 can also be clear visualized, which contacts 634

with N-helix of Tim29 in the mitochondrial matrix. A potential S-S bond between TM1 and 635

TM2 can be clearly visualized, which help us assign the sequence. A N-terminal plug bound 636

to Tim9/10a/10b hexamer was also manually built by COOT14

. 637

The final model of the TIM22 complex was refined against the overall cryo-EM maps 638

using PHENIX20

in real space with secondary structure restraints. Overfitting of the overall 639

model was monitored by refining the model in one of the two independent maps from the 640



https://doi.org/10.1101/869289


30

gold-standard refinement approach, and testing the refined model against the other map21

. The 641

structures of the TIM22 complex were validated through examination of the Molprobity 642

scores and statistics of the Ramachandran plots. Molprobity scores were calculated as 643

described22

. 644

645

References 646

647

1 Lu, P. et al. Three-dimensional structure of human gamma-secretase. Nature 512, 648

166-170 (2014). 649

2 Clayton, D. A. & Shadel, G. S. Isolation of Mitochondria from Tissue Culture Cells. 650

Cold Spring Harbor Protocols 2014, 1109-1111 (2014). 651

3 Sims, N. R. & Anderson, M. F. Isolation of mitochondria from rat brain using Percoll 652

density gradient centrifugation. Nat Protoc 3, 1228-1239 (2008). 653

4 Rehling, P. et al. Protein insertion into the mitochondrial inner membrane by a 654

twin-pore translocase. Science 299, 1747-1751 (2003). 655

5 Chari, A. et al. ProteoPlex: stability optimization of macromolecular complexes by 656

sparse-matrix screening of chemical space. Nat Methods 12, 859-865 (2015). 657

6 Wittig, I., Braun, H. P. & Schagger, H. Blue native PAGE. Nat Protoc 1, 418-428 658

(2006). 659

7 Mastronarde, D. N. Automated electron microscope tomography using robust 660

prediction of specimen movements. J Struct Biol 152, 36-51 (2005). 661

8 Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for 662

improved cryo-electron microscopy. Nat Methods 14, 331-332 (2017). 663

9 Zhang, K. Gctf: Real-time CTF determination and correction. J Struct Biol 193, 1-12 664

(2016). 665

10 Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM 666

structure determination with parallelisation using GPUs in RELION-2. Elife 5, 667

e18722 (2016). 668

11 Yan, C., Wan, R., Bai, R., Huang, G. & Shi, Y. Structure of a yeast activated 669

spliceosome at 3.5 A resolution. Science 353, 904-911 (2016). 670

12 Chen, S. et al. High-resolution noise substitution to measure overfitting and validate 671



https://doi.org/10.1101/869289


31

resolution in 3D structure determination by single particle electron cryomicroscopy. 672

Ultramicroscopy 135, 24-35 (2013). 673

13 Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, 674

absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol 675

Biol 333, 721-745 (2003). 676

14 Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta 677

Crystallogr D Biol Crystallogr 60, 2126-2132 (2004). 678

15 Stein, N. CHAINSAW: a program for mutating pdb files used as templates in 679

molecular replacement. J Appl Crystallogr 41, 641-643 (2008). 680

16 DiMaio, F., Tyka, M. D., Baker, M. L., Chiu, W. & Baker, D. Refinement of protein 681

structures into low-resolution density maps using rosetta. J Mol Biol 392, 181-190 682

(2009). 683

17 Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 684

1735-1742 (2013). 685

18 DiMaio, F. et al. Atomic-accuracy models from 4.5-A cryo-electron microscopy data 686

with density-guided iterative local refinement. Nat Methods 12, 361-365 (2015). 687

19 Wrobel, L., Sokol, A. M., Chojnacka, M. & Chacinska, A. The presence of disulfide 688

bonds reveals an evolutionarily conserved mechanism involved in mitochondrial 689

protein translocase assembly. Sci Rep 6, 27484 (2016). 690

20 Adams, P. D. et al. PHENIX: a comprehensive Python-based system for 691

macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221 692

(2010). 693

21 Amunts, A. et al. Structure of the Yeast Mitochondrial Large Ribosomal Subunit. 694

Science 343, 1485-1489 (2014). 695

22 Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins 696

and nucleic acids. Nucleic Acids Res 35, W375-383 (2007). 697

698

699



https://doi.org/10.1101/869289


32

ACKNOWLEDGMENTS 700

We thank Dr. Chang at Center of Cryo Electron Microscopy, Zhejiang University, for 701

assistance during data collection; and research associates at the Center for Protein Research 702

and Public Laboratory of Electron Microscopy, Huazhong Agricultural University, for 703

technical support. We thank the Tsinghua University Branch of China National Center for 704

Protein Sciences (Beijing) for providing the technical support on the Cryo-EM and 705

High-Performance Computation platforms. We thank Meng Han and protein chemistry 706

Facility at the Center for biomedical Analysis of Tsinghua University for sample mass-spec 707

analysis. We thank Ms. Wang Yan for model figure design. This work was supported by funds 708

from the Ministry of Science and Technology of China (2018YFA0507700), the National 709

Natural Science Foundation of China (31722017), the Fok Ying-Tong Education Foundation 710

(151021), and the Fundamental Research Funds for the Central Universities (2662017PY031). 711

This research was supported by Beijing Advanced Innovation Center for Structural Biology 712

(to Dr. Chuangye Yan). 713

AUTHOR CONTRIBUTIONS 714

P.Y. conceived the project. L.Q., Q.W., P.Y. designed all experiments. L.Q., Q.W., and Y.W. 715

performed the experiments. Q.W., Z.G., J.C., and X. Z collected the EM data. Z.G. and C. Y. 716

determined the structure. All authors analyzed the data and contributed to manuscript 717

preparation. P.Y wrote the manuscript. 718

719



https://doi.org/10.1101/869289


33

720

Extended Data Fig. 1 | Blue native PAGE analysis of the TIM22 complex. Purified TIM22 721

complex sample was analyzed by Blue native PAGE. The complex bands were visualized by 722

Coomassie blue staining (left panel) and western blot (right panel). 723

724



https://doi.org/10.1101/869289


34

725

726

Extended Data Fig. 2 | Flowchart for cryo-EM data processing of the human TIM22 727

complex. On the basis of the FSC value of 0.143, the final reconstruction has an average 728

resolution of 3.73 Å for the overall map, 3.53 Å for the inter-membrane region. Details are 729

presented in Methods. 730

731

732

733

734

735



https://doi.org/10.1101/869289


35

736

737

Extended Data Fig. 3 | Cryo-EM analysis of the human TIM22 complex. a, The average 738

resolutions for the TIM22 complex are estimated to be 3.73 Å and 3.53 Å for the overall and 739

inter-membrane region, respectively. The resolutions are reported on the basis of the FSC 740

criterion of 0.143. b, Angular distribution of the particles used for reconstruction of the 741

TIM22 complex. Each cylinder represents one view and the height of the cylinder is 742

proportional to the number of particles for that view. c, The local resolutions are color-coded 743

for the TIM22 complex (left panel) and the inter-membrane region (right upper panel). The 744

highest resolution of the EM maps reaches 3.2 Å. d, The FSC curves of the final refined 745

models of the TIM22 complex versus the overall maps that it is refined against (black); of the 746

model refined in the first of the two independent maps used for the gold-standard FSC versus 747

that same map (red/purple); and of the model refined in the first of the two independent maps 748

versus the second independent map (green/blue). The generally similar appearances between 749

the purple and blue, red and green curves indicate that the refinement of the atomic 750

coordinates did not suffer from severe over-fitting. 751

752

753

754

755

756



https://doi.org/10.1101/869289


36

757

758



https://doi.org/10.1101/869289


37

Extended Data Fig. 4 | The electron microscopy maps for the subunits of the TIM22 759

complex. a-c, The electron microscopy maps for the Tim9/10a/10b hexamer: Overview of 760

chaperone Tim9/10a/10b , composed of Tim9, Tim10a and Tim10b in a ratio of 2:3:1 (a); A 761

close up view for representative Tim9, Tim10a and Tim10b (b); Three segments 762

(helix-loop-helix) of Tim9, Tim10a and Tim10b (c). d, The electron microscopy maps for 763

chaperone Tim9/10a. e, The 4 transmembrane segments (TM1-4), intermembrane space 764

segments (24-50) and loop (142-154) of Tim22. f, AGK -1 (the predicted TM), 225-238 (the 765

membrane-anchored region), 73-102, 113-139, 162-185, and 403-415 of AGK. g, The 766

transmembrane segments (58-74) and intermembrane space segments (79-117, 131-167, 767

168-190, 201-215) of Tim29. 768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787



https://doi.org/10.1101/869289


38

788

789

Extended Data Fig. 5 | Topology diagrams of the TIM22 complex. a, Secondary elements 790

are labeled. Tim29, yellow; Tim22, orange; AGK, chartreuse; Tim9/10a, slate; Tim9/10a/10b, 791

blue; Tim10b, magenta. b, The topology diagram of AGK. 1, light pink; anchor, orange; 792

domain 1 (2~7, 2 and ~) and domain 2 (8~10 and ~793

794

795



https://doi.org/10.1101/869289


39

796

797

798

799

800

801

802



https://doi.org/10.1101/869289


40

Extended Data Fig. 6 | The N-terminal helix and disulfide bond in Tim22. a, Cartoon and 803

surface model of Tim22 and Tim9/10a/10b. Plug indicates the first -helix of Tim22. b, 804

Disulfide bond between C69 and C141 of Tim22. c, The sequences (Uniprot code: Homo 805

sapiens, Q9Y584; Mus musculus, Q9CQ85; Xenopus laevis, Q5U4U5; Drosophila 806

melanogaster, Q8IN78; Saccharomyces cerevisiae, Q12328; Neurospora crassa, Q9C1E8) 807

are aligned using ClustalW. Residues involved in the interaction with Tim9 and Tim10a are 808

highlighted with cyan and slate balls. Cysteines forming disulfide bond are highlighted using 809

yellow boxes. The secondary structural elements are indicated above the sequences. 810

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832



https://doi.org/10.1101/869289


41

833

834

Extended Data Fig. 7 | A phospholipid in the TIM22 complex and a new putative 835

chaperone recruiting interface of Tim29. a, A phospholipid, binding at the interface 836

between Tim29 and inner membrane. The aliphatic tails of the latter phospholipid may 837

interact with several hydrophobic residues (V125, L127, L129, W156 and F159). The 838

phosphate group likely to interact with several hydrophilic residues (Y134, and R162). b, The 839

electron microscopy maps for the phospholipid resembling to the structure of 840

Phosphatidylethanolamine (PDB code: PTY). c, d, Electrostatic surface of TIM29. A top view 841

(from IMS to matrix) of TIM22 complex is shown as colored surface or electrostatic surface 842

potential. The interface 1 is framed out using a slate circle. A putative interface for recruiting 843

another chaperone is framed out using a yellow dashed circle. Tim9/10a is omitted for clarity 844

in (D). AGK chartreuse; Tim22, orange; Tim9/10a/10b, blue; Tim9/10a, slate; Tim29, 845

electrostatic surface potential. 846



https://doi.org/10.1101/869289


42

847

848

Extended Data Fig. 8 | Center pore comparison of Tim9/10a and Tim9/10a/10b hexamer. 849

a, Two perpendicular views of Tim9/10a/10b hexamer. b, Structural alignment of Tim9/10 850

and Tim9/10a/10b. Tim9, cyan; Tim10a, slate or peal blue; Tim10b, magenta. c, A structure 851

alignment of chaperone Tim9/10a/10b with free chaperone Tim9/10a (PDB code: 2BSK). 852

Tim9, cyan; Tim10a, blue; Tim10b, magenta; free chaperone Tim9/10a, wheat. d, A structure 853

alignment of chaperone Tim9/10a/10b with free chaperone Tim9/10a (PDB code: 2BSK) are 854

shown as colored surface: inner helices of Tim9/10a/10b, yellow; outer helices of 855

Tim9/10a/10b and free Tim9/10a, slate; Tim10b, magenta. 856

857



https://doi.org/10.1101/869289


43

858

859

Extended Data Fig. 9 | Implication on carrier precursor insert into inner membrane. 860

Cartoon model of carrier precursor insertion into the membrane through The TIM22 complex. 861

The carrier precursor (red line, left panel) wraps around the Tim9/10a chaperone and then 862

inserts into the membrane (red stick, middle and right panel) through the cavity of AGK by a 863

“bending-in” mechanism. 864

865



https://doi.org/10.1101/869289


44

Extended data Table 1 | Statistics of Cryo-EM data collection and refinement of the 866

human TIM22 complex. 867

868

Data collection

EM equipment FEI Titan Krios

Voltage (kV) 300

Detector Gatan K2

Pixel size (Å) 1.014

Electron dose (e-/Å2) 60

Defocus range (m) -1.8 ~ -2.5

Reconstruction

Software RELION 3.0

Number of used Particles 482,959

Accuracy of rotation (˚) 1.446 (1.386)

Accuracy of translation (Å) 0.852 (0.785)

Final Resolution (Å) 3.73 (3.53)

Model building

Software COOT/ROSETTA

Refinement

Software PHENIX

Map sharpening B-factor (Å2) -162.8

Average Fourier shell correlation 0.827

R-factor 0.335

Model composition

Protein residues 1599

Lipid 1

Validation

R.m.s deviations

Bonds length (Å) 0.012

Bonds Angle (˚) 0.908

Ramachandran plot statistics (％)

Preferred 91.22

Allowed 8.45

Outlier 0.33

Molprobity score 14.08

869

870

871

872

873

874

875



https://doi.org/10.1101/869289


45

Extended data Table 2 | Summary of model building for the human TIM22 complex. 876

877

Length Domain Copies PDB code Modeling1 Resolution (Å) Chain ID

Tim22 194 24-194 1 De novo 3.5-6.0 A

AGK 422 23-415 1 2QV7 HM 3.5-4.2 B

Tim29 260 21-244 1 De novo 3.2-5.2 C

Tim9

89 5-82 5 2BSK RD 3.2-4.0 D-F, K, L

Tim10a 90 2-80 6 2BSK RD 3.2-3.8 G-I, M-O

Tim10b 103 3-85 1 2BSK HM 3.2-3.8 J

878 1Abbrivations in the “Modeling” column are as follows. HM, homology modeling; RD, rigid 879

docking and refinement; De novo, de novo model building. 880

881



https://doi.org/10.1101/869289


Documents

Cryo-EM Structure of the Human Mitochondrial Translocase … · The Tim9/10a/10b hexamer, similar to a 118 hub, was located at the center of TIM22 complex, and was encircled by the